Assay for determination of functional immunity against salmonella

Abstract

Method of performing SBA assay comprising: preparing media and buffer; preparing bacterial stock with a mother culture stock, media and buffer; diluting bacterial stock with buffer to obtain a dilute

Description

First Aspect: METHOD In one aspect, the present invention is directed to a method of performing Serum Bactericidal Activity Assay (hereinafter referred to as ‘SBA’ or ‘the assay’ interchangeably). The assay is a simple, cost effective, robust, less time consuming, consistent, precise and accurate method for evaluation of the functional antibody response induced by an immunogenic composition. In one aspect, the present invention is directed to a method of performing Serum Bactericidal Activity Assay, the method comprising: a. preparing a media and a buffer; b. preparing a bacterial stock with a mother culture stock, the media and the buffer, and diluting the bacterial stock with the buffer; c. adding a test serum, a complement, the bacterial stock and the buffer to form an assay solution; d. incubating the assay solution, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate; and e. calculating antibody titre to determine the serum bactericidal activity. In another aspect, the method for performing a SBA assay comprises: i. providing the media and the buffer; ii. preparing the bacterial stock with the mother culture stock, the media and the buffer; iii. diluting the bacterial stock with the buffer to obtain the diluted bacterial stock; iv. adding the test serum, the complement, the diluted bacterial stock, and the buffer to form the assay solution; v. incubating the assay solution, spotting the assay solution on the agar plate and incubating the agar plate; and vi. calculating antibody titre to determine the serum bactericidal activity. In yet another aspect, the method for performing a SBA assay comprises: i. providing the media and the buffer; ii. preparing the bacterial stock with the mother culture stock and the media; iii. diluting the bacterial stock with the buffer to obtain the diluted bacterial stock; iv. adding a test serum, the complement, the diluted bacterial stock, and the buffer to form the assay solution; v. incubating the assay solution, spotting the assay solution on the agar plate and incubating the agar plate; and vi. calculating antibody titre to determine the serum bactericidal activity. In an embodiment, the assay provides evaluation of functional antibodies induced in a subject administered with vaccines. The vaccines include conjugated and non-conjugated vaccines. The conjugate vaccines include monovalent, bivalent, trivalent, tetravalent, and / or multivalent vaccines. In another embodiment, the present invention relates to a method of performing Serum Bactericidal Activity Assay, wherein the assay includes: • preparation of the media and the buffer; • preparation of the bacterial stock with the mother culture stock for S. Typhi / S. Paratyphi / both, the media and the buffer; • preparation of the bacterial assay stock (also referred to herein as diluted bacterial stock) for titration for S. Typhi / S. Paratyphi / both; • performing assay for S. Typhi / S. Paratyphi / both with addition of the test serum, the complement, the bacterial stock to form an assay solution; • incubating the assay solution, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate; • calculating antibody Titre for assay solution for S. Typhi / S. Paratyphi / both; • optionally validating results. In another embodiment, the present invention relates to a method of determining antibody titre of serum against S. Typhi or S. Paratyphi, wherein the method comprises: • providing the media and the buffer; • preparing the bacterial stock with the mother culture stock for S. Typhi, the media and optionally the buffer; or preparing the bacterial stock with the mother culture stock for S. Paratyphi, the media and optionally the buffer; • diluting the bacterial stock with the buffer to obtain the diluted bacterial stock for S. Typhi or the diluted bacterial stock for S. Paratyphi; • adding a test serum, the complement, the diluted bacterial stock for S. Typhi, and the buffer to form an assay solution for determining antibody titre against S. Typhi; or adding a test serum, the complement, the diluted bacterial stock for S. Paratyphi, and the buffer to form an assay solution for determining antibody titre against S. Paratyphi; • incubating the assay solution, spotting the assay solution on an agar plate and incubating the agar plate; and • calculating antibody titre for the assay solution corresponding to S. Typhi to determine the serum bactericidal activity against S. Typhi or calculating antibody titre for the assay solution corresponding to S. Paratyphi to determine the serum bactericidal activity against S. Paratyphi. In another embodiment, the present invention relates to a method for performing a Serum Bactericidal Assay (SBA), comprising: i. preparing the bacterial stock with the mother culture stock and a media; ii. diluting the bacterial stock with the buffer to obtain the diluted bacterial stock that yields 50 to 250 colony forming units (cfus); iii. adding the test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. In another embodiment, the present invention relates to a method for determining antibody titre of a test serum, comprising: i. preparing the bacterial stock with the mother culture stock and the media; ii. diluting the bacterial stock with the buffer to obtain the diluted bacterial stock that yields 50 to 250 colony forming units (cfus); iii. adding a test serum, the complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on the agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. In another embodiment, the present invention relates to a method for performing a serum bactericidal activity assay, comprising: i. preparing and providing the bacterial stock; ii. diluting the bacterial stock with the buffer to obtain the diluted bacterial stock that yields 50 to 250 colony forming units (cfus); iii. adding a test serum, the complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on the agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. In another embodiment, the present invention relates to a method for performing a serum bactericidal activity assay, comprising: i. preparing and providing the bacterial stock; ii. diluting the bacterial stock 1:100 to 1:6000 with the buffer to obtain the diluted bacterial stock; iii. adding the test serum, the complement, the diluted bacterial stock, and the buffer to form the assay solution; iv. incubating the assay solution, spotting the assay solution on the agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. The steps of the above methods are described as herein below. PREPARING AND / OR PROVIDING MEDIA AND BUFFER In an embodiment, the SBA assay includes preparing / providing the media and the buffer. MEDIA The preparation of the media includes addition of the media in glass bottle to make a 1000 ml volume with de-ionized water. Media is mixed, dissolved, sterilized and stored at low temperature. In some embodiments, the media may be a sterile ready-to-use media. The media for the SBA assay includes lysogeny broth, Luria Bertani broth, Hi Soy (Hi Bacteriological media), tryptone, peptone, yeast extract, Brain Heart Infusion broth, Tryptic Soy broth, Todd-Hewitt Yeast Extract broth, nutrient broth etc. or combinations thereof. In a preferred embodiment, the media is lysogeny broth, or Luria Bertani broth. In another embodiment, an 80% glycerol is prepared by mixing de-ionized water and glycerol. The 80% glycerol is prepared and added to Luria Bertani broth for preparation of bacterial glycerol stocks. The glycerol is used as a cryo-protectant for long term storage of bacterial stocks at -70°C. In a preferred embodiment, 80% glycerol is prepared by mixing 20 ml of de-ionized water and 80 ml glycerol and sterilized at 121 °C for 15 minutes by autoclaving. In another embodiment, the present invention is directed to a method of Serum Bactericidal Activity Assay with an agar medium including a blood agar, Luria Bertani agar, etc. Bacterial growth on the blood agar is prolific and colonies merge easily and can lead to variation in count. Luria Bertani agar plates are associated with well-defined smaller colonies and are easier to count both by manual counting and automated colony counter. In an embodiment, the agar medium used for the Serum Bactericidal Activity Assay is a Luria Bertani agar. The Luria Bertani agar when used for plating is associated with growth of both S. Typhi and S. Paratyphi colonies that are round, opaque and legible. In an embodiment, pH for Luria Bertani broth, Luria Bertani agar and buffer used for assay as well as titration was neutral (7.0 pH). Media preparation is defined in more details in the METHODS section below. BUFFER In an embodiment, the present invention is directed to a method of performing Serum Bactericidal Activity Assay, wherein the assay is performed using the buffer, also referred to interchangeably as assay dilution buffer. The assay is performed with a buffer that does not interfere with the bacterial growth and is composed to facilitate proper killing / neutralization of the bacteria in the presence of complement and antibody. The establishment of an appropriate buffer is therefore important. In an embodiment, the buffer includes a buffer selected from Hanks' Balanced Salt Solution (HBSS); a HBSS with calcium (Ca) and magnesium (Mg), i.e. a HBSS (+) buffer; a HBSS with Calcium and Magnesium andwithout phenol red; a HBSS without Calcium and Magnesium, i.e. HBSS (-) buffer; a HBSS without calcium and magnesium and without phenol red; sodium chloride; acetate carbonate; citrate; lactate; gluconate; tartrate; phosphate buffer saline; borate; Histidine buffer; Succinate buffer; HEPES; TRIS or Citrate-phosphate; Gey’s balanced salt solution (GBSS) with / without BSA or FBS; PBS with / without calcium and magnesium; saline with stabilizers like BSA or 1% to 5 % FBS; HBSS with 0.1 to 1 % gelatine. In a preferred embodiment, the buffer includes saline; phosphate; phosphate buffered saline; bovine serum albumin; Hanks' balanced salt solution (HBSS); fetal bovine serum (FBS); or combinations thereof. In an embodiment, the HBSS buffer includes D glucose (1000 mg / L), KC1 (400 mg / L), KH2PO4 (60 mg / L), NaCl (8000 mg / L), NaHCO3 (350 mg / L) and NaHPO4 (48 mg / L). When HBSS buffer is with calcium and magnesium, i.e., HBSS (+) buffer, the HBSS (+) buffer includes the above-mentioned components and further includes CaCL 2H2O (185 mg / L) and MgSO4 (98 mg / L). In another preferred embodiment, the assay is performed with the HBSS(-) buffer for S.Typhi. The term HBSS(-) buffer refers to the HBSS buffer without calcium and magnesium, however, including Fetal Bovine Serum (FBS). The buffer composition facilitates better clearing of the bacteria, enhancing killing and generating a smooth and accurate curve, allowing a distinct difference between pre and post sera samples not obtained with regular positive (+) buffers. Smooth curve in the context of the invention refers to gradual killing curve wherein bacterial CFU are gradually increasing with the dilution of the positive sera, thus giving a proper sigmoidal curve wherein a single distinguishable titre is attained and no multiple readouts and abrupt counts of CFU’s are encountered. In a more preferred embodiment, the buffer includes HBSS and FBS. In another more preferred embodiment, the assay is performed with the HBSS (-) buffer (i.e., the HBSS buffer without calcium and magnesium, but, containing FBS). In a further more preferred embodiment, the FBS is in the range of 0.10% to 10.0% v / v, or in the range of 0.10 to 9.0% v / v, or in the range of 0.10 to 8.0%v / v, or in the range of 0.10 to 7.0 % v / v, or in the range of 0.10 to 6.0% v / v or in the range of 0.10 to 5.0% v / v or in the range of 0.10 to 4.0% v / v. In a further more preferred embodiment, the FBS is in the range of 0.10 to 3.0% v / v, or in the range of 0.20 to 3.0% v / v, or in the range of 0.30 to 3.0% v / v, or in the range of 0.40 to 3.0% v / v, or in the range of 0.50 to 3.0% v / v. In a further more preferred embodiment, the FBS is in the range of 0.50 to 2.0% v / v. In a further more preferred embodiment, the HBSS (-) buffer includes HBSS comprising 1.0% FBS v / v for determining the antibody titre of test serum comprising antibodies for S. Typhi and shows a good clearance, i.e. killing of S. Typhi. In the assay for S. Typhi, the buffer / the assay dilution buffer is the HBSS(-) with 1% FBS buffer, i.e. the HBSS buffer with 1 % FBS and without calcium and magnesium. In a preferred embodiment, the assay is performed with the HBSS (+) buffer for S. Paratyphi. In the assay for S. Paratyphi, the buffer / the assay dilution buffer is the HBSS(+) buffer, i.e. the HBSS buffer having calcium and magnesium. In an embodiment, when the HBSS(+) buffer is used, the HBSS(+) buffer has calcium and magnesium. In another embodiment, the HBSS(+) buffer includes calcium in form of CaCh in range of 100 mg / L to 200 mg / L. In an alternate embodiment, calcium in the buffer is calcium chloride anhydride at concentration of 140 mg / L. In another embodiment, the HBSS(+) buffer includes magnesium in form of MgSO4 in range of 50 mg / L to 150 mg / L. In an alternate embodiment, the magnesium sulphate is lOOmg / L of MgSO4, 7H2O. In an embodiment, pH for both buffers used (i.e. HBSS+ and HBSS-) is neutral (7.0 pH) which helps for desired growth of bacterial cells. PREPARING A BACTERIAL STOCK AND DILUTING THE BACTERIAL STOCK In an embodiment, the SBA assay includes preparing a bacterial stock and diluting the bacterial stock. In an embodiment, the mother culture stock vial is retrieved from freezer, streaked on a blood agar plate and incubated at temperature in range of 35°C to 39°C, preferably at temperature in range of 36°C to 38°C or more preferably at 37°C with 5% in CO2 incubator for 14-16 hours. Blood agar plate is used during bacterial stock preparation on day one to ensure use of highly enriched media. Once bacterial working stocks are prepared, Luria Bertani agar plates are used during actual SBA assay. See Methods below for detailed Bacterial stock preparation. In an embodiment, loopful of isolated colonies are transferred from the blood culture agar plate and inoculated in a sterile conical flask with the Luria Bertani Broth to form a bacterial suspension. Flask is incubated for 2 to 4 hours in a shaker incubator at a temperature of 35°C to 39°C, preferably at a temperature of 36°C to 38°C or more preferably at 37°C with 120-180 RPM until the absorbance of the culture broth has an absorbance of ODeoo of desired value. Further, in an embodiment, after achieving desired value of ODeoo, the bacterial suspension is added to the Luria Bertani broth (having 80% glycerol), mixed by vertexing and dispensed in aliquots. Aliquots of bacteria is dispensed in labelled sterile cryo-vials. GROWTH PATTERN In another embodiment, for the method of SBA assay as described, the bacterial stock is prepared with bacteria in initial log phase, or in exponential phase, or in late log phase, or in the stationary growth phase. The present invention is directed to a method of performing the Serum Bactericidal Activity Assay, comprising culturing the bacteria and understanding the growth pattern, along with checking of vulnerability for neutralization of bacteria during the assay procedure. In another embodiment, the assay includes testing growth characteristics with respect to capsule formation and maturity of capsule during the growth phase. The growth phase is associated with evasion / escapement from complement binding, leading to resistance towards killing during the assay for S. Typhi. This is a critical aspect for the S. Typhi SBA, as results with conventional assay wherein this particular factor of bacterial growth and capsule maturation is not considered, leads to partial or no response for the sample testing, with no major difference observed for pre and post sera analysis. In another embodiment, the assay includes killing the bacteria during an initial Log phase of growth. In an embodiment, the S. Typhi bacterial stock is prepared with bacteria in initial log phase, wherein the S. Typhi bacterial stock preparation includes incubating S. Typhi bacteria from the S. Typhi mother culture stock at 37°C in 5% CO2 incubator for 12 to 16 hours in a blood agar plate and wherein the S. Typhi bacterial stock preparation includes further incubating S. Typhi bacteria from the blood agar plate on the media for 2 to 4 hours at 37°C in shaker incubator. In another embodiment, for the method of SBA assay disclosed herein, the bacterial stock is prepared with bacteria in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm. In an embodiment, the S. Typhi bacterial stock is prepared with bacteria in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm, wherein the S. Typhi bacterial stock preparation includes incubating S. Typhi bacteria from the S. Typhi mother culture stock at 37°C in 5% CO2 incubator for 12 to 16 hours in a blood agar plate and wherein the S. Typhi bacterial stock preparation includes further incubating S. Typhi bacteria from the blood agar plate on the media for 2 to 4 hours at 37°C in shaker incubator. In a preferred embodiment, the bacteria is S. Typhi and the initial Log phase was associated with OD value in the range of 0.10 to 0.50, or in the range of 0.20 to 0.50, or in the range of 0.30 to 0.50 or in the range of 0.30 to 0.40. In a more preferred embodiment, the assay includes killing the S. Typhi in the initial Log phase with OD value in the range of 0.30 to 0.40. In an embodiment, the S. Typhi bacterial stock is prepared with bacteria in initial log phase with absorbance value in range of 0.30 to 0.40 for Optical Density at 600 nm, wherein the S. Typhi bacterial stock preparation includes incubating S. Typhi bacteria from the S. Typhi mother culture stock at 37°C in 5% CO2 incubator for 12 to 16 hours in a blood agar plate and wherein the S. Typhi bacterial stock preparation includes further incubating S. Typhi bacteria from the blood agar plate on the media for 2 to 4 hours at 37°C in shaker incubator. In yet another preferred embodiment, the S. Typhi is susceptible to killing only during the initial Log phase and becomes completely resistant to killing during the stationary phase. In another embodiment, the assay includes killing the bacteria during late Log phase to stationary phase of growth. In an embodiment, the S. Paratyphi bacterial stock is prepared with bacteria in late log phase, wherein the S. Paratyphi bacterial stock preparation includes incubating S. Paratyphi bacteria from the S. Paratyphi mother culture stock at 37°C in 5% CO2 incubator for 12 to 16 hours in a blood agar plate and wherein the S. Paratyphi bacterial stock preparation includes further incubating S. Paratyphi bacteria from the blood agar plate on the media for 2 to 4 hours at 37°C in shaker incubator. In another embodiment, for the method of SBA assay disclosed herein, the bacterial stock is prepared with bacteria in late Log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm. In an embodiment, the S. Paratyphi bacterial stock is prepared with bacteria in late log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm, wherein the S. Paratyphi bacterial stock preparation includes incubating S. Paratyphi bacteria from the S. Paratyphi mother culture stock at 37°C in 5% CO2 incubator for 12 to 16 hours in a blood agar plate and wherein the S. Paratyphi bacterial stock preparation includes further incubating S. Paratyphi bacteria from the blood agar plate on the media for 2 to 4 hours at 37°C in shaker incubator. In a preferred embodiment, the bacteria is S. Paratyphi and the killing is associated with late Log phase to stationary phase of growth and OD value is in the range of 0.60 to 1.20, or in the range of 0.70 to 1.20, or in the range of 0.80 to 1.20 or in the range of 0.90 to 1.20. In another embodiment the OD value is in the range of 0.90 to 1.10 or in the range of 0.90 to 1.00. In a more preferred embodiment, the assay includes killing the S. Paratyphi with late Log phase to stationary phase of growth and OD value in the range of 0.90 to 1.10. In an embodiment, the S. Paratyphi bacterial stock is prepared with bacteria in late log phase with absorbance value in range of 0.90 to 1.10 for Optical Density at 600 nm, wherein the S. Paratyphi bacterial stock preparation includes incubating S. Paratyphi bacteria from the S. Paratyphi mother culture stock at 37°C in 5% CO2 incubator for 12 to 16 hours in a blood agar plate and wherein the S. Paratyphi bacterial stock preparation includes further incubating S. Paratyphi bacteria from the blood agar plate on the media for 2 to 4 hours at 37°C in shaker incubator. In another preferred embodiment the S. Paratyphi is associated with the formation of no capsular structure and antibodies are able to directly target the LPS on the cell wall and the effect of different growth stages (early log, log and stationary phase) does not affect the bactericidal activity. Preparation of the Bacterial stock for both S. Typhi and S. Paratyphi is listed below in METHODS section. DILUTING THE BACTERIAL STOCK In an embodiment, the method of Serum Bactericidal Activity assay includes dilution of the bacterial stock with the buffer. In an embodiment, the preparation of bacterial stock includes preparing frozen glycerol stock with specific growth phase of bacteria and performing bacterial titration to know the exact dilution required to achieve required CFU in each well. In another embodiment, the frozen assay stock of the desired strain from freezer is thawed. Frozen assay stock is added to the buffer or the assay dilution buffer (HBSS (-) +1% FBS for S. Typhi and HBSS (+) for S. Paratyphi A), mixed and aliquots are prepared for further dilution. Further dilutions are made if needed to obtain 1:2000, 1:3000, 1:4000, 1:5000, 1:6000 and other dilutions. Spots of aliquots from each well is taken on Media plate in duplicate and incubated overnight at 37°C in 5% CO2 incubator. Colonies are counted in each growth and average count for each dilution is taken. Ideal colonies for assay was taken between 50 to 250 colony forming units. The method of Serum Bactericidal Activity assay as disclosed includes dilution of the bacterial stock, wherein the bacterial stock is diluted with the buffer to a dilution in range of 1:100 to 1:6000. The preparation of bacterial stock enables usage of the frozen stock directly on the day of the assay thus reducing total analysis time to only 2 days instead of 3 days for conventional method. The preparation of the bacterial stock allowed 5 days for assay work / week as opposed to 4 days of assay with normal method. The preparation of bacterial stocks with known dilution for use in assay, results in attaining SBA titres in 2 working days instead of 3 working days, thus facilitating 5 working days / week instead of 4 working days / week. Assay solves the issue related to overall time required for completion of SBA’s (any conventional SBA requires at-least 3 days) as Bacterial stock preparation facilitates accelerated and accurate analysis. Bacterial stocks are titrated for required dilution to yield appropriate colonies in control wells. Stocks prepared are used on day 1 with determined dilution factor and results for SBA are obtained on day 2. In an embodiment, a method for performing a serum bactericidal activity assay, comprises i. preparing and providing a bacterial stock; ii. diluting the bacterial stock with a buffer to obtain a diluted bacterial stock that yields 50 to 250 colony forming units (cfus); iii. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. In another embodiment, a method for performing a serum bactericidal activity assay, comprises i. preparing and providing a bacterial stock; ii. diluting the bacterial stock 1:100 to 1:6000 with a buffer to obtain a diluted bacterial stock; iii. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. In another embodiment, a method for performing a serum bactericidal activity assay, comprises i. preparing and providing a bacterial stock; ii. diluting the bacterial stock 1:100 to 1:6000 with a buffer to obtain a diluted bacterial stock that yields 50 to 250 colony forming units (cfus); iii. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; and calculating antibody titre to determine the serum bactericidal activity. In these embodiments, a frozen vial of bacterial stock (prepared from bacteria harvested at a desired growth phase or desired OD value) is provided / taken and the bacterial stock is diluted with a buffer to a pre-determined dilution factor that yields 50 to 250 colony forming units (cfus) or the bacterial stock is diluted 1:100 to 1:6000 with a buffer to obtain a diluted bacterial stock. This diluted bacterial stock is mixed with the complement, the test serum, and the buffer to perform the assay. Providing the bacterial assay stock harvested at a desired growth phase or desired OD value reduces the assay time from 3 days to 2 days as the assay can start by diluting the provided bacterial stock to a pre-determined dilution factor that yields 50 to 250 colony forming units. Frozen assay stock of the desired strain is titrated for calculating the dilution factor required to yield 50 to 250 cfu in the control wells. For this purpose, different dilutions of the frozen stock are prepared (i.e. 1:1000 dilution, 1: 2000, 1: 3000, 1: 4000, 1: 5000, 1: 6000 and further dilutions). Spotting of each individual dilution is carried out onto agar plates and incubated overnight at 37°C. Dilution factor yielding colonies(cfu) in the range of 50 to 250 is selected and used in the assay. Preparing the bacterial stock helps to complete the assay in 2 days duration, thus faster results are generated implying high throughput ability. Analysis is achieved with cells cultivated from a homogenous mixture which are from the same pool, thus negates the use of preparing bacteria and adjusting O.D on each day, thus eliminating variation by using the conventional method. In the conventional method, usually bacterial suspension is adjusted at a particular O.D for enumeration before addition to the assay plate in SBA and this means that cells taken could be from different growth stages, which can lead to improper analysis and false results in conventional method of SBA. BACTERIAL STOCK In an embodiment, the bacterial stock is prepared. In another embodiment, for the SBA assay as disclosed, the bacterial stock is prepared for bacteria causing bacterial infections including bacteria for gastro-intestinal infections, invasive, cause systematic infection, hepatobiliary colonization and chronic carriage. Bacterial infection includes infection of non-salmonella serovars and Salmonella serovars. Salmonella serovars include Typhoidal Salmonella and non-Typhoidal Salmonella. Typhoidal Salmonella includes invasive and non-invasive Salmonella. Typhoidal Salmonella includes Salmonella enterica subsp. enterica serovar Typhi or Salmonella Typhi or Eberth’s Bacillus (herein after Typhi), Salmonella Paratyphi A, B, and C (Paratyphi), S. Typhi Ty2, S. Typhi Ty21a. In another embodiment, for the SBA assay as disclosed, the bacterial stock is prepared for Typhoidal Salmonella, or S. Typhi or S. Paratyphi. The Salmonella serovar strains include S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition may include: Salmonella enterica serovar typhi TY2 strain with "tviB" gene specific for Vi polysaccharide; Salmonella enterica serovar typhi deposited at NCMR-NCCS Accession No. MCC 0193; Strain designation- PDL-1, 5. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC 9150, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis Rll; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219;S. typhimurium: S. typhimurium 2192; ATCC 14208; 5. typhimurium 2189; 5. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2 / SL134 (ST19)); S. typhimurium 177(ST19) CDC 6516-60; ATCC 700720. Any attenuated Salmonella serovar strain (X typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used for the preparation of the immunogenic composition for evaluation under the SBA assay. In another embodiment, for the SBA assay as disclosed, the bacterial stock is prepared for bacteria including Salmonella enterica serovar typhi, Salmonella enterica serovar paratyphi A, Salmonella enterica serovar paratyphi B, Salmonella enterica serovar paratyphi C, Salmonella enterica serovar typhimurium, Salmonella enterica serovar enteritidis, Salmonella enterica serovar choleraesuis, Salmonella enterica serovar Dublin. In a preferred embodiment, the bacterial stock is diluted with the buffer / the assay dilution buffer, i.e. the HBSS (-) buffer for S. Typhi and HBSS (+) buffer for S. Paratyphi, where HBSS (-) includes FBS and HBSS (+) includes calcium and magnesium. In an embodiment, the bacterial stock for S. Typhi or S. Paratyphi or both is prepared. In an embodiment, the bacterial assay stock for titration for S. Typhi or S. Paratyphi or both is prepared. ADDING TEST SERUM, COMPLEMENT, BACTERIAL STOCK In an embodiment, the SBA assay includes adding the test serum, the complement, and the bacterial stock to form an assay solution. The assay dilution buffer (HBSS(-) +1% FBS for S. Typhi and HBSS(+) for S. Paratyphi A) is added to the wells of flat bottom plate. The flat bottom plate includes a 96 well plate. The test serum and a reference serum are added to the wells. 2-fold serial dilution of the test and the reference serum is made. Wells (columns) are reserved for complement controls. Based on the bacterial titration results, suitable dilutions of bacteria that give required number of colonies are made and bacteria is added in each well. Complement is added to the plate and the final assay volume is adjusted with the dilution buffer to form the assay solution in the plate. Heat inactivated complement source and the dilution buffer are added to the reference column. In an alternate embodiment, the test serum, the complement and the bacterial stock are added to form the assay solution in an assay vial. The heat inactivated complement source and the dilution buffer are added to a reference vial. In an embodiment, the SBA assay provides assessment of complement mediated killing achievable for the immune response. In another embodiment, the assay estimates antibodies against S. Typhi, S. Paratyphi or both. In an embodiment, the assay for S. Typhi or S. Paratyphi or both includes adding the buffer, test serum, complement and the bacterial stock in the buffer on a plate. TEST SERUM The “Test serum” is the serum collected from subjects involved in studies used for vaccine evaluation. The vaccine is injected into the trial subjects and blood is collected on two separated days (Pre-vaccination-Day 0 and Post-vaccination Day 28). Immunogenic response (i.e. antibody titre) is checked on both the days and a four-fold rise in antibody titres at Day 28 (Post vaccination) over the Day 0 (Pre-vaccination) is considered as threshold for immunogenicity. The subjects or hosts can include animal and human subjects based on stages of preclinical and clinical trials. In an embodiment, for the method of SBA assay disclosed, the test serum is associated with an immunogenic composition for bacteria. In another embodiment, for the method of the SBA assay disclosed, the test serum is associated with immunogenic composition for Salmonella, Salmonella enterica, typhoidal Salmonella, non-Typhoidal Salmonella, Salmonella Typhi, and Salmonella Paratyphi. The term “the test serum is associated with an immunogenic composition” or “the test serum is associated with a vaccine” means that the test serum comprises antibodies formed on administration of the immunogenic composition or the vaccine against a bacteria. The immunogenic composition or the vaccine against bacteria, when administered to a subject, induces an antibody response in the subject. The SBA assay measures the antibody titre of sera obtained from the immunized subject to determine the level of antibody response induced by the immunogenic composition or the vaccine. In another embodiment the SBA assay provides evaluation of the vaccines / immunogenic compositions for bacterial infections including bacteria for gastro-intestinal infections, invasive, that cause systematic infection, hepatobiliary colonization and chronic carriage. Bacterial infection includes infection of non-Salmonella serovars and Salmonella serovars. Salmonella serovars include Typhoidal Salmonella and non-Typhoidal Salmonella. Typhoidal Salmonella includes invasive and non-invasive Salmonella. Typhoidal Salmonella includes Salmonella enterica subsp. enterica serovar Typhi or Salmonella Typhi or Eberth’s Bacillus (herein after Typhi), Salmonella Paratyphi A, B, and C (Paratyphi), S. Typhi Ty2, S. Typhi Ty21a, or combinations thereof. In another embodiment, the assay is used for evaluation of the vaccines / immunogenic compositions for Typhoidal Salmonella, or S. Typhi or S. Paratyphi. The Salmonella serovar strains include S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition may include: Salmonella enterica serovar typhi TY2 strain with "tviB" gene specific for Vi polysaccharide; Salmonella enterica serovar typhi deposited at NCMR-NCCS Accession No. MCC 0193; Strain designation- PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); 5. paratyphi A: ATCC 9150, CMCC50073, CMCC50973; 5. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis Rll; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219;5. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2 / SL134 (ST19)); S.typhimurium 177(ST19) CDC 6516-60; ATCC 700720, or combinations thereof. Any attenuated Salmonella serovar strain (A typhi, S. paratyphi A, S. enteritidis and S. typhimurium), or combinations thereof may be used for the preparation of the immunogenic composition for evaluation under the SBA assay. In another embodiment, for the SBA Assay, the test serum comprises antibodies formed on administration of the vaccines / immunogenic composition comprising: i) at least one antigen selected from: a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate; b) Salmonella enterica serovar paratyphi A saccharide- carrier protein conjugate; c) Salmonella enterica serovar paratyphi B saccharide- carrier protein conjugate; d) Salmonella enterica serovar paratyphi C saccharide- carrier protein conjugate; e) Salmonella enterica serovar typhimurium saccharide- carrier protein conjugate; f) Salmonella enterica serovar enteritidis saccharide- carrier protein conjugate; g) Salmonella enterica serovar choleraesuis saccharide- carrier protein conjugate; h) Salmonella enterica serovar dublin saccharide- carrier protein conjugate; or combination (including, bivalent, trivalent, tetravalent) thereof, and ii) optionally an antigen selected from a group comprising of Salmonella (non-typhoidal), Diphtheria toxoid (D), Tetanus toxoid (T), Whole cell pertussis (wP), hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-Carrier protein conjugate (Hib), Haemophilus influenzae (a, c, d, e, f serotypes and the unencapsulated strains), Polio virus, Conjugate comprising of N. meningitidis antigens (A, B, C, D, W135, X, Y, Z and 29E), Streptococcus Pneumoniae antigen(s) (1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11 A, 1 IF, 11B, 11C, 11D, HE, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F , 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48), Group A Streptococcus spp , Group B Streptococcus (group la, lb, II, III, IV, V, VI, VII, VIII, andIX.) Neisseria meningitidis B bleb or antigen(s)(fHbp , PorA, chimeric fHbp-porA), Staphylococcus aureus antigen(s), Anthrax, BCG, Hepatitis (A, C, D, E, F and G strains) antigen(s), Human papillomavirus, HIV, acellular pertussis, modified adenylate cyclase, Malaria Antigen (RTS,S), Measles, Mumps, Rubella, Dengue, Zika, Ebola, Chikungunya, Japanese encephalitis, Rotavirus, Diarrheal antigens (E. coli spp., Shigella spp., Campylobacter spp. Vibrio cholera), Flavivirus, smallpox, yellow fever, Shingles, Varicella virus antigens, or combinations thereof. In another embodiment, for the SBA Assay, the test serum comprises antibodies formed on administration of the vaccines / immunogenic composition with a Salmonella antigen conjugated with carrier protein (CP) selected from the group comprising of tetanus toxin, tetanus toxoid (TT), fragment of tetanus toxoid, diphtheria toxoid (DT), CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, 8MTT (Genetically detoxified tetanus toxin), Cholera toxin B subunit, fHbp, PorA, Clostridium perfringens toxoid, E.coli LT, E. coli ST, Escherichia coli heat-labile toxin - B subunit, Neisseria meningitidis outer membrane complex, rEPA, protein D of H. influenzae, Flagellin FliC, Horseshoe crab Haemocyanin, exotoxin A from Pseudomonas aeruginosa, outer membrane complex C (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, Staphylococcus aureus derived proteins Hla, ClfB, and IsdB, Group B Streptococcus pili proteins GBS80 and GBS67 protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S.agalactiae proteins with or without linker and fragments, derivatives, modifications thereof, or combinations thereof. In an embodiment, the assay provides assessment of complement mediated killing achievable for the immune response. In one embodiment of the method of performing the SBA assay, the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella Typhi. In one embodiment of the method of performing the SBA assay, the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella Paratyphi. In an embodiment, the SBA method determines a functional IgG response of an immunogenic composition or a vaccine comprising Vi-TT and OSP-DT, wherein the test serum comprises antibodies formed on administration of the Vi-TT or OSP-DT polysaccharide protein conjugate. In a preferred embodiment, the SBA method determines a functional IgG response of an immunogenic composition or a vaccine comprising Vi-TT, wherein the test serum comprises antibodies formed on administration of the Vi-TT polysaccharide protein conjugate. In another preferred embodiment, the SBA method determines a functional IgG response of an immunogenic composition or a vaccine comprising OSP-DT, wherein the test serum comprises antibodies formed on administration of the OSP-DT polysaccharide protein conjugate. COMPLEMENT CONCENTRATION: Complement is the main bioactive reagent in the SBA, which is responsible for the killing of bacteria in presence of the specific antibody. Intrinsic killing (killing caused due to complement alone) against any complement should be kept to the minimum in order to estimate accurate bactericidal titre. Complement concentration evaluation facilitates determining the concentration of the complement required for desired bactericidal effect. Failing to determine the desired complement concentration could lead to errors in SBA titre estimation. The assay is associated with lower intrinsic killing percentage of below 15.0 %. The intrinsic killing is the difference between Heat Inactivated Complement (HIC) control and the C’-complement control wells. The intrinsic killing is associated with gradual clearing of bacteria enabling distinct titre footprint. In another embodiment, for the method of performing the SBA assay as disclosed herein, the complement is at a concentration of 2.0 % to 20.0% v / v with respect to the volume of the final assay solution. For example, if the volume of the final assay solution is 40 pl, 0.8 pl to 8 pl of the complement can be added to the assay. The complement includes Baby Rabbit and Human complements. The present invention is directed to a method of performing a Serum Bactericidal Activity Assay, wherein the assay is performed using a complement concentration in the range of 2.0 to 20.0 %. In an embodiment, the complement concentration in the assay for S. Typhi is in the range of 2.0 to 20.0%. In a preferred embodiment, the complement concentration in the assay for S. Typhi is in range of 2.0 to 18.0% or in the range of 2.0 to 16.0% or in the range of 2.0 to 14.0% or in the range of 2.0 to 12.0% or in the range of 2.0 to 10.0% or in the range of 2.0 to 8.0% or in the range of 2.0 to 6.0%. In another preferred embodiment, the complement concentration in the assay for S. Typhi is in the range of 3.0 to 6.0% or in the range of 4.0 to 6.0%. In a more preferred embodiment, the complement concentration in the assay for S. Typhi is 5.0%. The complement concentration of 5.0% is associated with 2 pl content. The 2 pl of complement is present in the final volume of 40 pl which corresponds to 5 % of total volume of assay. In an embodiment, the complement concentration in the assay for S. Paratyphi is in the range of 2.0 to 20.0 %. In a preferred embodiment, the complement concentration in the assay for S. Typhi is in range of 2.0 to 19.0%, or in the range of 2.0 to 18.0%, or in the range of 2.0 to 17.0%, or in the range of 2.0 to 16.0 %, or in the range of 2.0 to 15.0%, or in the range of 2.0 to 4.0%. In another preferred embodiment, the complement concentration in the assay for S.Paratyphi is in the range of 3.0 to 14.0%, or in the range of 4.0 to 14.0%, or in the range of 5.0 to 14.0%, or in the range of 6.0 to 14.0%, or in the range of 7.0 to 14.0%, or in the range of 8.0 to 14.0%, or in the range of 9.0 to 14.0%, or in the range of 10.0 to 14.0%, or in the range of 11.0 to 14.0%. In a more preferred embodiment, the complement concentration in the assay for S. Paratyphi is 12.50%. The complement concentration of 12.50% is associated with 5 pl content. In a preferred embodiment, the complement is a baby rabbit complement or a human complement. In a more preferred embodiment, the complement is a human complement. In a more preferred embodiment, the complement is a baby rabbit complement (BRC). COMBINATIONS OF MEDIA, BUFFER, AND COMPLEMENT CONCENTRATIONS: In another embodiment, for the method of performing the SBA assay as disclosed herein, the media is the Luria Bertani broth, and the buffer includes the HBSS, other buffers, supplements, or combinations thereof. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, and the buffer includes the HBSS without phenol red, other buffers, supplements, or combinations thereof. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS, other buffers, supplements, or combinations thereof, and the complement is in concentration of 2.0% to 20.0%, wherein the HBSS is without phenol red; and wherein the test serum comprises antibodies formed on administration of an immunogenic composition against typhoidal Salmonella. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS, the complement is in concentration of 2.0% to 20.0%, and the test serum comprises antibodies formed on administration of an immunogenic composition against typhoidal Salmonella. In another embodiment, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium or with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 20.0%; wherein the test serum comprises antibodies formed on administration of an immunogenic composition against typhoidal Salmonella and the bacterial stock is in initial log phase or in late log phase. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS, the complement is in concentration of 2.0% to 15.0%. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS and the complement is in concentration of 2.0% to 15.0%, and the test serum comprises antibodies formed on administration of an immunogenic composition against typhoidal Salmonella. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium or with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 20.0%. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium or with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium or with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%, and the test serum comprises antibodies formed on administration of the immunogenic composition against typhoidal Salmonella. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 20.0%, and the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%, and the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella Typhi. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 20.0%. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%. In another embodiment, for the method of performing the SBA assay as disclosed herein, the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%, and the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella Paratyphi. In an embodiment, the assay solution or final assay volume for both S. Typhi and S. Paratyphi A SBA is kept at 40pl, with composition for S. Typhi SBA: 20 pl sera (50%) + 10 pl bacteria (25%) + 2 pl complement (5%) + 8 pl buffer (20%) and S. Paratyphi A SBA: 20 pl sera (50%) + 10 pl bacteria (25%) + 5 pl complement (12.5%) + 5 pl buffer (12.5%). INCUBATION In an embodiment, the plate is incubated. The plate including the assay solution is incubated at temperature in range of 35°C to 39°C, preferably at temperature in range of 36°C to 38°C or more preferably at 37°C for 60 mins without shaking and spotting is done from each well onto the media plate. Plate is incubated for 10 to 14 hours at temperature in range of 3 5 °C to 39°C, preferably at temperature in range of 36°C to 38°C or more preferably at 37°C, 5% CO2. The incubator includes static incubator or shaker incubator. In an alternate embodiment, the assay vial and the reference vial are incubated at temperature in range of35°C to 39°C, preferably at temperature in range of36°C to 38°C or more preferably at 37°C for 60 mins without shaking and spotting is done from assay vial and the reference vial onto the media plate. The plate is incubated for 10 to 14 hours at temperature in range of35°C to 39°C, preferably at temperature in range of 36°C to 38°C or more preferably at 37°C, 5% CO2. CALCULATING ANTIBODY TITRE The method of Serum Bactericidal Activity includes calculating antibody titre. In an embodiment, the antibody titre is calculated for antibodies formed on administration of immunogenic composition against bacteria. In a preferred embodiment, the colony count is obtained first in the Heat Inactivated Complement Control (HIC). The SBA titre for the test serum in the assay solution is calculated as the highest serum dilution where the colony count is less than or equal to 50% of the complement control (HIC). The bacterial count difference for C’ (complement control) and HIC (Heat Inactivated complement) wells is not kept more than 30%. The calculation of the antibody titre for test serum in the assay solution is calculated as highest serum dilution where colony count is less than or equal to 50% of complement control (HIC). In another preferred embodiment, repetition with higher dilution is taken in-case last dilution has colonies less than 50 % of the HIC control. In a more preferred embodiment, internal positive controls created from a pool of serum from individuals 90 days post-challenge with bacteria are run on each plate. The impact of pre-existing tetanus and diphtheria antibodies on immune response of typhoid and paratyphoid antigens is assessed. Baseline and Day 29 serum samples are tested for quantitative estimation of anti-tetanus and anti-diphtheria IgG antibodies using CLASSIC SERION IgG ELISA kits. In another embodiment, the antibody titre is calculated for antibodies formed on administration of the immunogenic composition against Salmonella. In an embodiment, the antibody titre is calculated for antibodies formed on administration of the immunogenic composition against Typhoidal Salmonella, or S. Typhi or S. Paratyphi. The Salmonella serovar strains include S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition may include: Salmonella enterica serovar typhi TY2 strain with "tviB" gene specific for Vi polysaccharide; Salmonella enterica serovar typhi deposited at NCMR-NCCS Accession No. MCC 0193; Strain designation- PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); 5. paratyphi A: ATCC 9150, CMCC50073, CMCC50973; 5. enteritidis: ATCC 4931; ATCC 13076; 5. enteritidis Rll; 5. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis rV3453219;5. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2 / SL134 (ST19)); S.typhimurium 177(ST19) CDC 6516-60; ATCC 700720, Salmonella enterica, L-6895-1 MG, Salmonella typhi (Ty2), bacteria with Salmonella typhi Vi Polysaccharide, etc. Any attenuated Salmonella serovar strain (5. typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used for the preparation of the immunogenic composition for evaluation under the SBA assay. In another embodiment, the antibody Titre is calculated for antibodies formed on administration of the immunogenic composition against Typhoidal Salmonella, or S. Typhi or S. Paratyphi. The Salmonella serovar strains include S. typhi; and S. paratyphi A. In an embodiment, S. Typhi is Typhi MCC-0193, a Clinical isolate from patient at Villoo Poonawalla hospital, India. In another embodiment, Salmonella. Paratyphi A ATCC 9150 (Salmonella enterica subsp. enter ica (ex-Kaufftnann and Edwards) Le Minor and Popoff serovar Paratyphi A) is procured from American Type Culture Collection, USA. The SBA titre for each individual serum sample is calculated as the highest serum dilution giving >50% killing as compared to the CFU count obtained in the corresponding heat inactivated complement (HIC) only condition wells. In a preferred embodiment, the colonies are counted manually. In a preferred embodiment, the colonies are quantified using a semi-automated colony counter. In another preferred embodiment, the colonies are counted using automated colony counter. In a more preferred embodiment, the automated colony counter includes a ProtoCOL 3 make from Synbiosis, UK. In another embodiment, the method of SBA assay for S. Typhi comprises: a. preparing the Luria Bertani broth and the HBSS buffer supplemented with FBS, i.e. the HBSS (-) buffer, the HBSS (-) buffer is without calcium, magnesium and phenol red; b. preparing the Salmonella Typhi bacterial stock with the Salmonella Typhi mother culture stock, the Luria Bertani broth and the HBSS (-) buffer; c. diluting the Salmonella Typhi bacterial stock with the HBSS (-) buffer to obtain a diluted Salmonella Typhi bacterial stock; d. adding the test serum comprising antibodies formed on administration of the immunogenic composition against Salmonella Typhi, the complement in concentration of 2.0% to 20.0%, the diluted Salmonella Typhi bacterial stock and the HBSS (-) buffer to form the assay solution e. incubating the assay solution at 35°C to 39°C for 50 to 90 mins; spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and f. calculating antibody titre; wherein the Salmonella Typhi bacterial stock is in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm. In another embodiment, the method of SBA assay for S. Typhi comprises: a. preparing the Luria Bertani broth and the HBSS buffer supplemented with FBS, i.e. the HBSS (-) buffer, the HBSS (-) buffer is without phenol red; b. preparing the Salmonella Typhi bacterial stock with the Salmonella Typhi mother culture stock, the Luria Bertani broth and the HBSS (-) buffer and diluting the Salmonella Typhi bacterial stock with the HBSS (-) buffer to obtain a diluted Salmonella Typhi bacterial stock; c. adding the test serum comprising antibodies formed on administration of the immunogenic composition against Salmonella Typhi, the complement is in concentration of 2.0% to 20.0%, the diluted Salmonella Typhi bacterial stock and the HBSS (-) buffer to form the assay solution d. incubating the assay solution at 35°C to 39°C for 50 to 90 mins, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and e. calculating antibody titre; wherein the Salmonella Typhi bacterial stock is in initial log phase with absorbance value in range of 0.30 to 0.40 for Optical Density at 600 nm. In another embodiment, the method of SBA assay for S. Typhi comprises: a. providing the Luria Bertani broth and the HBSS buffer supplemented with FBS, i.e. the HBSS (-) buffer, wherein the HBSS (-) buffer is without calcium, magnesium and phenol red; b. providing Salmonella Typhi bacterial stock prepared from the Salmonella Typhi mother culture stock and the Luria Bertani broth; c. diluting the Salmonella Typhi bacterial stock with the HBSS (-) buffer to obtain a diluted Salmonella Typhi bacterial stock that yields 50 to 250 colony forming units (cfus) or diluting the Salmonella Typhi bacterial stock 1:100 to 1:6000 with the HBSS (-) buffer to obtain a diluted Salmonella Typhi bacterial stock; d. adding the test serum comprising antibodies against the immunogenic composition having Salmonella Typhi, the complement in concentration of 2.0% to 20.0%, the diluted Salmonella Typhi bacterial stock and the HBSS (-) buffer to form the assay solution e. incubating the assay solution at 35°C to 39°C for 50 to 90 mins; spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and f. calculating antibody titre; wherein the Salmonella Typhi bacterial stock is in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm. In an embodiment, the method of SBA assay for S. Paratyphi comprises a. preparing the Luria Bertani broth and the HBSS buffer supplemented with Calcium and Magnesium, i.e. the HBSS (+) buffer, wherein the HBSS (+) buffer is without phenol red; b. preparing the Salmonella Paratyphi bacterial stock with the Salmonella Paratyphi mother culture stock, the Luria Bertani broth and the HBSS (+) buffer; c. diluting the Salmonella paratyphi bacterial stock with the HBSS (+) buffer to obtain a diluted Salmonella Paratyphi bacterial stock; d. adding the test serum, the complement in concentration of 2.0% to 20.0%, the Salmonella paratyphi bacterial stock and the HBSS (+) buffer i.e. HBSS with Calcium and Magnesium to form the assay solution; e. incubating the assay solution at 35°C to 39°C for 50 to 90 mins; spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and f. calculating antibody titre; wherein the Salmonella Paratyphi bacterial stock is in late log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm. In an embodiment, the method of SBA assay for S. Paratyphi comprises a. preparing the Luria Bertani broth and the HBSS buffer supplemented with Calcium and Magnesium, i.e. the HBSS (+) buffer, wherein the HBSS (+) buffer is without phenol red; b. preparing the Salmonella Paratyphi bacterial stock with the Salmonella Paratyphi mother culture stock, the Luria Bertani broth and the HBSS (+) buffer and diluting the Salmonella paratyphi bacterial stock with the HBSS (+) buffer to obtain a diluted Salmonella Paratyphi bacterial stock; c. adding the test serum, the complement in concentration of 2.0% to 20.0%, the diluted Salmonella paratyphi bacterial stock and the HBSS (+) buffer, i.e. HBSS with Calcium and Magnesium to form the assay solution; d. incubating the assay solution at 35°C to 39°C for 50 to 90 mins, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and e. calculating antibody titre; wherein the Salmonella Paratyphi bacterial stock is in late log phase with absorbance value in range of 0.90 to 1.10 for Optical Density at 600 nm. In an embodiment, the method of SBA assay for S. Paratyphi comprises a. providing the Luria Bertani broth and the HBSS buffer supplemented with Calcium and Magnesium, i.e. the HBSS (+) buffer, wherein the HBSS (+) buffer is without phenol red; b. providing the Salmonella Paratyphi bacterial stock prepared from the Salmonella Paratyphi mother culture stock and the Luria Bertani broth; c. diluting the Salmonella paratyphi bacterial stock with the HBSS (+) buffer to obtain a diluted Salmonella Paratyphi bacterial stock that yields 50 to 250 colony forming units (cfus) or diluting the Salmonella Paratyphi bacterial stock 1:100 to 1:6000 with the HBSS (-) buffer to obtain a diluted Salmonella Paratyphi bacterial stock; d. adding the test serum, the complement in concentration of 2.0% to 20.0%, the Salmonella Paratyphi bacterial stock and the HBSS (+) buffer to form the assay solution; e. incubating the assay solution at 35°C to 39°C for 50 to 90 mins; spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and f. calculating antibody titre; wherein the Salmonella Paratyphi bacterial stock is in late log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm. The method of performing the SBA assay or the method for determining the antibody titre according to the present disclosure is a non-diagnostic method. The term “diagnosis” refers to a confirmation by a doctor / clinician / radiologist that a person has a specific condition / disorder / disease. The methods of the present disclosure do not detect or identify any specific condition / disorder / disease. The methods are employed to determine the effectiveness of an immunogenic composition or a vaccine. When an immunogenic composition or a vaccine is administered to a subject, the immunogenic composition or the vaccine induces an antibody response in the subject against the antigen present in the immunogenic composition or the vaccine. The methods of the present disclosure determine the antibody titre of serum from an immunized subject. ANOTHER ASPECT: VACCINE In another aspect, the present invention is directed to the immunogenic composition as tested as per the SBA Assay method, the method comprising: preparing the media and the buffer; preparing the bacterial stock with the mother culture, the media and the buffer and diluting the bacterial stock; adding the test serum, the complement and the bacterial stock to form an assay solution; incubating the assay solution, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate; and calculating antibody titre; wherein the test serum comprises antibodies formed on administration of the immunogenic composition against Typhoidal Salmonella. In an embodiment, the immunogenic composition is tested as per the SBA Assay method, wherein the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella Typhi. In an embodiment, the immunogenic composition as tested as per the SBA Assay method, the method comprising: preparing the Luria Bertani media and the HBSS (-) buffer; preparing the S.Typhi bacterial stock with the S.typhi mother culture, the Luria Bertani media and the HBSS (-) buffer and diluting the S. Typhi bacterial stock; adding the test serum for S.Typhi, the complement and the S. Typhi bacterial stock to form the assay solution; incubating the assay solution, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate; and calculating antibody titre; wherein the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella typhi. In an embodiment, the immunogenic composition as tested as per the SB A Assay method, the method comprising: preparing the Luria Bertani media and the HBSS (+) buffer; preparing the S.Paratyphi bacterial stock with the S.Paratyphi mother culture, the Luria Bertani media and the HBSS (+) buffer and diluting the S.Paratyphi bacterial stock; adding the test serum for S.Paratyphi, the complement and the S.Paratyphi bacterial stock to form the assay solution; incubating the assay solution, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate; and calculating antibody titre; wherein the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella Paratyphi. In another embodiment, the immunogenic composition is tested as per the SBA Assay method, wherein the test serum comprises antibodies formed on administration of the immunogenic composition against Salmonella Paratyphi. ANOTHER ASPECT: KIT In another aspect, the present invention is directed to a kit for performing the Serum Bactericidal Activity (SBA) assay as described herein, wherein the kit comprises a media, a buffer, a bacterial stock, and a complement. ANOTHER ASPECT: SYSTEM In another aspect, the present invention is directed to a system for performing the Serum Bactericidal Activity (SBA) assay as disclosed hereinabove, wherein the system includes the media, the buffer, the bacterial stock, and the complement. In an embodiment, the present invention is directed to the system for performing the Serum Bactericidal Activity (SBA) assay as disclosed hereinabove, wherein the system includes the Luria Bertani broth, the HBSS (-) buffer, the S. Typhi bacterial stock, and the complement. In another embodiment, the present invention is directed to the system for performing the Serum Bactericidal Activity (SBA) assay as disclosed hereinabove, wherein the system includes the Luria Bertani broth, the HBSS (+) buffer, the S. Paratyphi bacterial stock, and the complement. ANOTHER ASPECT: APPLICATION In another aspect, the present invention is directed to the method of performing the SBA Assay method as disclosed herein, as and when required to determine antibody titre of an immunogenic composition / a vaccine. Bactericidal Activity (SBA) assay, wherein the serum sample has been taken from a human aged 0-55 years, 1 month - 20 years, 2 months - 16 years, 6 months - 10 years, 8 months - 7 years, 10 months - 4 years, 0-2 years, 6 months - 2 years, or 1-2 years. METHODS MEDIA PREPARATION: a. Luria Bertani broth (Media) is prepared in a 1 litre glass bottle with addition of 25 g of Luria Bertani broth to make up to 1000 ml with de-ionised water. Solution is mixed until all components have completely dissolved. Broth is sterilized at 121°C for 15 minutes by autoclaving. Media is stored at 2 to 8°C. b. 80% glycerol is prepared by mixing 20 ml of de-ionised water and 80 ml glycerol and sterilized at 121 °C for 15 minutes by autoclaving. c. Luria Bertani agar plate (LBA plate): In a 1000 ml autoclavable flask, 16 g of the Luria Bertani agar powder is added to 400 ml of de-ionised water, autoclaved (sterilized at 121°C for 15 minutes by autoclaving) and cooled to 56°C in water bath. 12 ml of solution is added to the square petri plate. d. Columbia blood agar plate with 5% horse blood: In a 1000 ml conical flask, 17.6 g of Columbia blood agar base is added to 400 ml of de-ionised water, autoclaved (sterilized at 121°C for 15 minutes by autoclaving) and cooled to 56°C in a water bath. 12 ml of solution is added to the square petri plate. e. Luria Bertani overlay agar: In a 250 ml autoclavable flask, 2 g of Luria Bertani broth and 1.5 g of Bacto agar is added to 100 ml of de-ionised water, autoclaved (sterilized at 121°C for 15 minutes by autoclaving). f. TTC stock (2,3,5 Triphenyl Tetrazolium Chloride): Prepared as 25 mg / ml (1000X) stock solution in water by adding 1.25 g of TTC to 40 ml de-ionised water. Volume adjusted to 50 ml with addition of de-ionised water and sterile filtered using 0.2pl filter to obtain a yellow coloured solution. g. Assay dilution buffer (Hanks Balanced Salt solution): HBSS-ve (without Ca and Mg) along with 1% FBS is used as assay buffer for S. Typhi SBA while HBSS+ve (with Ca and Mg) is used as assay dilution buffer for S. Paratyphi A SBA. PREPARATION OF BACTERIAL STOCK S. TYPHI: a. Retrieve the mother culture stock vial from the freezer, streak it onto a blood agar plate and incubate the plate 12-16 hours at 37°C with 5% in CO2 incubator. b. Transfer one loopful of isolated colonies from culture and inoculate in a sterile conical flask containing 50 ml of Luria Bertani broth. Incubate flask for 2-4 hours, in shaker incubator (37°C) with 150 RPM until the absorbance of the culture broth has an ODeoo of 0.3 to 0.4. c. After achieving desired OD, 10 ml of bacterial suspension, 10 ml of fresh Luria Bertani broth, and 4 ml of sterile 80% glycerol were mixed by vertexing and dispense 0.5 ml aliquots of bacteria into properly labeled sterile cryo-vials. BACTERIAL ASSAY STOCK TITRATION FOR S. TYPHI a. Allow a frozen assay stock of the desired strain from the -70°C freezer to thaw at room temperature for 2 min. b. Take 100 pl and add to 0.9 ml assay dilution buffer (this is a 10 4 dilution). Mix properly and aliquot of 100 pl from the 10'1 tube and added to 0.9 ml assay dilution buffer (this becomes a 10'2 dilution). c. Repeat the procedure to generate a 10'3. i.e. 1:1000 dilution. This was further diluted to obtain 1: 2000, 1: 3000, 1: 4000, 1: 5000, 1: 6000 and further dilutions. Each of the test dilutions is further diluted: 30 pl of assay dilution buffer + 10 pl of the bacterial dilution. d. Spot of 10 pl aliquots from each well on Luria Bertani agar plate in duplicate and incubate plate 10-14 hours at 37°C in 5% CO2 Incubator. e. Count the colonies in each streak of growth and find the average count for each dilution. The ideal number of colonies for assay is taken between 50 to 250 cfu. SERUM BACTERICIDAL ACTIVITY (SBA) ASSAY PROTOCOL S. TYPHI: a. Add 20 pl of assay dilution buffer (HBSS-ve with 1% FBS) to each well of a flat bottom 96-well plate. b. Add 20 pl of diluted or undiluted test serum to the wells of column Al-Fl. G1 and Hl are reserved for Reference sera (serum with a known Titre). c. Make 2-fold Serial dilution of test and Reference serum (rows 1 to 9). Column 10 and 11 which is reserved for the complement controls. d. Based on the bacterial titration results make a suitable dilution of bacteria that gives ideal colony number / Colony Forming Units (CFU) of specific bacteria (here S.Typhi) and add 10 pl of bacteria to each well. Use bacteria from a frozen stock (5. Typhi 0.4 OD) in assay dilution buffer. e. Add 2 pl complement (Baby Rabbit Serum) to the plate through column 1 to 10. Final assay volume 40 pl was adjusted by adding 8 pl dilution buffer to form the assay solution. f. 2 pl Heat inactivated complement source and 8 pl dilution buffer was added in column 11. g. Incubate the plate having the assay solution at 37°C for 60 min without shaking and 10 pl spotting was done from each well onto Luria Bertani agar plates. h. Incubate the plates overnight at 37°C, 5% CO2. Counting of plates was done after 10 to 14 hours incubation manually or using ProtoCOL 3 Automated colony counter. PREPARATION OF BACTERIAL STOCK S. PARATYPHI A: a. Retrieve the mother culture stock vial from the freezer, streak it onto a blood agar plate and incubate the plate 12-16 hours at 37°C with 5% in CO2 incubator. b. Transfer one loopful of isolated colonies from culture and inoculate in a sterile conical flask containing 50 ml of Luria Bertani broth. Incubate flask for 2-4 hours, in shaker incubator (37°C) with 150 RPM until the absorbance of the culture broth has an ODeoo of 1.0 to 1.5. c. After achieving desired OD, 10 ml of bacterial suspension, 10 ml of fresh Luria Bertani broth, and 4 ml of sterile 80% glycerol were mixed by vertexing and dispensed 0.5 ml aliquots of bacteria into properly labeled sterile cryo-vials. BACTERIAL ASSAY STOCK TITRATION FOR S. PARATYPHI A: a. Allow a frozen assay stock of the desired strain from the -70°C freezer to thaw at room temperature for 2 min. b. Take 100 pl and add to 0.9 ml assay dilution buffer (this is a 10 4 dilution). Mix properly and aliquot of 100 pl from the 10'1 tube and add to 0.9 ml assay dilution buffer (this becomes a 10'2 dilution). c. Repeat the procedure to generate a 10'3 i.e. 1:1000 dilution. This was further diluted to obtain 1: 2000, 1: 3000, 1: 4000, 1: 5000, 1: 6000 and further dilutions. Each of the test dilutions is further diluted: 30 pl of assay dilution buffer + 10 pl of the bacterial dilution. d. Spot of 10 pl aliquots from each well on Luria Bertani agar plate in duplicate and incubate plate 10-14 hours at 37°C in 5% CO2 Incubator. e. Count the colonies in each streak of growth and find the average count for each dilution. The ideal number of colonies for assay is taken between 50 to 250 cfu. SERUM BACTERICIDAL ACTIVITY (SBA) ASSAY PROTOCOL S. PARATYPHI A: a. Add 20 pl of assay dilution buffer (HBSS +ve buffer) to each well of a flat bottom 96-well plate. b. Add 20 pl of diluted or undiluted test serum to the wells of column Al-Fl. G1 and Hl are reserved for Reference sera (serum with a known Titre). c. Make 2-fold Serial dilution of test and Reference serum (rows 1 to 9). Column 10 and 11 which is reserved for the complement controls. d. Based on the bacterial titration results make a suitable dilution of bacteria that gives ideal colony number / Colony Forming Units (CFU) of specific bacteria (here S. Paratyphi) and added 10 pl of bacteria to each well. Used bacteria from a frozen stock (S. Paratyphi A 1.0 OD) in assay dilution buffer. e. Add 5 pl complement to the plate through column 1 to 10. Final assay volume 40 pl was adjusted by adding 5 pl dilution buffer to form the assay solution. f. Heat inactivated complement source and 5 pl dilution buffer was added in column 11. g. Incubate the plate having the assay solution at 37°C for 60 min without shaking and 10 pl spotting was done from each well onto Luria Bertani agar plates. h. Incubate the plates overnight at 37°C, 5% CO2. Counting of plates was done after 10 to 14 hours incubation manually or using ProtoCOL 3 Automated colony counter. CALCULATION OF ANTIBODY TITRES: a. Colony counts obtained in the Heat Inactivated Complement Control (HIC) are taken. The SBA Titre is calculated as the highest serum dilution where the colony count is less than or equal to 50% of the complement control (HIC). b. The bacterial count difference for C’ (complement control) and HIC (Heat Inactivated complement) wells should not be more than 30%. c. In case if the dilution scheme falls short to give an appropriate titre (when the last dilution also has colonies less than 50 % of the HIC control) then the serum sample should be repeated with a higher dilution scheme. EMBODIMENTS The present invention is illustrated in more detail by the following embodiments and combinations of embodiments which result from the corresponding dependency references and links: I. A method of performing Serum Bactericidal Activity (SBA) assay, the method comprising: a. preparing a media and a buffer; b. preparing a bacterial stock with a mother culture stock, the media and the buffer, and diluting the bacterial stock with the buffer; c. adding a test serum, a complement, the bacterial stock, and the buffer to form an assay solution; d. incubating the assay solution, spotting the assay solution on an agar plate and incubating the agar plate; and e. calculating antibody titre to determine the serum bactericidal activity. II. The method of SBA assay as disclosed in embodiment I, wherein the media includes lysogeny broth, Luria Bertani, Hi Soy (Hi Bacteriological media), tryptone, peptone, yeast extract, or combinations thereof. III. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media is lysogeny broth, or Luria Bertani broth. IV. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the buffer includes saline, phosphate, phosphate buffered saline, bovine serum albumin, Hanks' balanced salt solution (HBSS), fetal bovine serum (FBS), or combinations thereof. V. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the buffer includes HBSS. VI. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the buffer includes HBSS and FBS. VII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the buffer includes HBSS, and calcium and magnesium. VIII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the agar plate is Luria Bertani agar plate. IX. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the bacterial stock is prepared with bacteria in initial log phase, or in exponential phase, or in late Log phase, or in the stationary growth phase; optionally, wherein the bacterial stock preparation includes incubating bacteria from the mother culture stock at 37°C in for 12 to 16 hours in a blood agar plate and optionally, wherein the bacterial stock preparation includes further incubating bacteria from the blood agar plate on the Luria Bertani broth for 2 to 4 hours at 37°C in shaker incubator. X. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the bacterial stock is prepared with bacteria in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical density at 600 nm. XI. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the bacterial stock is prepared with bacteria in late Log phase with absorbance value in range of 0.60 to 1.20 for Optical density at 600 nm. XII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the bacterial stock is titrated and diluted using the buffer. XIII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the bacterial stock is diluted with the buffer to provide the diluted bacterial stock that yields 50 to 250 colony forming units (cfus). XIV. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the bacterial stock is diluted to a dilution factor of 1:100 to 1:6000 with the buffer to provide the diluted bacterial stock. XV. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the test serum comprises antibodies against an immunogenic composition for bacteria, Salmonella, typhoidal Salmonella, non-Typhoidal Salmonella, Salmonella Typhi, and Salmonella Paratyphi. XVI. The method of SBA assay as disclosed in embodiment XV, wherein the bacteria is Salmonella selected from a group comprising: typhoidal Salmonella, non-Typhoidal Salmonella, Salmonella Typhi, and Salmonella Paratyphi. XVII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the test serum comprises antibodies against the immunogenic composition for Salmonella Typhi. XVIII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the test serum comprises antibodies against the immunogenic composition for Salmonella Paratyphi. XIX. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the complement is in concentration of 2.0 % to 20.0% optionally wherein the complement includes Human complement and Rabbit complement. XX. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media is the Luria Bertani media, and the buffer includes the HBSS, optionally, wherein the HBSS is without phenol red; and optionally wherein the test serum comprises antibodies against an immunogenic composition for typhoidal Salmonella. XXI. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, and the buffer includes the HBSS without phenol red. XXII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS, and the complement is in concentration of 2.0% to 20.0%. XXIII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS, the complement is in concentration of 2.0% to 20.0%, and the test serum comprises antibodies against an immunogenic composition for typhoidal Salmonella. XXIV. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS, and the complement is in concentration of 2.0% to 15.0%. XXV. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS and the complement is in concentration of 2.0% to 15.0%, and the test serum comprises antibodies against an immunogenic composition for typhoidal Salmonella. XXVI. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium or with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 20.0%; optionally wherein the test serum comprises antibodies against an immunogenic composition for typhoidal Salmonella; optionally wherein the bacterial stock is in initial log phase or in late log phase. XXVII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS is supplemented with Calcium and Magnesium or with FBS, the HBSS is optionally without phenol red, and the complement is in concentration of 2.0% to 15.0%. XXVIII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS is supplemented with Calcium and Magnesium or with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%, and the test serum comprises antibodies against the immunogenic composition for typhoidal Salmonella. XXIX. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 20.0%, and the test serum comprises antibodies against the immunogenic composition for Salmonella. XXX. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS is supplemented with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%. XXXI. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS is supplemented with FBS, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%, and the test serum comprises antibodies against the immunogenic composition for Salmonella Typhi. XXXII. The method of SBA assay as disclosed in any one of the previous embodiments, the method comprising a. preparing the Luria Bertani broth and the HBSS buffer supplemented with Calcium and Magnesium, i.e. the HBSS (+) buffer, wherein the HBSS (+) buffer is without phenol red; b. preparing the Salmonella Paratyphi bacterial stock with the Salmonella Paratyphi mother culture stock, the Luria Bertani broth and the HBSS (+) buffer and diluting the Salmonella paratyphi bacterial stock with the HBSS (+) buffer; c. adding the test serum, the complement in concentration of 2.0% to 20.0%, the Salmonella paratyphi bacterial stock and the HBSS buffer supplemented with Calcium and Magnesium to form the assay solution; d. incubating the assay solution at 35°C to 39°C for 50 to 90 mins, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and e. calculating antibody titre; wherein the Salmonella Paratyphi bacterial stock is in late log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm. XXXIII. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS is supplemented with Calcium and Magnesium, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 20.0%. XXXIV. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS is supplemented with Calcium and Magnesium, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%. XXXV. The method of SBA assay as disclosed in any one of the previous embodiments, wherein the media includes the Luria Bertani broth, the buffer includes HBSS is supplemented with Calcium and Magnesium, the HBSS is optionally without phenol red, the complement is in concentration of 2.0% to 15.0%, and the test serum includes the immunogenic composition for Salmonella Paratyphi. XXXVI. The method of SBA assay as disclosed in any one of the previous embodiments, the method comprising: a. preparing the Luria Bertani broth and the HBSS buffer supplemented with FBS, i.e. the HBSS (-) buffer, the HBSS (-) buffer is without phenol red; b. preparing the Salmonella Typhi bacterial stock with the Salmonella Typhi mother culture stock, the Luria Bertani broth and the HBSS (-) buffer and diluting the Salmonella Typhi bacterial stock with the HBSS (-) buffer c. adding the test serum for immunogenic composition having Salmonella Typhi, the complement is in concentration of 2.0% to 20.0%, the Salmonella Typhi bacterial stock and the HBSS (-) buffer to form the assay solution d. incubating the assay solution at 35°C to 39°C for 50 to 90 mins, spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and e. calculating antibody titre; wherein the Salmonella Typhi bacterial stock is in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm. XXXVII. The immunogenic composition as tested as per the SBA Assay method of any one of the previous embodiments, the method comprising: a. preparing the media and the buffer; b. preparing a bacterial stock with a mother culture stock, the media and the buffer, and diluting the bacterial stock; c. adding the test serum, the complement, and the bacterial stock to form an assay solution; d. incubating the assay solution; and e. calculating antibody titre wherein the test serum is for Typhoidal Salmonella. XXXVIII. The immunogenic composition as tested as per the SBA Assay method of any one of the previous embodiments, the method comprising: a. preparing the media and the buffer; b. preparing a bacterial stock with the mother culture stock, the media and the buffer, and diluting the bacterial stock; c. adding the test serum, the complement and the bacterial stock to form an assay solution; d. incubating the assay solution; and e. calculating antibody titre wherein the test serum is for Salmonella Typhi. XXXIX. The immunogenic composition as tested as per the SBA Assay method of any one of the previous embodiments, the method comprising: a. preparing the media and the buffer; b. preparing the bacterial stock with the mother culture stock, the media and the buffer, and diluting the bacterial stock; c. adding the test serum, the complement and the bacterial stock to form an assay solution; d. incubating the assay solution; and e. calculating antibody titre f. wherein the test serum is for Salmonella Paratyphi. XL. A kit for performing the Serum Bactericidal Activity (SBA) assay of any one of the previous embodiments, wherein the kit comprises a media, a buffer, a bacterial stock, and a complement. XLI. The method of embodiment I to determine antibody titre of an immunogenic composition / a vaccine. XLII. A system for performing the Serum Bactericidal Activity (SBA) assay of any one of the previous embodiments, wherein the system comprises a media, a buffer, a bacterial stock, and a complement. XLIII. The method of Serum Bactericidal Activity assay as disclosed in any one of previous embodiments, wherein the bacterial stock is diluted with the buffer to a dilution in range of 1: 100 to 1:6000. XLIV. The method of Serum Bactericidal Activity assay as disclosed in any one of previous embodiments, wherein SBA titre for test serum in the assay solution is calculated as highest serum dilution where colony count is less than or equal to 50% of complement control (HIC). XLV. A method for performing Serum Bactericidal Activity (SBA) assay, the method comprising: i. providing a media and a buffer; ii. preparing a bacterial stock with a mother culture stock and the media; iii. diluting the bacterial stock with the buffer to obtain a diluted bacterial stock; iv. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; v. incubating the assay solution, spotting the assay solution on an agar plate and incubating the agar plate; and vi. calculating antibody titre to determine the serum bactericidal activity. XLVI. A method for performing a Serum Bactericidal Assay (SBA), comprising: i. preparing a bacterial stock with a mother culture stock and a media; ii. diluting the bacterial stock with the buffer to obtain a diluted bacterial stock that yields 50 to 250 colony forming units (cfus); iii. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. XLVII. A method for determining antibody titre of a test serum, comprising: i. preparing a bacterial stock with a mother culture stock and a media; ii. diluting the bacterial stock with a buffer to obtain a diluted bacterial stock that yields 50 to 250 colony forming units (cfus); iii. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. XLVIII. A method for performing a serum bactericidal activity assay, comprising: i. preparing and providing a bacterial stock; ii. diluting the bacterial stock with a buffer to obtain a diluted bacterial stock that yields 50 to 250 colony forming units (cfus); iii. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. XLIX. A method for performing a serum bactericidal activity assay, comprising: i. preparing and providing a bacterial stock; ii. diluting the bacterial stock 1:100 to 1:6000 with a buffer to obtain a diluted bacterial stock; iii. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution; iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; and v. calculating antibody titre to determine the serum bactericidal activity. L. The method as disclosed in any one of embodiments XLV-XLIX, wherein the media comprises lysogeny broth, Luria Bertani broth, Hi Soy (Hi Bacteriological media), tryptone, peptone, yeast extract, or any combinations thereof. LI. The method as disclosed in any one of embodiments XLV-L, wherein the media comprises Luria Bertani broth. LII. The method as disclosed in any one of embodiments XLV-LI, wherein the buffer comprises saline, phosphate, phosphate buffered saline, bovine serum albumin, Hanks' balanced salt solution (HBSS), fetal bovine serum (FBS), or any combinations thereof. LIII. The method as disclosed in any one of embodiments XLV-LII, wherein the buffer comprises Hanks' balanced salt solution (HBSS) comprising calcium and magnesium. LIV. The method as disclosed in any one of embodiments XLV-LII, wherein the buffer comprises Hanks' balanced salt solution (HBSS) comprising fetal bovine serum. LV. The method as disclosed in any one of embodiments LIII or LIV, wherein the HBSS buffer does not comprise phenol red. LVI. The method as disclosed in any one of embodiments XLV-LV, wherein the agar plate is Luria Bertani agar plate. LVII. The method as disclosed in any one of embodiments XLV-LVI, wherein the bacterial stock is prepared with bacteria in initial log phase, in exponential phase, in late Log phase, or in a stationary growth phase. LVIII. The method as disclosed in any one of embodiments XLV-LVII, wherein the preparation of the bacterial stock comprises incubating bacteria from the mother culture stock at 35°C to 39°C for 12 to 16 hours on a blood agar plate. LIX. The method as disclosed in any one of embodiments XLV-LVII, wherein the preparation of the bacterial stock comprises: a. incubating bacteria from the mother culture stock at 35°C to 39°C for 12 to 16 hours on a blood agar plate; and b. incubating bacteria from the blood agar plate in Luria Bertani broth for 2 to 4 hours at 37°C in shaker incubator. LX. The method as disclosed in any one of embodiments XLV-LIX, wherein the bacterial stock is prepared with bacteria in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm. LXI. The method as disclosed in any one of embodiments XLV-LIX, wherein the bacterial stock is prepared with bacteria in late Log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm. LXII. The method as disclosed in any one of embodiments XLV-LXI, wherein the test serum comprises antibodies against bacteria. LXIII. The as disclosed in any one of embodiments XLV-LXII, wherein the bacteria is Salmonella selected from a group comprising typhoidal Salmonella, non-typhoidal Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella Typhi, Salmonella Paratyphi, and any combinations thereof. LXIV. The method as disclosed in any one of embodiments XLV-LXIII, wherein the complement is at a concentration of 2.0 % to 20.0%, optionally wherein the complement includes Human complement and Rabbit complement. LXV. The method as disclosed in any one of embodiments XLV-LXIV, wherein the media is the Luria Bertani broth, and the buffer includes the HBSS; optionally, wherein the HBSS is without phenol red; and optionally wherein the test serum comprises antibodies against typhoidal Salmonella. LXVI. The method as disclosed in any one of embodiments XLV-LXIV, wherein the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with Calcium and Magnesium or with FBS, the HBSS is without phenol red, the complement is at a concentration of 2.0% to 20.0%; optionally wherein the test serum comprises antibodies against typhoidal Salmonella, optionally wherein the bacterial stock is in initial log phase or in late log phase. LXVII. The method as disclosed in any one of embodiments XLV-LXIV, comprising: i. providing the Luria Bertani broth and the HBSS buffer supplemented with FBS, i.e. the HBSS (-) buffer, wherein the HBSS (-) buffer is without phenol red; ii. providing Salmonella Typhi bacterial stock prepared from the Salmonella Typhi mother culture stock and the Luria Bertani broth; iii. diluting the Salmonella Typhi bacterial stock with the HBSS (-) buffer to obtain a diluted Salmonella Typhi bacterial stock that yields 50 to 250 colony forming units (cfus) or diluting the Salmonella Typhi bacterial stock 1:100 to 1:6000 with the HBSS (-) buffer to obtain a diluted Salmonella Typhi bacterial stock; iv. adding the test serum comprising antibodies against the immunogenic composition having Salmonella Typhi, the complement in concentration of 2.0% to 20.0%, the diluted Salmonella Typhi bacterial stock and the HBSS (-) buffer to form the assay solution v. incubating the assay solution at 35°C to 39°C for 50 to 90 mins; spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and vi. calculating antibody titre; wherein the Salmonella Typhi bacterial stock is in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm. LXVIII. The method as disclosed in any one of embodiments XLV-LXIV, comprising: i. providing the Luria Bertani broth and the HBSS buffer supplemented with Calcium and Magnesium, i.e. the HBSS (+) buffer, wherein the HBSS (+) buffer is without phenol red; ii. providing the Salmonella Paratyphi bacterial stock prepared from the Salmonella Paratyphi mother culture stock and the Luria Bertani broth; iii. diluting the Salmonella paratyphi bacterial stock with the HBSS (+) buffer to obtain a diluted Salmonella Paratyphi bacterial stock that yields 50 to 250 colony forming units (cfus) or diluting the Salmonella Paratyphi bacterial stock 1:100 to 1:6000 with the HBSS (-) buffer to obtain a diluted Salmonella Paratyphi bacterial stock; iv. adding the test serum, the complement in concentration of 2.0% to 20.0%, the Salmonella Paratyphi bacterial stock and the HBSS (+) buffer to form the assay solution; v. incubating the assay solution at 35°C to 39°C for 50 to 90 mins; spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; and vi. calculating antibody titre; wherein the Salmonella Paratyphi bacterial stock is in late log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm. LXIX. The method as disclosed in any one of embodiments XLV-LXVIII, wherein the step of calculating antibody titre comprises calculating number of bacterial colonies and determining the antibody titre based on the highest serum dilution where the number of bacterial colonies is less than or equal to 50% of heat-inactivated complement control. LXX. The method as disclosed in any one of embodiments XLV-LXIX, wherein the method determines antibody titre as a measure of effectiveness of an immunogenic composition or a vaccine. LXXI. The method as disclosed in any one of embodiments XLV-LXX, wherein the method determines functional IgG response to an immunogenic composition or a vaccine comprising Vi-TT and OSP-DT polysaccharide protein conjugate, and wherein the test serum comprises antibodies against the Vi-TT or OSP-DT polysaccharide protein conjugate. EXAMPLES The present invention is further illustrated in combination with the following examples. These examples are provided to exemplify the present invention but are not intended to restrict the scope of the presently claimed invention in any way. The terms and abbreviations in the examples have their common meanings. For example, “%”, “Eq. wt”, “Eq”, "° C", “wt. %”, "% w / w", “% w / v” and "gm" represent “percentage”, “Equivalent Weight”, “Equivalents”, "degree Celsius", “percent by weight”, "percent weight by weight", “percent weight by volume” and "gram" respectively. BIOLOGICAL MATERIALS • Minimum volume of sera sample required for analysis: 20 pl, Sera samples are heat inactivated (Treated at 56°C for 30 mins) prior to use in the assay. If any initial dilution is required for a particular sera sample, then appropriate buffer is used for making the required dilution. Process of sera sample is disclosed in “Test Serum” described hereinabove. • Strains in general: Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition may include: Salmonella enterica serovar typhi TY2 strain with "tviB" gene specific for Vi polysaccharide; Salmonella enterica serovar typhi deposited at NCMR-NCCS Accession No. MCC 0193; Strain designation-PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC 9150, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis Rll; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219;S. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2 / SL134 (ST19)); S.typhimurium 177(ST19) CDC 6516-60; ATCC 700720. Any attenuated Salmonella serovar strain (S. typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used for the preparation of the immunogenic composition for evaluation under the SBA assay. • Mother culture S.Typhi: Salmonella.Typhi strain (MCC-0193). S.typhi strain(MCC-0193) was originally isolated from stool sample of patient from Villo Poonawalla Hospital at SIIPL, further antigen structure was analysed and confirmed at CRI(Central Research Institute, Kasauli). Strain was further deposited to National Centre for Microbial Resource(NCMR) in 2020 and Accession number MCC-0193 and Strain designation number PDL-1, was obtained. • Mother culture S.Paratyphi: S.Paratyphi A strain (ATCC 9150) Salmonella enterica subsp. Enterica (ex-Kauffmann and Edwards) Le Minor and Popoff serovar Paratyphi A, procured from American Type Culture Collection, USA. • BABY Rabbit Complement: Rabbit Complement 3-4 week sterile / Cat # 31061-3 / Pel -Freez Biologicals, Rogers, AR, USA • Fetal Bovine Serum FBS: Fetal Bovine Serum Sterile Filtered / Moregate Biotech / 54 Banya Street, Bulimba, Australia • HBSS: - Hanks’ Balanced Salt Solution with Ca and Mg / Cat # 14025 / Life technologies Corporation / 3175 Staley Rd, Grand Island, New York, USA - Hanks’ Balanced Salt Solution without Calcium chloride, Magnesium chloride and magnesium sulfate / Cat # 14175 / Life technologies Corporation / 3175 Staley Rd, Grand Island, NY / USA • Columbia blood agar base: Columbia blood agar base / Cat # 279240 / Becton Dickinson and company / Sparks, MD 21152 / USA Horse blood agar plate: 5% Horse blood agar plates were inhouse prepared at SIIPL using Columbia blood agar base / Cat # 279240 / Becton Dickinson and company / Sparks, MD 21152 / USA • Human serum: • WHO International Standard 1st IS for Anti-Typhoid capsular Vi polysaccharide IgG (Human) / NIBSC code: 16 / 138 / National Institute for Biological Standards and Control / Potters Bar, Hertfordshire / United Kingdom • Rat, Rabbit and Mice serum: All animal sera samples (rat, rabbit and mice) were collected from animals used in the preclinical studies at Serum Institute of India Pvt Ltd (SIIPL), India. The studies followed national guidelines and institutional protocols / policies of SIIPL (41 / PO / RcBi / S / 99 / CPCSEA). The preclinical GLP-toxicity studies were performed in rodent and non-rodent models at GLP-certified facility (Reliance Life Sciences, Mumbai). The protocols were reviewed and approved by the Institutional Animal Ethics Committee (approval number 06-21) as per CPCSEA guidelines published in The Gazette of India, 2018. MATERIALS AND REAGENTS • Luria Bertani broth: Luria Bertani Broth, Miller / M1245-500G / HiMedia Laboratories Pvt. Ltd. / Reg.off:23, Vadhani Ind.Est, LBS marg, Mumbai / India • Luria Bertani agar: Luria Bertani agar, Miller / Ml 151 -500G / HiMedia Laboratories Pvt. Ltd. / Reg.off:23, Vadhani Ind. Est, LBS marg, Mumbai / India • Glycerol: Glycerol / Cat # G7893-500ML / Sigma-Aldrich / Co.,3050 Spruce Street, St Louis, MO 63103 / USA • 2,3,5 Triphenly Tetrazolium chloride stock: 2,3,5-Triphenyltetrazolium chloride / Cat # T8877-25G / Sigma-Aldrich / Co.,3050 Spruce Street, St Louis, MO 63103 / USA • Water: De-ionised or Water for injection (WFI) EXAMPLE 1: MEDIA PREPARATION The media for Luria Bertani Broth, the bacterial stock for S. Typhi and S. Paratyphi, Bacterial Assay stock titration for S. Typhi and S. Paratyphi were prepared as per METHODS SECTION listed hereinabove. Media Preparation: Luria Bertani broth (Media) was prepared in a 1 litre glass bottle with addition of 25 g of Luria Bertani broth to make up to 1000 ml with de-ionised water. Solution was mixed until all components have completely dissolved. Broth was sterilized at 121°C for 15 minutes by autoclaving. Media was stored at 2 to 8 °C. 80% glycerol was prepared by mixing 20 ml of deionised water and 80 ml glycerol and sterilized at 121°C for 15 minutes by autoclaving. Luria Bertani agar plate (LB A plate): In a 1000 ml autoclavable flask, 16 g of the Luria Bertani agar powder was added to 400 ml of de-ionised water, autoclaved (sterilized at 121°C for 15 minutes by autoclaving) and cooled to 56°C in water bath. 12 ml of solution was added to the square petri plate. Columbia blood agar plate with 5% horse blood: In a 1000 ml conical flask, 17.6 g of Columbia blood agar base was added to 400 ml of de-ionised water, autoclaved (sterilized at 121°C for 15 minutes by autoclaving) and cooled to 56°C in a water bath. 12 ml of solution was added to the square petri plate. Luria Bertani overlay agar: In a 250 ml autoclavable flask, 2 g of Luria Bertani broth and 1.5 g of Bacto agar was added to 100 ml of de-ionised water, autoclaved (sterilized at 121°C for 15 minutes by autoclaving). TTC stock (2,3,5 Triphenyl Tetrazolium Chloride): Prepared as 25 mg / ml (1000X) stock solution in water by adding 1.25 g of TTC to 40 ml de-ionised water. Volume adjusted to 50 ml with addition of de-ionised water and sterile filtered using 0.2pl filter to obtain a yellow coloured solution. Assay dilution buffer (Hanks Balanced Salt solution): HBSS-ve (without Ca and Mg) along with 1% FBS was used as assay buffer for S. Typhi SB A while HBSS+ve (with Ca and Mg) was used as assay dilution buffer for S. Paratyphi A SB A. Preparation of Bacterial Stock: S. Typhi: Retrieved the mother culture stock vial from the freezer, streak it onto a blood agar plate and incubate the plate 12-16 hours at 37°C with 5% in CO2 incubator. Transferred one loopful of isolated colonies from culture and inoculate in a sterile conical flask containing 50 ml of Luria Bertani broth. Incubated flask for 2-4 hours, in shaker incubator (37°C) with 150 RPM until the absorbance of the culture broth has an OD600 of 0.3 to 0.4. After achieving desired OD, 10 ml of bacterial suspension, 10 ml of fresh Luria Bertani broth, and 4 ml of sterile 80% glycerol were mixed by vertexing and dispense 0.5 ml aliquots of bacteria into properly labelled sterile cryo-vials. Preparation of bacterial stock S. Paratyphi A: Retrieved the mother culture stock vial from the freezer, streaked it onto a blood agar plate and incubate the plate 12-16 hours at 37°C with 5% in CO2 incubator. Transferred one loopful of isolated colonies from culture and inoculate in a sterile conical flask containing 50 ml of Luria Bertani broth. Incubate flask for 2-4 hours, in shaker incubator (37°C) with 150 RPM until the absorbance of the culture broth has an OD600 of 0.9 to 1.10. After achieving desired OD, 10 ml of bacterial suspension, 10 ml of fresh Luria Bertani broth, and 4 ml of sterile 80% glycerol were mixed by vertexing and dispensed 0.5 ml aliquots of bacteria into properly labelled sterile cryo-vials. Bacterial assay stock titration for S. Typhi Allowed a frozen assay stock of the desired strain from the -70°C freezer to thaw at room temperature for 2 min. Took 100 pl and add to 0.9 ml assay dilution buffer (this is a 10 4 dilution). Mixed properly and aliquoted of 100 pl from the 10'1 tube and added to 0.9 ml assay dilution buffer (this becomes a 10'2 dilution). Repeat the procedure to generate a 10'3. i.e. 1:1000 dilution. This was further diluted to obtain 1: 2000, 1: 3000, 1: 4000, 1: 5000, 1: 6000 and further dilutions. Each of the test dilutions was further diluted: 30 pl of assay dilution buffer + 10 pl of the bacterial dilution. Spot of 10 pl aliquots from each well on Luria Bertani agar plate in duplicate was made and incubated plate 10-14 hours at 37°C in 5% CO2 Incubator. Count the colonies in each streak of growth and find the average count for each dilution. The ideal number of colonies for assay was taken between 50 to 250 cfu. Bacterial assay stock titration for S. Paratyphi A: Allowed a frozen assay stock of the desired strain from the -70°C freezer to thaw at room temperature for 2 min. Took 100 pl and add to 0.9 ml assay dilution buffer (this is a 10 -1 dilution). Mixed properly and aliquot of 100 pl from the 10-1 tube and add to 0.9 ml assay dilution buffer (this becomes a 10-2 dilution). Repeated the procedure to generate a 10-3 i.e. 1:1000 dilution. This was further diluted to obtain 1: 2000, 1: 3000, 1: 4000, 1: 5000, 1: 6000 and further dilutions. Each of the test dilutions was further diluted: 30 pl of assay dilution buffer + 10 pl of the bacterial dilution. Spot of 10 pl aliquots from each well on Luria Bertani agar plate in duplicate was made and incubated plate 10-14 hours at 37°C in 5% CO2 Incubator. Counted the colonies in each streak of growth and find the average count for each dilution. The ideal number of colonies for assay was taken between 50 to 250 cfu. EXAMPLE 2: SB A ASSAY The SBA Assay was performed for both S. Typhi and S. Paratyphi based on the methods as disclosed in the methods section. Serum bactericidal activity (SBA) assay protocol S. Typhi: Added 20 pl of assay dilution buffer (HBSS-ve with 1% FBS) to each well of a flat bottom 96-well plate. Added 20 pl of diluted or undiluted test serum to the wells of column Al-FL G1 and Hl are reserved for Reference sera (serum with a known Titre). Made 2-fold Serial dilution of test and Reference serum (rows 1 to 9). Column 10 and 11 which was reserved for the complement controls. Based on the bacterial titration results made a suitable dilution of bacteria that gives ideal colony number / Colony Forming Units (CFU) of specific bacteria (here S.Typhi) and added 10 pl of bacteria to each well. Used bacteria from a frozen stock (S. Typhi 0.4 OD) in assay dilution buffer. Added 2 pl complement (Baby Rabbit Serum) to the plate through column 1 to 10. Final assay volume 40 pl was adjusted by adding 8 pl dilution buffer to form the assay solution. 2 pl Heat inactivated complement source and 8 pl dilution buffer was added in column 11. Incubated the plate having the assay solution at 37°C for 60 min without shaking and 10 pl spotting was done from each well onto Luria Bertani agar plates. Incubated the plates overnight at 37°C, 5% CO2. Counting of plates was done after 10 to 14 hours incubation manually or using ProtoCOL 3 Automated colony counter. Serum bactericidal activity (sba) assay protocol S. Paratyphi A: Added 20 pl of assay dilution buffer (HBSS +ve buffer) to each well of a flat bottom 96-well plate. Added 20 pl of diluted or undiluted test serum to the wells of column Al -F1. G1 and Hl are reserved for Reference sera (serum with a known Titre). Made 2-fold Serial dilution of test and Reference serum (rows 1 to 9). Column 10 and 11 which is reserved for the complement controls. Based on the bacterial titration results make a suitable dilution of bacteria that gives ideal colony number / Colony Forming Units (CFU) of specific bacteria (here S.Paratyphi) and added 10 pl of bacteria to each well. Used bacteria from a frozen stock (S. Paratyphi A 1.0 OD) in assay dilution buffer. Added 5 pl complement to the plate through column 1 to 10. Final assay volume 40 pl was adjusted by adding 5 pl dilution buffer to form the assay solution. Heat inactivated complement source and 5 pl dilution buffer was added in column 11. Incubated the plate having the assay solution at 37°C for 60 min without shaking and 10 pl spotting was done from each well onto Luria Bertani agar plates. Incubated the plates overnight at 37°C, 5% CO2. Counting of plates was done after 10 to 14 hours incubation manually or using ProtoCOL 3 Automated colony counter. Calculation of antibody titres: Colony counts obtained in the Heat Inactivated Complement Control (HIC) were taken. The SBA Titre was calculated as the highest serum dilution where the colony count was less than or equal to 50% of the complement control (HIC). The bacterial count difference for C’ (complement control) and HIC (Heat Inactivated complement) wells were not be more than 30%. In case if the dilution scheme fell short to give an appropriate titre (when the last dilution also has colonies less than 50 % of the HIC control) then the serum sample were repeated with a higher dilution scheme. EXAMPLE 3: GROWTH PATTERN &STOCK PREPARATION METHOD For the Serum Bactericidal Activity Assay against both S. Typhi and S. Paratyphi A, bacterial growth pattern was evaluated and vulnerability of bacteria was checked for neutralization during assay. Growth characteristics was tested with respect to capsule formation and maturity of capsule during the growth phase. Growth phase is associated with evasion / escapement from complement binding, leading to resistant towards killing during the assay for S. Typhi. Stock preparation was done by 2 methods that facilitated accelerated and accurate analysis. Bacteria were prepared by regular / conventional method involving freezing (glycerol stock) bacteria in healthy condition. The bacteria were then used for streaking on to enriched medium on day 1, again steaked on day 2, for confluent growth, to access bacteria, in exponential phase for using them in SB A on day 2, the results are obtained on day 3. In another method, i.e. O.D based method, bacteria were prepared by broth culturing in Luria Bertani broth for about 2-4 hours till a particular O. D (600nm) was achieved. S. Typhi was grown till O.D(600nm) value observed was of, 0.3-0.4, and S. Paratyphi was grown till O.D (600nm) observed was 0.9-1.0 for S.Paratyphi A. Glycerol stocks were prepared and frozen. Bacterial stocks were titrated to determine a dilution required to yield appropriate colonies in control wells. Stocks prepared were used on day 1 with determined dilution factor and results for SBA are obtained on day 2. The assay using Luria Bertani broth as per the O.D based method was completed in 2 days duration. The O.D based method was thus faster than conventional method and imply high throughput ability. Since, analysis was achieved with cells cultivated from a homogenous mixture from the same pool, requirement of preparing bacteria and adjusting O.D on each day is removed and possible variations in the conventional method are significantly reduced. S. Typhi was susceptible to killing only during the initial Log phase (0.3-0.4 O.D). S. Typhi became completely resistant to killing during the stationary phase. The late Log to stationary phase was associated with formation of the capsule for A Typhi bacteria. S. Typhi once matured and with capsule is able to evade bactericidal activity. Since S. Paratyphi A did not show any effect of different growth phases, late log to stationary phase (0.9-1.0 O.D) was used. S. Paratyphi A has no capsular structure and antibodies are directly targeted to the LPS on the cell wall, thus the effect of different growth stages (early log, log, and stationary phase) doesn’t affect the bactericidal activity. Multiple stocks were prepared for both S. Typhi and S. Paratyphi A to check the effect of growth phase on bacterial killing S. Typhi: Stock was harvested at 600nm OD with absorbance of 0.3-0.4 and 0.8-0.9 &1.2 -1.4 TABLE 1 : S.TYPHI BACTERIAL STOCKS AT DIFFERENT OD INTERVALS S. Typhi Stoc < 0.4 OD Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:10) 0 0 2 2 4 1 6 6 21 23 53 >10240 16 / 138 (1:100) 3 2 5 8 3 7 16 23 19 18 42 >102400 S. Typhi Stoc <0.8 OD Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:10) 64 64 60 58 64 51 62 63 56 52 50 NA 16 / 138 (1:100) 57 65 59 48 65 48 57 60 58 70 58 NA S. Typhi Stoc < 1.2 OD Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:10) 71 57 51 53 55 53 79 56 56 49 55 NA 16 / 138 (1:100) 59 50 62 66 53 50 42 39 47 43 61 NA TABLE 2: COMPARISON OF S. TYPHI BACTERIAL STOCKS AT DIFFERENT OD INTERVALS Dilution 4 8 16 32 64 128 256 512 1024 C HIC 0.4 OD Stock 0 0 2 2 4 1 6 6 21 23 53 0.8 OD Stock 64 64 60 58 64 51 62 63 56 52 50 1.2 OD Stock 71 57 51 53 55 53 79 56 56 49 55 Overall Inference: No antibody Titre was obtained for bacteria harvested at 0.8 OD &1.2 OD (mid log phase to stationary phase) because of capsule generation that helps bacteria to evade killing while positive reaction was seen for bacteria harvested at early log phase 0.4 OD. See FIG. 1 for reference. S. Paratyphi A: Stock was harvested at 600nm OD with absorbance of 0.3-0.4 and 0.9-1.0 &1.2-1.4 TABLE 3: S. PARATYPHI A BACTERIAL STOCKS AT DIFFERENT OD INTERVALS S. Paratyphi A Stock 0.4 OD Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:10) 4 4 2 3 7 18 37 72 64 65 82 2560 16 / 138 (1:100) 0 8 20 45 74 82 61 68 88 77 106 3200 S. Paratyphi A Stock 0.9 OD Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:10) 0 0 0 2 18 38 76 75 64 81 105 1280 16 / 138 (1:100) 1 23 41 64 66 74 76 80 75 72 98 1600 S. Paratyphi A Stock 1.2 OD Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:10) 0 0 0 0 11 32 63 60 84 78 109 1280 16 / 138 (1:100) 1 10 52 57 88 82 81 84 78 82 112 1600 SB A titer is calculated at the highest dilution of the sera sample yielding 50% les CFU’s as compared to CFU count in the HIC well. HIC well CFU count in this example is 82, 50% of this would be 41, Hence highest dilution of sera 16 / 138 standard yielding CFU close of 41 or less is calculated as SBA titer. In this example 256 is having CFU count of 37(< 41) hence the titer calculated is 2560 (as initial dilution factor of sera is 1:10, hence 256 x 10 = 2560). TABLE 4: COMPARISON OF S. PARATYPHI A BACTERIAL STOCKS AT DIFFERENT OD INTERVALS Dilution 4 8 16 32 64 128 256 512 1024 C HIC 0.4 OD Stock 0 8 20 45 74 82 61 68 88 77 106 0.9 OD Stock 1 23 41 64 66 74 76 80 75 72 98 1.2 OD stock 1 10 52 57 88 82 81 84 78 82 112 Overall Inference: Antibody Titre was obtained for bacteria harvested at 0.4 OD, 0.9 OD &1.2 OD shows that there is no effect of growth phase on bacterial killing as S. Paratyphi A has no capsule and antibody is directed against LPS of the bacterial cell wall. 0.9-1.0 O.D bacteria was chosen in-order to show killing against completely matured and developed bacteria. See FIG. 2 for reference. EXAMPLE 4: BUFFER COMPOSITIONS Hanks balanced salt solution, HBSS+ and HBSS- buffer with and without stabilizers like BSA (Bovine Serum Albumin) or FBS (Fetal Bovine Serum) were used. The SBA for S. Paratyphi A showed excellent results with HBSS+ buffer, whereas S. Typhi, had an observable reaction with multiple buffer systems but showed good clearing / killing of bacteria with HBSS-containing 1% FBS. Buffer selection for S. Typhi with inclusion of 1% FBS has facilitated better clearing of bacteria thus enhancing the effective killing and generating a smooth curve, allowing a distinct difference between Pre and Post sera samples, which is otherwise not obtained with regular positive buffers. Final Assay volume for both S. Typhi and S. Paratyphi A SB A was kept at 40pl, with composition for S. Typhi SB A: 20 pl sera+ 10 pl bacteria + 2 pl complement + 8 pl buffer and S. Paratyphi A SB A: 20 pl sera+ 10 pl bacteria + 5 pl complement + 5 pl buffer. These compositions keep the assay format easier and dilution scheme starts with 1:4 dilution. Combinations of buffer system were checked. For S. Typhi, buffer systems included: HBSS + HBSS- supplemented with 5 % BSA and HBSS- supplemented with 1 % FBS TABLE 5: S. TYPHI USING HBSS+ BUFFER S. Typhi HBSS+ Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 79 87 73 67 73 77 63 61 77 84 90 2 16 / 138 85 84 74 75 91 71 70 80 73 75 85 2 Inference: No antibody Titre obtained for positive control sera sample (NIBSC 16 / 138- Human QC sera) in HBSS+ buffer TABLE 6: S. TYPHI USING HBSS - WITH 5 % BSA BUFFER S. Typhi HBSS - with 5 % BSA Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:10) 7 0 3 5 9 13 19 40 53 86 114 >10240 16 / 138 (1:100) 8 10 19 21 50 55 80 83 88 84 110 12800 Bacteria control 18 17 20 22 16 13 20 21 15 13 12 NA Bacteria control 14 17 16 20 18 13 18 20 21 20 26 NA Inference: Antibody Titre obtained for positive sera sample (NIBSC 16 / 138) in HBSS-supplemented with 5 % BSA but in the bacteria control wells the bacteria alone could not survive till the assay duration of 60 mins. Hence needed better supplementation. TABLE 7: S. TYPHI USING HBSS - WITH 1 % FBS BUFFER S. Typhi F BSS - with 1 % FBS Dilution 4 8 16 32 6 4 12 8 25 6 51 2 102 4 C HI C Titre 16 / 138 (1:10) 0 0 0 0 0 0 1 3 18 10 3 124 >1024 0 16 / 138 (1:100) 0 0 0 0 5 20 62 84 83 10 0 126 25600 Bacteria control 10 1 11 1 11 3 10 0 9 5 10 0 92 98 99 10 5 98 NA Bacteria control 10 0 98 86 95 9 7 99 10 5 10 2 95 99 98 NA Inference: Antibody Titre obtained for positive sera sample (NIBSC 16 / 138) in HBSS-supplemented with 1 % FBS S. Typhi SB A showed better killing curve for standard NIBSC 16 / 138 with HBSS- and 1% FBS in buffer matrix, these imply that supplementation of buffer matrix with 1% FBS helped better growth of bacteria and also facilitated better killing curve for the S. Typhi bacteria. TABLE 8: S. TYPHI USING ALL BUFFER MATRIX Dilution 4 8 16 32 64 128 256 512 1024 C HIC HBSS + BUFFER 85 84 74 75 91 71 70 80 73 75 85 HBSS - with 5% BSA 8 10 19 21 50 55 80 83 88 84 110 HBSS - with 1% FBS 0 0 0 0 5 20 62 84 83 100 126 Overall Inference: Using HBSS- buffer supplemented with 1 % FBS for S. typhi showed better clearing of bacteria and passing all the assay acceptance criteria. Hence, the same buffer was employed for performing the SB A assays for S. typhi. See FIG. 3 for Effect of Buffer matrix on killing (for HBSS+, HBSS- with 5% BSA and HBSS-with 1% FBS). S. Paratyphi A: TABLE 9: S. PARATYPHI A USING HBSS+ BUFFER S. Paratyphi A HBSS + Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:4) 2 2 0 3 6 29 52 79 83 117 139 1024 16 / 138 (1:8) 0 0 2 5 18 47 89 90 93 121 130 1024 Inference: Antibody Titre obtained for positive control sera sample (NIBSC 16 / 138) in HBSS+ buffer. SB A titers are obtained for NIBSC 16 / 138 standard for S. Paratyphi A with use of HBSS+ buffer alone. TABLE 10: S. PARATYPHI A USING HBSS - WITH 1 % FBS BUFFER S. Paratyphi A HBSS - with 1 % FBS Dilution 4 8 16 32 64 128 256 512 1024 C HIC Titre 16 / 138 (1:4) 4 0 0 0 5 27 70 95 99 115 131 512 16 / 138 (1:8) 2 0 3 9 35 78 99 100 106 125 142 512 Inference: Antibody Titre obtained for positive control sera sample (NIBSC 16 / 138) in HBSS-supplemented with 1 % FBS buffer. SB A titers are obtained for NIBSC 16 / 138 standard for S. Paratyphi A with use of HBSS- with 1% FBS buffer. TABLE 11: S. PARATYPHI A USING ALL BUFFER MATRIX Dilution 4 8 16 32 64 128 256 512 1024 C HIC HBSS + BUFFER 0 0 2 5 18 47 89 90 93 121 130 HBSS - withl% FBS 4 0 0 0 5 27 70 95 99 115 131 Overall Inference: Since there was no difference in the Titre obtained by both buffer system the HBSS+ buffer was employed for performing the SB A assays for S. Paratyphi A. See FIG. 4 for Effect of Buffer matrix on killing (for HBSS+ and HBSS- with 1% FBS). EXAMPLE 5: CONCENTRATION OF COMPLEMENT Complement concentrations of different ranges were checked to obtain a lower killing percentage of below 15% of intrinsic killing. Intrinsic killing is difference between HIC-heat inactivated complement control and C’- complement control wells. Intrinsic killing is associated with gradual clearing of bacteria enabling distinct Titre footprint. A 2 (5 %) pl complement concentration for the S. Typhi assay and 5 (12.5 %) pl for S. Paratyphi A provided desired results. S. Typhi Complement concentration evaluation was done in different buffer system viz., HBSS + BUFFER, HBSS - buffer with 5% BSA and HBSS - Buffer with 1% FBS: • Different complement concentrations 6.25%, 12.5 %, 18.75% and 25% was tested for HBSS + buffer and HBSS- buffer with 5% BSA. • HBSS - Buffer with 1% FBS was tested for 5%, 6.25%, 12.5 %, 18.75% and 25% complement concentrations. HBSS + BUFFER: TABLE 12: S. TYPHI VARYING COMPLEMENT CONCENTRATION USING HBSS + BUFFER S. Typhi varying complement concentration Buffer Complement concentration HBSS + Buffer 2.5 pl (6.25%) 5 pl (12.5%) 7.5 pl (18.75%) 10 pl (25%) C 86.25 75.25 67.5 55.5 HIC 97.5 91.5 93.75 101 % Killing 11.5 17.8 28.0 45.0 Note: Values indicating for C’, HIC and % Killing are average count of bacterial CFU. See FIG. 5 for reference. S. TYPHI VARYING COMPLEMENT CONCENTRATION USING HBSS + BUFFER • HBSS - buffer with 5% BSA: TABLE 13: S. TYPHI VARYING COMPLEMENT CONCENTRATION USING HBSS - BUFFER WITH 5% BSA S. Typhi varying complement concentration Buffer Complement concentration HBSS -with 5% BSA 2.5 pl (6.25%) 5 pl (12.5%) 7.5 pl (18.75%) 10 pl (25%) C 87.5 56.5 27.5 18.5 HIC 98 111.5 93.5 85.5 % Killing 10.7 49.3 70.6 78.4 Note: Values indicating for C’, HIC and % Killing are average count of bacterial CFU. See FIG. 6 for reference. S. TYPHI VARYING COMPLEMENT CONCENTRATION USING HBSS - BUFFER WITH 5% BSA • HBSS- Buffer with 1 % FB S: TABLE 14: S. TYPHI VARYING COMPLEMENT CONCENTRATION USING HBSS - BUFFER WITH 1% FBS S. Typhi varying complement concentration Buffer Complement concentration HBSS - with 1% FBS 2 pl (5%) 2.5 pl (6.25%) 5 pl (12.5%) 7.5 pl (18.75%) 10 pl (25%) C 93 81 53 30 17 HIC 101 100 106 98 85 % Killing 7 19 50 70 80 Note: Values indicating for C’, HIC and % Killing are average count of bacterial CFU. See FIG.7 for reference. S. TYPHI VARYING COMPLEMENT CONCENTRATION USING HBSS - BUFFER WITH 1% FBS Over all Inference: Different Complement concentrations were analyzed for different buffer systems among all HBSS - buffer with 1% FBS result indicated that 2 ul (5%) complement showed better % killing and clearing of bacteria. Complement concentration evaluation is a critical aspect in determining the amount of complement required for a desired bactericidal effect, failing to determine this could lead to errors in SBA titer estimation. Various complement concentrations were checked to obtain a lower killing percentage of below 15% of intrinsic killing (Difference between HIC-heat inactivated complement control and C’- complement control wells) along with gradual clearing of bacteria enabling distinct titer footprint. A 2 (5 %) pl complement concentration for the S. Typhi assay and 5 (12.5 %) pl for S. Paratyphi A provided desired results. Complement Concentration for S. Paratyphi A: • HBSS + Buffer: TABLE 15: S. PARATYPHI A VARYING COMPLEMENT CONCENTRATION USING HBSS + BUFFER S. Paratyphi A varying complement concentration Buffer Complement concentration HBSS + Buffer 2.5 pl (6.25%) 5 pl (12.5%) 7.5 pl (18.75%) 10 pl (25%) C 125 110 87.75 73.5 HIC 128 128 124 117.25 % Killing 2.3 14.1 29.2 37.3 Note: Values indicating for C’, HIC and % Killing are average count of bacterial CFU. See FIG. 8 for reference. S. PARATYPHI A VARYING COMPLEMENT CONCENTRATION USING HBSS + BUFFER Inference: Different Complement concentrations were analyzed wherein results indicated that 2.5ul showed better results for % killing which increased with increasing complement concentration but clearing and Titres were seen at 5ul (12.5%) complement. EXAMPLE 6: EFFECT OF SHAKING VS NON-SHAKING SBA’s were performed using either a shaking step for plates at 150 rpm or were kept stationary (non-shaking) during 1 hr. incubation period. No major difference was observed in the Titres (by using either of the variation) indicating no additional benefit for shaking step and hence proper mixing of well contents prior to incubation step was adequate for killing / neutralization of bacteria during the SB A. EXAMPLE 7: STANDARDIZATION USING INTERNAL / INTERNATIONAL QC’S Various usage was done of Quality controls sera with positive and negative sera from different species along with different assay controls during the experiments. All controls gave acceptable results. Median SBA Titres with specific range have been established for all the quality controls (QC). EXAMPLE 8: USE OF DIFFERENT AGAR MEDIUM FOR SPOTTING PURPOSE Different agar medium used for spotting purpose were assessed using Blood agar (Columbia blood agar base with 5% horse blood / Columbia blood agar base / Cat # 279240 / Becton Dickinson and company / Sparks, MD 21152 / USA) and Luria Bertani agar plates. Growth of bacteria on blood agar plates was too prolific and colonies would merge easily thus causing error while counting, Luria Bertani agar plates gave well defined smaller colonies which were easier to count both manually and on automated counter (ProtoCOL 3, Synbiosis, UK). EXAMPLE 9: VALIDATION: • Validation parameters were studied for the SBA’s involving various parameters such as Precision, performed with quality control serums (Human, Rat, and Rabbit). • Good precision was of >90% for various control sera tested was observed. • Specificity was observed for the both SBA’s wherein at least >4-fold inhibition was seen for homologous and <2-fold inhibition for heterologous reaction were observed. • Linearity was assessed including different test sera and results obtained indicate good assay linearity for both the serogroups with r2 values of >0.9 were achieved for all the occasions with the slope lying between -1 to - 1.3. • For accuracy, control sera were tested in duplicates and each run was repeated twice. An accuracy of 100 % was achieved for both serogroups. • Robustness was assessed with different (30, 60 and 90 mins) incubation intervals, 60 and 90 mins did not have a significant change on the SBA Titre, whereas reduced Titres (<+ 1 SBA Titres) were observed for S. Paratyphi A at 30 mins, thus indicating, incubation period of more than 30 mins is required for reproducible Titres for both assays. Robustness involving repeat free thaw cycles for sera samples were also tested, with no loss of functionality till 10 cycles of freeze thaw. WCB (Working Cell Bank) were prepared for both of the Salmonella Serovars (S.Typhi and S.Paratyphi A) with 45 glycerol stock vials prepared for each serovar and stored at -70°C until further use. Bacterial titration was done in order to find a desired dilution of the bacteria to be used in the assay. Baby rabbit complement Lot# 02943-A which was utilized in the developmental activities of the assay was utilized for the entire validation process. All other reagents like buffer (HBSS+, make- Invitrogen, FBS-Fetal bovine serum, make Moregate) and media (Luria Bertani agar, Luria Bertani broth, make Hi-media) used for assay, were of the same lot. For use of standard sera (Quality control sera) for validating the method, we made use of sera from different species (Human, Rat and Rabbit). Overall conclusion: The SBA’s for both the serovars (S.Typhi and S.Paratyphi A) are measured to be precise, linear, accurate, robust and specific from the results shown above for each of the individual parameter tested. Acceptance criteria are meet for each of the parameter. Both assays are robust for 60 and 90-mins incubation time interval as for 30 mins incubation time, lower SBA titers are seen as compared to median titer. Bacterial strains and stock preparation. Two separate methods of bacterial stock preparation were used, a) Bacterial cultures of S. Typhi (referred as ‘ST’) and S. Paratyphi A (referred as ‘SPT’) were grown on 5% horse blood agar plates overnight before being transferred to BHI broth. A thick bacterial suspension was used for the preparation of glycerol stocks and bacteria was adjusted to an OD (OD650) of 0.1 (UV-1800 spectrophotometer, Shimadzu) giving 1-5 x 104 cells / well and was used in the assay, b) Bacterial cultures of both serogroups grown overnight on 5% horse blood agar plates were transferred to Luria Bertani broth and were incubated at 37°C with 150 rpm shaking (incubator shaker-Thermo Scientific, MaxQ6000). Bacteria were harvested at different optical densities at 600nm (OD600). S. Typhi and S. Paratyphi A were harvested, at OD of 0.4 and 1.0 respectively. Bacterial glycerol (80%) working stocks, prepared from harvested stocks, were frozen at -70°C until use. Working cell banks were titrated in order to find desired dilution. A range of 10'1 to 10'6 dilution was used. A dilution yielding 400 CFU per well were selected for performing the assays. Sera samples. Positive and negative controls for each species were used in the test experiments and validation. All animal sera samples (rat, rabbit and mice) were collected from animals used in the preclinical studies. Human sera used were procured from NIB SC (NIB SC 16 / 138) and Pel-Freez Biologicals (Human IgG and IgM depleted serum, Pel-Freez Biologicals Cat # 34010). TABLE 16: SERA SAMPLE DETAILS Source Sample Description Type of Sample Human NIBSC 16 / 138 Positive control (ST, SPT) D28 sera (Post-vaccination) Positive control (ST) IgG-IgM depleted sera Negative control (ST, SPT) Rat D28, D42 sera post-BTCV vaccination Positive control (ST, SPT) Pre-vaccination sera Negative control (ST, SPT) Mice D28, D42 sera post-BTCV vaccination Positive control (ST, SPT) Pre-vaccination sera Negative control (ST, SPT) Rabbit D28, D42 sera post-BTCV vaccination Positive control (ST, SPT) Pre-vaccination sera Negative control (ST, SPT) ST: S.Typhi; SPT: S.ParatyphiA Quality Control Sera (QC Sera) Validation of SBA’s required defined quality control sera, prepared in-house Rat (Rat 25pg) and Rabbit (D28) QC sera by pooling sera obtained from healthy vaccinated rat and rabbit with various in-house formulation. Sera from individual rat and rabbits was pooled for preparation of the QC sera. Pool of sera thus prepared was aliquoted(500pl) into 1.5 ml centrifuge tubes, labelled and stored at -70°C. Human Quality control standard was procured from NIBSC (NIBSC 16 / 138). Preclinical studies and ethical statement: The preclinical studies and GLP-tox studies were performed in accordance with concerned ethics committee approvals. The preclinical development studies were performed in various small animal models such as mice, rat, and rabbit. The studies were in compliance with national guidelines and institutional protocols / policies of SIIPL (41 / PO / RcBi / S / 99 / CPCSEA). The preclinical GLP-toxicity studies were performed in rodent and non-rodent models at GLP-certified facility (Reliance Life Sciences, Mumbai). The protocols were reviewed and approved by the Institutional Animal Ethics Committee (approval number 06-21) as per CPCSEA guidelines published in The Gazette of India, 2018. All animal sera used in assay validation were derived from mice, rat, and rabbit immunization experiments. The sera samples from GLP-tox study were analyzed for ELISA and SBA titer using validated methods. Approval details for Tox study in preclinical models • Study location: Laboratory Animal Research Services, Reliance Life Sciences Pvt. Ltd. 212 / 2, DALC, R-282, Thane- Belapur Road, Rabale, Navi Mumbai, MH 400701 INDIA. Approval details for In-house preclinical studies • study approval number is 13 / 26-06 / 2021-R Validation Procedure: To validate the estimation of serum bactericidal titer, following Parameters were analyzed: • Precision • Linearity and Accuracy • Specificity • Robustness EXAMPLE 10: PRECISION Intermediate precision is the degree of agreement of SBA results when serum specimens are tested multiple times (i.e., on different days or by different analysts). This parameter was assessed by the following method. Parameters: 5 replicates, intra-assay and inter-assay precision, performed by 3 analysts. Criteria: Median titers for each of the standard sera tested for both SP and SPT were calculated. Acceptance criteria were set with >85 % of repetitions tested should be in agreement (SBA titers between +2-fold of the median titer). Repeatability: • A panel of standard controls (NIBSC 16 / 138, Rat 25pg and Rabbit D28) sera were analysed repeatedly. • Analyst 1 performed each quality control sera 5 times per plate and 3 runs were performed by analyst 1. • A total 15 results for each Quality Control serum were obtained. Ruggedness: • Two different Analysts (2 and 3) performed the same assay for quality controls sera as done by analyst 1. Results comparison • Results obtained by analyst 1 were considered for calculation of median titer and results obtained by analyst 2 and 3 were compared to analyst 1. • Combined comparison of results obtained by all three analysts was done. Acceptance criteria: • The acceptance criteria for this parameter are that the results for >70% of the specimens in the panel, be in agreement. Agreement is defined as a titer within two-fold of the established median SBA titer. For example, if the median titer is 512, then titers of 256, 512, and 1,024 would be in agreement. • >70% of the results obtained by Analyst 2 and Analyst 3 must be within 2-fold of the established median titer obtained by Analyst 1 The precision was evaluated via forty-five data points (FIG. 9) for both ST and SPT SBA. For ST SBA, the median titer obtained for NIBSC human sera, rat QC sera and rabbit QC sera was 8192, 4096 and 2048 respectively with a frequency % for median titer of 100, 100 and 97.7 respectively. For SPT SBA, the median titer obtained for NIBSC human sera, rat QC sera and rabbit QC sera was 1024, 1024 and 512 respectively with a frequency % for median titer of96, 100 and 100 respectively. Both the ST (FIG. 9, A) and SPT SB As (FIG. 9, B) sufficed the criteria for precision (with>85% of titers within 2-fold of median titers). TABLE 17: PRECISION RESULTS S.typhi SBA precision NIBSC 16 / 138 Rabbit D28 Rat 25 pg Median Titer Analyst 1 8192 2048 4096 Analyst 2 (% Agreement) 15 / 15 (100%) 15 / 15 (100%) 15 / 15 (100%) Analyst 3 (% Agreement) 15 / 15 (100%) 14 / 15 (93.3%) 15 / 15 (100%) S.Paratyphi A SBA precision NIBSC 16 / 138 Rabbit D28 Rat 25 pg Median Titer Analyst 1 1024 512 1024 Analyst 2 (% Agreement) 15 / 15 (100%) 15 / 15 (100%) 15 / 15 (100%) Analyst 3 (% Agreement) 15 / 15 (100%) 15 / 15 (100%) 15 / 15 (100%) Table 17. shows Median titer calculated based on the 15 titers results for each of the quality control sera (NIBSC 16 / 138, Rabbit D28 and Rat 25pg) performed for both the serovars (S.Typhi and S.Paratyphi A). Also, the results obtained by Analyst 2 and 3 are compared to the median titer obtained by Analyst 1. Result’s obtained by both the analyst (2 and 3) are in acceptable limits. See FIG. 9 for the Precision results (Graphl) FIG. 9 (Graph 1) shows total repetition performed by all 3 analysts (total 45 SBA titres / quality control sera) on the X axis and SBA titer on the Y axis. Precision assessment for S Typhi (A) and S Paratyphi A (B) using three different standards namely NIBSC 16 / 138, Rabbit QC, and Rat QC assay performed with multiple repetitions. X axis represents the number of repetitions while the Y axis is plotted with SBA titers. Conclusion for Precision: Median titers were calculated for repetitions performed by Analyst 1 and are mentioned in the Table 1 for each of the QC sera for both the serovar tested. Results for Analyst 2 and 3 were found to be within acceptable limits and overall precision (>70% results) is seen as all reading are within acceptable limits. EXAMPLE 11: LINEARITY AND ACCURACY Linearity is defined as the ability of the SBA to yield titers that are directly proportional to the concentration of serotype-specific Salmonella antibody in the specimen. Accuracy is the closeness of agreement of a value obtained from the assay to the reference or known value. An approach involving the spiking of different amounts of SBA-positive serum into negative sample (buffer) was used for accuracy. Experiment design: 1. For both Linearity and Accuracy common experiment were performed. For linearity: Serogroup specific SBA was performed with five different initial dilutions of all 3 Quality control serum, ranging from Neat to 1:512. All assays were performed twice (Run 1 and Run 2) for all three QC sera. SBA titers obtained and dilution used for each serum were Log2 transformed before plotting. Linear regression analysis was performed for the data and r2 values were calculated. 2. For Accuracy: An approach involving spiking of different amount of SBA positive sera into negative sample (Assay buffer) was used to estimate accuracy. SBA titer obtained for each of the dilution were compared with the established median titer from precision experiment to prove the accuracy of methodology. Acceptance criteria: For Linearity: • Acceptable linearity was obtained if the calculated Linear regression (r2) had a value of 0.9 and above, which describes the relationship between the measured SBA titers and the concentrations of the test serum sample. Parameters: 2 runs, samples in duplicates, 5 initial dilutions Criteria: Titer and serum dilutions were log2 transformed. Regression (r2) value >0.9 and slope range -0.65 to -1.35. • The criterion for acceptable assay accuracy is that the observed SBA titer of the spiked serum specimen be within two-fold of the expected SBA titer (Median titer). Overall agreement for each of the dilution checked should be above 80%. Parameters: Two dilutions of positive sera were used per standard, spiking SBA positive sera with SBA negative sera. Criteria: The spiked sera titer should be within +2-fold of the median SBA titer. See FIG. 10 for Linearity results: Graph 2: S.Typhi See FIG. 11 for Linearity results: Graph 3: S.Paratyphi A FIG. 10 (Graph 2) and FIG. 11 (Graph 3) shows Linearity experimentation done for both serovars (S.Typhi and S.Paratyphi A) for all 3 QC sera. Log2 transformed dilution are plotted on the X axis and corresponding Log2 transformed SBA titers are plotted on the Y axis, rZ values for each of the results are displayed on each individual graph. See FIG. 12 for Linearity profile. FIG. 12 (Figure (A, C) denotes the linearity profile for ST SBA, while B and D denotes linearity profile for SPT SBA with respective SBA titer on Y-axis and serial dilutions on X-axis. The initial serial dilutions and total number of dilutions were selected from the median SBA titers for each of the control sera. Both the assays for ST and SPT, across all six runs, displayed excellent linearity with different sera samples tested with r2 values >0.9with slope ranging between -0.65 and -1. 35. FIG. 12 denotes Serogroup-specific SBA was performed with different dilutions of each sera sample and resulting titers were compared by linear correlation analysis. The sera dilution and the titer (converted into log2 form) were plotted with SBA titers on Y-axis and dilutions on X-axis. Conclusion for Linearity: SBA titer obtained for each of the QC sera were checked by linear regression analysis and assay for both serovars was found to be very linear as rZ values of >0.9 were obtained throughout. See TABLE 18 below for Accuracy results The Accuracy and Robustness of ST and SPT SBA complied with variety of sera matrices. The rat, rabbit and human (NIBSC) QC sera was subjected to different dilution ranges. For ST SBA, the dilution of 32 to 512, 4 to 64 and 16 to 256 were employed for rat, rabbit and human (NIBSC) QC sera respectively. For both SBAs, at least 5 dilutions were tested for each serum and dilutions were made after spiking these sera with negative sera for twenty data points. The ST SBA exhibited a median titer of 4096, 2048 and 8192 for rat, rabbit and human QC sera respectively while SPT SBA had a median titer of 1024, 512 and 1024 for rat, rabbit and human QC sera respectively. The spiking was compliant in all (100%) samples tested in both ST and SPT SBAs demonstrating high accuracy for the method. Table 1 summarizes these observations for both ST and SPT serotypes. TABLE 18: ACCURACY RESULTS Sample Serotype No. of run Dilution Titre Min Titre Max Expected Median Titre Agreement NIBSC 16 / 138 ST Run 1 1:16 to 1:256 4096 8192 8192 20 / 20 NIBSC 16 / 138 ST Run 2 1:16 to 1:256 8192 16384 8192 20 / 20 NIBSC 16 / 138 SPT Run 1 1:2 to 1:32 1024 2048 1024 20 / 20 NIBSC 16 / 138 SPT Run 2 1:2 to 1:32 2048 2048 1024 20 / 20 Rabbit D28 ST Run 1 1:4 to 1:64 1024 4096 2048 20 / 20 Rabbit D28 ST Run 2 1:4 to 1:64 1024 2048 2048 20 / 20 Rabbit D28 SPT Run 1 Neat: 1:16 256 512 512 20 / 20 Rabbit D28 SPT Run 2 Neat: 1:16 512 512 512 20 / 20 Rat 25 pg ST Run 1 1:32 to 1:512 2048 4096 4096 20 / 20 Rat 25 pg ST Run 2 1:32 to 1:512 2048 8192 4096 20 / 20 Rat 25 pg SPT Run 1 1:2 to 1:32 1024 1024 1024 20 / 20 Rat 25 pg SPT Run 2 1:2 to 1:32 1024 2048 1024 20 / 20 Over all agreement 100% Legend: ST: S.typhi and SPT: S.Paratyphi A Table 18 explains the results obtained for different dilutions (5 per sera / serovar) for accuracy wherein the minimum and maximum titer obtained is given. Variation (within + 2-fold variation) is taken into consideration before calculating the individual QC sera agreement and also the overall agreement. Accuracy results for S. Typhi: for all three (NIBSC 16 / 138, Rabbit QC, and Rat QC) sera samples were found to be within the expected median titer and agreement of 20 / 20 for RUN1 and RUN2. Conclusion for Accuracy: SBA titer obtained for each of the 5-initial dilution for all 3 QC sera were found to be in agreement (within + 2-fold) with the median titer already established for each of the QC sera. EXAMPLE 12: ROBUSTNESS Robustness is expressed as the tolerability of the assay to different assay conditions like incubation time. We checked the effect of varying the incubation time period on assay titers. Incubation period of 30 mins, 60 mins and 90 mins were analysed. Each assay was performed twice (Runl and Run 2) for this parameter. Parameter: Effect of different incubation times intervals of 30, 60, and 90 minutes. Criteria: Titer in altered incubation should be within +2-fold of the median SBA titer. Acceptance criteria: The Serum bactericidal titer for both serovar should be within agreement for varying incubation time intervals of 30 mins 60 min and 90 mins. The assay is considered robust till it yields consistent results for different time interval. The robustness was evaluated with altered incubations times (30, 60 and 90 minutes) as compared to the traditional SBA protocol. The median titer was assessed across various QC sera. Figure 14 illustrates robustness from two separate runs for ST SBA [rat QC sera; Figure 14A] and SPT SBA [human (NIBSC) QC sera; Figure 41B], At 60 min and 90 min of incubation, titer within agreement to the median titers were obtained, whereas at 30 min lower titer were observed for both ST and SPT serogroups, indicating assay robustness at 60 and 90 min. This also suggested that an incubation time of 30 mins is inadequate for an observable reaction. TABLE 19: ROBUSTNESS RESULTS Serotype Quality control Runs Incubation time 30 mins Incubation time 60 mins Incubation time 90 mins Median titer S. Typhi NIBSC 16 / 138 Run 1 768 6144 12288 8192 S. Typhi NIBSC 16 / 138 Run 2 1024 4096 4096 8192 S. Typhi Rat 25 pg Run 1 2560 4096 4096 4096 S. Typhi Rat 25 pg Run 2 1536 3072 4096 4096 S. Typhi Rabbit D28 Run 1 384 2048 4536 2048 S. Typhi Rabbit D28 Run 2 512 1536 1024 2048 S.Paratyphi NIBSC 16 / 138 Run 1 256 2048 2048 1024 S.Paratyphi NIBSC 16 / 138 Run 2 512 2048 2048 1024 S.Paratyphi Rat 25 pg Run 1 256 1024 1024 1024 S.Paratyphi Rat 25 pg Run 2 256 1024 1024 1024 S.Paratyphi Rabbit D28 Run 1 128 512 512 512 S.Paratyphi Rabbit D28 Run 2 128 512 512 512 Table 19 shows the SBA titer obtained for each of the different incubation time interval (i.e 30, 60 and 90 mins) Median titer from precision experiment are mentioned on the right side, for comparison purpose. See FIG. 13 for Robustness results (Graph 4) FIG. 13 (Graph 4) shows SBA titers obtained, plotted on the Y axis and different incubation time interval(30,60 and 90 min) plotted on the X axis. Conclusion for Robustness: SBA titer obtained at different incubation time interval (60 and 90mins) were found to be in agreement (within + 2-fold) with the median titer already established for each of the sera. SBA titers at Incubation time interval of 30 mins were consistently <4 fold as compared to median titers established and thus it shows that 30 mins is insufficient time for the complete reaction (i.e- complement activation and killing of bacterial cells) to happen and hence assay results are robust only for Incubation time interval of 60 and 90 mins. See FIG. 14 for Robustness using Rat QC FIG. 14 shows S. Typhi (A) Robustness using Rat QC and for S. Paratyphi A (B) NIBSC 16 / 138 as a standard was analyzed respectively. Two Runs (Run 1 (a) and Run 2 (b)) at 30, 60, and 90 min time intervals. EXAMPLE 13: SPECIFICITY Investigation of assay specificity was done by neutralizing a quality control serum with homologous PS (50pg), a pool of heterologous PS (50pg), or with no PS (Buffer) and an inert PS (non related PS e.g. Meningococcal PS-50pg) before being tested in SBA. Heterologous PS pools contained PS from other serovar (i.e S.Typhi as heterologous for S.Paratyphi A and vice versa).Note: In case of S.Paratyphi A polysaccharide, Conjugate OSP-DT (O-antigen specific polysaccharide-Diphtheria toxoid)was used. Parameters: Neutralizing sera for 1 hour at 370C with 50 pg / ml of respective specific / homologues PS [for SP / SPT] and heterologous PS, non-specific PS (MnPS C) and No PS (only buffer) before testing in SBA. Criteria: Specific / homologues PS should inhibit SBA titer for the respective sera by >4-fold and heterologous, non-specific and no PS should result in no inhibition (titers within + 2-fold). Acceptance criteria: Homologous inhibitions are indicated by lack of SBA titers or SBA titers of <4-fold of median titer (i.e reduction of >4-fold in titers) when sera samples were incubated with homologous polysaccharide, whereas the inhibition should not be observed in all the other combinations when sera samples were incubated with no PS, heterologous polysaccharides and Inert PS. See FIG. 15 for Specificity results(Graph 5) The ST and SPT SBA were found to be specific for the respective serotypes; an inhibition with respective PS were checked to observe assay specificity. For both ST and SPT SBA, the homologous inhibition was clearly evident via reduced colonies and low SBA titer (lack of killing or titers >4-fold lower as compared to median titers). This was observed only when sera samples were incubated with homologous PS for respective serotypes (Figure 15A and 15B; leftmost bar). On the contrary, the inhibition was not observed (titers within + 2-fold) in incubations across all three treatment combinations wherein sera samples were incubated with ‘No’ PS, mixed heterologous PS and non-specific PS (Mn PS). This clearly indicated assay specificity as the susceptibility of the target bacterial strains was affected only in the presence of homologous PS. FIG. 15 Specificity of S. Typhi and S. Paratyphi A: Results show expected homologous inhibitions (a) indicated by lack of killing (reduced colonies) for homologous polysaccharide, whereas, the inhibition is not observed in all other combinations when sera samples were incubated with ‘No’ PS (c) or mixed heterologous polysaccharides (b) and non-related Mn PS (d) polysaccharides for both Serotypes. FIG. 15 (Graph 5) shows SB A titers obtained for each of the individual sera samples upon different treatments (Homologous PS incubation, Heterologous PS incubation, No PS and Inert PS (MnPS) incubation) prior to be used in the assay. Conclusion for Specificity: SBA titer obtained for Homologous PS treatment show reduction in titer (> 4-fold) as compared to median titer for each of the 3 QC sera whereas no inhibition of SBA titer is seen for any of the other 3 treatment groups (Heterologous PS, No PS and Inert PS), thus the specificity of the assay is proved. EXAMPLE 14: S. TYPHI AND S. PARATYPHI A SBA ASSAY STABILITY REPEATABILITY AND CONSISTENCY Quality control standards: 1) Human quality control NIB SC 16 / 138 2) Inhouse Rabbit (D28) Quality control sera TABLE 20: Median titer for S. Typhi and S. Paratyphi A from precision parameter of validation Serotype Standard Median titer Range S. Typhi NIBSC 16 / 138 8192 4096 to 16384 RABBIT D28 2048 1024 to 4096 S. Paratyphi NIBSC 16 / 138 1024 512 to 2048 RABBIT D28 512 256 to 1024 • Two quality control sera viz., Human Quality control standard (WHO standard NIB SC 16 / 138) and Rabbit D28 (Prepared In-house at SIIPL) were used in various studies since last year and showed consistent SBA titer within the range specified above. These results suggest that the assay is highly stable over period of 1 to 2 years thus proving repeatability and consistency. • Please find below the comparative NIBSC quality control sera titre for S. Typhi and S. Paratyphi A: TABLE 21: NIBSC 16 / 138 QUALITY CONTROL SERA TITRE S. Typhi S. Paratyphi A STUDY DATE REPETATI ON TITE R STUDY DATE REPETATI ON TITE R S001 02.01.2 024 1 4096 S001 DO 03.01.2 024 1 1024 2 4096 2 1024 3 8192 3 1024 4 8192 4 1024 5 8192 5 1024 6 4096 6 1024 7 8192 7 1024 8 8192 8 1024 S001D28 04.01.2 024 9 8192 S001D28 05.01.2 025 9 1024 10 8192 10 1024 11 8192 11 1024 12 8192 12 1024 13 1638 4 13 1024 14 8192 14 512 15 8192 15 512 16 8192 16 512 S001D42 06.01.2 024 17 4096 S001D28 REP 09.01.2 024 17 1024 18 8192 18 2048 19 8192 19 2048 20 8192 20 2048 21 8192 S001D42 13.01.2 024 21 1024 22 8192 22 1024 23 8192 23 1024 24 8192 24 2048 S002 D28 03.04.2 024 25 8192 25 2048 26 8192 26 512 27 1638 4 S002 D28 22.03.2 024 27 2048 28 8192 28 2048 04.04.2 024 29 8192 29 2048 30 8192 30 2048 31 8192 S003 DO 10.07.2 024 31 1024 32 8192 32 1024 S003 DO 25.07.2 024 33 1638 4 33 2048 34 1638 4 34 2048 35 1638 4 35 1024 36 1638 4 27.12.2 024 36 2048 31.07.2 024 37 8192 37 2048 38 8192 S004 DO 13.11.2 024 38 2048 39 1638 4 39 1024 40 1638 4 40 512 S003 D28 01.08.2 024 41 1638 4 41 1024 42 1638 4 S004 D28 05.12.2 024 42 2048 43 8192 43 1024 44 1638 4 44 2048 45 8192 45 2048 S003 D28 REP 06.08.2 024 46 1638 4 46 2048 47 1638 4 47 512 48 1638 4 48 1024 49 1638 4 49 1024 S003 D42 14.08.2 024 50 1638 4 S004 D42 13.12.2 024 50 1024 51 8192 51 2048 52 1638 4 52 2048 53 1638 4 53 2048 54 1638 4 54 1024 55 1638 4 55 2048 S004 DO 13.11.2 024 56 8192 56 1024 57 8192 57 2048 58 1638 4 59 4096 S004 D28 05.12.2 024 60 8192 61 8192 62 8192 63 1638 4 S004 D42 18.12.2 024 64 1638 4 65 1638 4 66 8192 67 1638 4 68 1638 4 69 1638 4 70 1638 4 71 1638 4 See FIG. 16 for Repeatability date for SB A assay for S. Typhi using NIB SC quality control (NIBSC 16 / 138 for S. Typhi) See FIG. 17 for Repeatability date for SB A assay for S. Paratyphi using NIBSC quality control (NIBSC 16 / 138 for S. Paratyphi A) TABLE 22. IN-HOUSE RABBIT (D28) CONTROL SERA S. Typhi S. Paratyphi A STUDY DATE REPETATI ON TITE R STUDY DATE REPETATI ON TITE R S001 02.01.2 024 1 2048 S001 DO 03.01.2 024 1 512 2 2048 2 512 3 4096 3 512 4 4096 4 512 5 4096 5 512 6 2048 6 512 7 1024 7 512 8 2048 8 512 S001D28 04.01.2 024 9 4096 S001D28 05.01.2 025 9 512 10 2048 10 512 11 2048 11 512 12 2048 12 512 13 2048 13 512 14 1024 14 256 15 2048 15 512 16 4096 16 512 S001D42 17 2048 17 512 06.01.2 024 18 4096 S001D28 REP 09.01.2 024 18 512 19 2048 19 512 20 2048 20 512 21 2048 S001D42 13.01.2 024 21 512 22 2048 22 512 23 2048 23 512 24 2048 24 1024 S002 D28 03.04.2 024 25 2048 25 512 26 2048 26 512 27 4096 S002 D28 22.03.2 024 27 512 28 2048 28 512 04.04.2 024 29 2048 29 512 30 1024 30 512 31 2048 S003 DO 10.07.2 024 31 512 32 2048 32 1024 S003 DO 25.07.2 024 33 2048 33 1024 34 4096 34 1024 35 2048 35 1024 36 2048 27.12.2 024 36 1024 31.07.2 024 37 2048 37 1024 38 2048 S004 DO 13.11.2 024 38 1024 39 1024 39 1024 40 2048 40 512 SOO3 D28 01.08.2 024 41 4096 41 256 42 4096 S004 D28 05.12.2 024 42 256 43 2048 43 512 44 2048 44 512 45 2048 45 512 SOO3 D28 REP 06.08.2 024 46 2048 46 512 47 2048 47 256 48 4096 48 256 49 2048 49 256 SOO3 D42 14.08.2 024 50 1024 S004 D42 13.12.2 024 50 256 51 1024 51 256 52 2048 52 256 53 2048 53 512 54 2048 54 256 55 2048 55 256 S004 DO 13.11.2 024 56 4096 56 256 57 4096 57 256 58 1024 59 2048 S004 D28 05.12.2 024 60 4096 61 2048 62 2048 63 2048 S004 D42 18.12.2 024 64 2048 65 2048 66 2048 67 2048 68 2048 69 1024 70 2048 71 4096 See FIG. 18 for Repeatability date for SBA assay for S. Typhi using Rabbit quality control (In house Rabbit D28 for S. Typhi) See FIG. 19 for Repeatability date for SBA assay for S. Paratyphi using Rabbit quality control (In house Rabbit D28 for S. Paratyphi A) Repeatability and consistency of both S. Typhi and S. Paratyphi A SBA’s were checked over a period of 1-2 years using different quality standard sera samples (i.e. NIB SC 16 / 13 8-human standard and Rabbit D28- animal standard sera). Median titers for both the standards have been calculated and mentioned in Table 20. From Fig 18 and Fig 19, it can be seen that all individual repetitions performed over period of 1-2 years show tires within the range mentioned in Table 20, thus proving repeatability and consistency for the assay. SBA titers of immune sera were highly correlated with anti- Vi IgG hence SBA titers can be used as a good biomarker for clinical evaluation of typhoid vaccines. The preparation and enumeration or titration of bacteria was performed. The various methods for S. Typhi and S. Paratyphi A adapted earlier wherein bacteria were frozen at a particular OD were evaluated. We found that S. Typhi is susceptible to killing only during the Log phase (0.4 OD) and not in the stationary phase. S. Paratyphi A, in the contrary, did not have any effect of different phases. Hence stationary phase (OD 1.0) bacteria were used for S. Paratyphi A. This supports the finding by Boyd et.al. (2014) where the capsule formation (in case of S. Typhi) evaded bactericidal activity during the assay. S.Paratyphi A lacks capsular structure and hence the antibodies directly targeted the LPS on the cell wall across different growth stages thereby did not have effect on the overall bactericidal activity. The establishment of an appropriate buffer system is a critical parameter in the SBA. The SBA for S. Paratyphi A showed a good gradual killing curve for the positive control and no killing for the negative control with normal HBSS+ buffer whereas S. Typhi exhibited the required clearing with HBSS- and 1% FBS buffer combination. The suitable make and required complement concentration, triggering a killing percentage of <15%, was investigated for both the SB As (Fig.l). As seen in Example 5, a 5 % complement concentration for the S. Typhi assay and 12.5 % for S. Paratyphi A showed desired results. The experiments also involved use of various quality control sera (Tablel6) from variety of origin species, including both positive and negative sera along with different assay controls (Example 11 onwards). All controls mentioned gave acceptable results. The choice of agar, for spotting purpose, involved comparison of Blood agar (5% horse blood) and Luria Bertani agar. The LB agar plates exhibited smaller and well-defined smaller colonies than blood agar plates. These colonies were easier to count by either manual or automated counter (ProtoCOL3, Synbiosis, UK). Additionally, the plates were subjected to variable shaking conditions and “no shaking” approach provided superior growth conditions than “shaking” approach. Validation parameters studied for the SB A’s involved precision, performed with quality control serums (human, rat, and rabbit) demonstrated. A precision of >90% for various control sera (FIG 9). Specificity was observed for both SBA’s was at least >4-fold inhibition seen for homologous and <2-fold inhibition for heterologous reaction (FIG. 15). Linearity assessment indicated good assay linearity for both SP and SPT serogroups with r2 values of >0.9 and slope ranging between -1 to -1.3 (FIG. 12). An accuracy of 100 % was achieved for both serogroups (Tablel8). Similarly, robustness was observed wherein different incubation times (60 and 90 minutes) did not have a significant change on the titer of the sera tested, whereas reduced titers (<+ 1 SBA titers) were observed for S. Paratyphi A. This result implied that an incubation period of >30 minutes is required for obtaining reproducible titers for both ST and SPT assays. Serum Bactericidal Activity (SBA) assay disclosed hereinabove are performed for both S. Typhi and S. Paratyphi A. The assays (for both S.Typhi and S.Paratyphi) are precise, linear, accurate, specific, and robust. The assay facilitates accurate and reproducible results fortesting. Development of OPA for understanding cell mediated response and comparability with SBA: OP A was developed for S. Paratyphi A for testing cell mediated humoral immune response using differentiated HL-60 cells as effector cells. Various parameters were checked i.e. Effect of shaking speed, complement concentration, different sets of controls i.e. Complement control, bacteria control and sera control were checked. Results for, with and without shaking showed antibody Titres within two folds hence assay was performed without shaking during opsonization step. To check the performance of assay various Rat, Rabbit and QC Standards samples were analysed. Results obtained were compared to SBA Titres of same sample and a 6-8-fold increase in Titres was observed for OPA as compared to SBA. EXAMPLE 15: HIGH THROUGHPUT APPROACH USING COLONY COUNTER: Luria Bertani agar plates were used to allow growth of both S. Typhi and S. Paratyphi A, as proper round, opaque and legible colonies, which were easy to count manually, in order to facilitate high throughput analysis, Colony counter (ProtoCOL 3) was tested to get comparable Titres estimation as manual counting. This allowed ease of performing multiple plates by single analyst as arduous and tedious task of manual colony counting is avoided. Manual counting of bacterial colony forming units (CFUs) on agar plates is laborious, time consuming and error-prone. The SBA method as disclosed was also run on an automated colony counter which reliably detected, provided bacterial counts and colony size on agar plates. Colony counter system provides results with an accuracy that discriminates bacterial colonies accurately on agar plates helped to overcome the disadvantages of manual counting for high throughput results. Various changes were made in the settings of colony counter instrument like the choice of illumination, exposure time, sensitivity, colony appearance, size of colony inclusion and exclusion. Normal setting is generalized for SBA test in the colony counter, few minor adjustments are made like exposure time= 0.25 sec, sensitivity = auto along with background and colony colour classification were set as per our protocol (pale yellow= background and white opaque= colonies) For comparison and shifting to automated colony counting as opposed to manual counting, compared CFU’s and SBA titer estimated from both the methods. Around 30 sera samples (Rat QC sera, Rabbit QC sera and NIB SC QC sera) were included in the study to check effect on antibody titer. A good correlation was observed between two methods. The correlation coefficient observed for S.Typhi is 0.9 &for S.Paratyphi A is 0.8 calculated using statistical analysis. See FIG. 20 and 21 for comparison of the manual and automated colony counting for S. Typhi and S. Paratyphi respectively. Fig 20 for S.Typhi and Fig 21 for S.Paratyphi A, shows SBA titer comparison between colony counting methodologies (i.e. manual vs automated colony counting), It is observed that using either of the method gives consistent and within acceptable range(+ / - 2 fold titers) for both the methodologies, thus proving use of colony counter would help in high throughput assessment for the SBA’s. EXAMPLE 16: TITER DETERMINATION IN PRECLINIC AL SAMPLES FROM GLP-TOXICITY STUDY The OD values observed in the preclinical sera samples, from rats / rabbits treated with the vehicle (formulation buffer alone), were used to establish a cut-off for determination of antibody titer. This method is used for calculation of titer for both anti-Vi IgG and anti-OSP IgG levels in the sera samples from rats and rabbits treated with the test item. This approach is used as there does not exist any international reference sera standard of rat sera for anti-Vi and or anti-OSP IgG antibodies. The result of ELISA data is presented in two different methods, (a) Geometric mean of ELISA OD value from individual animals, (b) End point antibody titer in form of Geometric Mean Titer. SBA Titer determination was performed for all different groups of sera samples tested. Titers were represented as GMT (Geometric Mean Titers) and Fold Rise (Fold difference between Pre-Immune and Different Day of bleeding) was calculated for each of the testing groups. The OD values observed in the preclinical sera samples, from rats / rabbits treated with the vehicle (formulation buffer alone), were used to establish a cut-off for determination of antibody titer. This method is used for calculation of titer for both anti-Vi IgG and anti-OSP IgG levels in the sera samples from rats and rabbits treated with the test item. This approach is used as there does not exist any international reference sera standard of rat sera for anti-Vi and or anti-OSP IgG antibodies. The result of ELISA data is presented in two different methods, (a) Geometric mean of ELISA OD value from individual animals, (b) End point antibody titer in form of Geometric Mean Titer. SBA Titer determination was performed for all different groups of sera samples tested. Titers were represented as GMT (Geometric Mean Titers) and Fold Rise (Fold difference between Pre-Immune and Different Day of bleeding) was calculated for each of the testing groups. Table 23: ELISA GMT of anti-Vi-TT IgG and anti-OSP-DT IgG at IX dose (25.0pg) in rabbit sera samples Formulation Anti-Vi TT IgG Anti-OSP DT IgG D14 D28 D42 D14 D28 D42 GMT Opg (Placebo) 200 200 200 143 202 160 GMT IX Dose (25.Opg) 161270 203187 228070 3564 12699 25398 Fold Rise (for IX Dose) 806 1016 1140 25 63 159 The table indicates GMT values calculated for each treatment group; Fold rise indicates the rise in titer post -treatment at different days of bleeding. Table 24: SBA GMT of anti-Vi-TT and anti-OSP-DT at IX dose (25.0pg) in rabbit sera samples. Formulation Anti-Vi TT SBA Anti-OSP DT SBA D14 D28 D42 D14 D28 D42 GMT Opg (Placebo) 161 203 181 14 11 18 GMT IX Dose (25.0pg) 1549 2299 813 57 522 362 Fold Rise (for IX Dose) 10 11 4 4 46 20 The table indicates GMT values calculated for each treatment group; fold rise indicates the rise in titer post -treatment at different days of bleeding. The SBA titer for both anti-Vi TT and anti-OSP DT across all analytical time points of Day 14, 28 and 42 (post-intramuscular administration) was >4 suggesting a strong “bivalent” response by the vaccine. The GMT for IgG and SBA are illustrated in graphical manner in Figure 9 and 10 respectively. The data from half of 0.5X SHD and 2X SHD showed a similar immunogenicity potential across both ELISA and SBA platforms (data not shown). Inference: Table 23 and Table 24 show ELISA and SBA GMT data respectively obtained during toxicological evaluation of SIIPL’s Bivalent Typhoid Conjugate Vaccine performed in Rabbits at IX (Human Dose). Values obtained for placebo and for the IX dose are compared in terms of fold rise (increase in titers from placebo to IX dose). A fold rise of 4 and above is considered as positive immunogenic reaction demonstrating observable antibodies raised against the vaccine in the animal model. Results clearly demonstrate good immunogenic response for SIIPL’s BTCV titers of >4-fold at D14, D28 and D36 and the SBA for S. Typhi and S. Paratyphi A are able to successfully evaluate response for the vaccine. Development of OPA for understanding cell mediated response and comparability with SBA: OP A was developed for S.Paratyphi A for testing cell mediated humoral immune response using differentiated HL-60 cells as effector cells. Various parameters were checked i.e. Effect of shaking speed, complement concentration, different sets of controls i.e. Complement control, bacteria control and sera control were checked. Results for, with and without shaking showed antibody titers within two folds hence assay was performed without shaking during opsonization step. To check the performance of assay various Rat, Rabbit and QC Standards samples were analysed. Results obtained were compared to SBA titers of same sample and a 6-8-fold increase in titers was observed for OPA as compared to SBA. KEY FEATURES: • SBA for estimation of antibodies against S. Typhi and S. Paratyphi A for assessment of cell mediated killing achievable for the immune response. • The method involves preparing the media and the buffer; preparing a bacterial stock and diluting the bacterial stock; adding the buffer, a test serum, a complement and the bacterial stock in the media on the plate; incubating the plate and calculating antibody titre. • Assay includes preparing specific Media i.e. Luria Bertani Broth and a specific buffer, i.e. Hanks balanced salt solution. • Buffer used is without phenol red. • Buffer includes HBSS supplemented with Ca &Mg or Fetal Bovine Serum. • Buffer facilitated proper killing / neutralization of the bacteria. • Assay includes a complement concentration of 2.0% to 20.0%. • Assay includes specific step of bacterial stock preparation at an initial Log phase (with absorbance of 0.3 to 0.4 at optical density of 600 nm for S. Typhi) or at late Log phase (with absorbance of 0.9 to 1.0 at optical density of 600 nm for S. Paratyphi A). Bacterial harvesting is performed based on the growth phase and optical density (O.D.) ADVANTAGES: • Assay is applicable for both S. Typhi and S. Paratyphi. • Assay includes bacterial stock in initial growth phase (for S. Typhi) and late Log phase (for S. Paratyphi). Accordingly, the assay is associated with accelerated and accurate analysis as time is saved in bacterial stock preparation. • Assay needs less complement concentration compared to any other SBA assays. • Bacterial stocks are titrated for required dilution to yield appropriate colonies in control wells. • Assay is associated with the Titre estimation that is consistent and precise. • Assay is able to provide Titre estimation in robust and repeatable manner. Also, the assay is sensitive enough to provide Major or distinguishable difference between pre and post sera samples. • Assay is simple and repeatable for Toxicity studies / immunogenicity analysis. • Use of frozen glycerol stock directly on the day of the assay with specific growth phase of bacteria as determined by bacterial titration, with exact dilution reduces total analysis time. • Bacterial stocks are titrated for a dilution required to yield appropriate number of colonies. • A colony counting system performing multiple plates in less time. APPLICATIONS: • Assay is able to provide functional antibody Titre estimation. • Assay being able to provide repeatable results for pre-clinical testing, clinical testing, Controlled Human Infection Model testing. • Assay being simple and easy to perform is replicable with different animal models including mice, rat, rabbits. • Assay is cost effective, simple, consistent and repeatable for vaccine candidates including conjugate vaccine candidates for e.g. Typhoid Conjugate Vaccines. • Assay is universal in nature and provides consistent results for immunogenicity analysis of any candidate typhoidal vaccine against S. Typhi and S. Paratyphi A. • The assay fulfills the validation criteria of robustness, specificity, accuracy, linearity, and precision (repeatability and intermediate precision)

Claims

1. A method of performing Serum Bactericidal Activity (SB A) assay, the method comprising: a. preparing a media and a buffer;b. preparing a bacterial stock with a mother culture stock, the media and the buffer;c. diluting the bacterial stock with the buffer to obtain a diluted bacterial stock;d. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution;e. incubating the assay solution, spotting the assay solution on an agar plate and incubating the agar plate; andf. calculating antibody titre to determine the serum bactericidal activity.

2. The method of SB A assay as claimed in claim 1, wherein the media includes lysogeny broth, Luria Bertani broth, Hi Soy (Hi Bacteriological media), tryptone, peptone, yeast extract, or combinations thereof.

3. The method of SB A assay as claimed in any one of claims 1 or 2, wherein the buffer includes saline, phosphate, phosphate buffered saline, bovine serum albumin, Hanks' balanced salt solution (HBSS), fetal bovine serum (FBS), or combinations thereof.

4. The method of SBA assay as claimed in any one of claims 1 to 3, wherein the buffer includes HBSS, optionally comprising calcium and magnesium.

5. The method of SBA assay as claimed in any one of claims 1 to 3, wherein the buffer includes HBSS and FBS.

6. The method of SBA assay as claimed in any one of claims 1 to 5, wherein the agar plate is Luria Bertani agar plate.

7. The method of SBA assay as claimed in any one of claims 1 to 6, wherein the bacterial stock is prepared with bacteria in initial log phase, in exponential phase, in late Log phase, or in a stationary growth phase;optionally, wherein the bacterial stock preparation includes incubating bacteria from the mother culture stock at 35°C to 39°C for 12 to 16 hours on a blood agar plate andoptionally, wherein the bacterial stock preparation includes further incubating bacteria from the blood agar plate on the Luria Bertani broth for 2 to 4 hours at 37°C in shaker incubator.

8. The method of SB A assay as claimed in any one of claims 1 to 7, wherein the bacterial stock is prepared with bacteria in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm.

9. The method of SBA assay as claimed in any one of claims 1 to 7, wherein the bacterial stock is prepared with bacteria in late Log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm.

10. The method of SBA assay as claimed in any one of claims 1 to 9, wherein the bacterial stock is titrated and diluted using the buffer.

11. The method of SBA assay as claimed in any one of claims 1 to 10, wherein the bacterial stock is diluted 1:100 to 1:6000 with the buffer to provide the diluted bacterial stock.

12. The method of SBA assay as claimed in any one of claims 1 to 11, wherein the test serum comprises antibodies against an immunogenic composition for bacteria., Salmonella, typhoidal Salmonella, non Typhoidal Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella Typhi, and Salmonella Paratyphi.

13. The method of SBA assay as claimed in claim 12, wherein the bacteria is Salmonella including typhoidal Salmonella, non-typhoidal Salmonella, Salmonella enteritidis, Salmonella typhimurium, Salmonella Typhi, and Salmonella Paratyphi.

14. The method of SBA assay as claimed in any one of claims 1 to 13, wherein the complement is in concentration of 2.0 % to 20.0%, optionally wherein the complement includes Human complement and Rabbit complement.

15. The method of SBA assay as claimed in any one of claims 1 to 14, wherein the media is the Luria Bertani broth, and the buffer includes the HBSS;optionally, wherein the HBSS is without phenol red; andoptionally wherein the test serum comprises antibodies against the immunogenic composition for typhoidal Salmonella.

16. The method of SB A assay as claimed in any one of claims 1 to 15, wherein the media includes the Luria Bertani broth, the buffer includes HBSS supplemented with / without Calcium and Magnesium or with FBS, the HBSS is without phenol red, the complement is in concentration of 2.0% to 20.0%;optionally wherein the test serum comprises antibodies against the immunogenic composition for typhoidal Salmonella;optionally wherein the bacterial stock is in initial log phase or in late log phase.

17. The method of SB A assay as claimed in any one of claims 1 to 16, the method comprising a. preparing the Luria Bertani broth and the HBSS buffer supplemented with Calcium and Magnesium, (the HBSS (+) buffer), wherein the HBSS (+) buffer is without phenol red;b. preparing the Salmonella Paratyphi bacterial stock with the Salmonella Paratyphi mother culture stock, the Luria Bertani broth and the HBSS (+) buffer;c. diluting the Salmonella Paratyphi bacterial stock with the HBSS (+) buffer to obtain a diluted Salmonella Paratyphi bacterial stock;d. adding the test serum, the complement in concentration of 2.0% to 20.0%, the diluted Salmonella Paratyphi bacterial stock and the HBSS (+) buffer, i.e. HBSS with Calcium and Magnesium to form the assay solution;e. incubating the assay solution at 35°C to 39°C for 50 to 90 mins;spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; andf. calculating antibody titre;wherein the Salmonella Paratyphi bacterial stock is in late log phase with absorbance value in range of 0.60 to 1.20 for Optical Density at 600 nm.

18. The method of SBA assay as claimed in any one of claims 1 to 16, the method comprising: a. preparing the Luria Bertani broth and the HBSS buffer supplemented with FBS, (the HBSS (-) buffer), wherein the HBSS (-) buffer is without calcium, magnesium and phenol red;b. preparing the Salmonella Typhi bacterial stock with the Salmonella Typhi mother culture stock, the Luria Bertani broth and the HBSS (-) buffer;c. diluting the Salmonella Typhi bacterial stock with the HBSS (-) buffer to obtain a diluted Salmonella Typhi bacterial stock;d. adding the test serum comprising antibodies formed on administration of the immunogenic composition against Salmonella Typhi, the complement in concentration of 2.0% to 20.0%, the diluted Salmonella Typhi bacterial stock and the HBSS (-) buffer to form the assay solution;e. incubating the assay solution at 35°C to 39°C for 50 to 90 mins;spotting the assay solution on a Luria Bertani agar plate and incubating the Luria Bertani agar plate for 10 to 14 hours; andf. calculating antibody titre;wherein the Salmonella Typhi bacterial stock is in initial log phase with absorbance value in range of 0.10 to 0.50 for Optical Density at 600 nm.

19. The method of SB A assay as claimed in any one of claims 1 to 18, wherein the step of calculating antibody titre comprises calculating number of bacterial colonies and determining the antibody titre based on the highest serum dilution where the number of bacterial colonies is less than or equal to 50% of heat-inactivated complement control.

20. The method of SB A assay as claimed in claim 1 to 19, wherein the method determines antibody titre as a measure of effectiveness of an immunogenic composition or a vaccine.

21. The method of SB A assay as claimed in claim 1 to 20, wherein the method determines functional IgG response to an immunogenic composition or a vaccine comprising Vi-TT and OSP-DT polysaccharide protein conjugate, and wherein the test serum comprises antibodies to the Vi-TT or OSP-DT polysaccharide protein conjugate.

22. A kit for performing the Serum Bactericidal Activity (SBA) assay as claimed in any one of claims 1-21, wherein the kit comprises:- the media, the buffer, the bacterial stock, and the complement as recited in any one of claims 1 to 21; and- optionally, a package insert or label providing instructions to perform the SBA assay.

23. A system for performing the Serum Bactericidal Activity (SBA) assay as claimed in any one of claims 1-21, wherein the system includes:- the media, the buffer, the bacterial stock, and the complement as recited in any one of claims 1 to 21; and- optionally, a package insert or label providing instructions to perform the SBA assay.

24. A method for performing a Serum Bactericidal Assay (SBA), comprising:i. preparing a bacterial stock with a mother culture stock and a media;ii. diluting the bacterial stock 1:100 to 1:6000 with a buffer to obtain a dilutedbacterial stock;iii. adding a test serum, a complement, the diluted bacterial stock, and the buffer to form an assay solution;iv. incubating the assay solution, spotting the assay solution on an agar plate, and incubating the agar plate; andv. calculating antibody titre to determine the serum bactericidal activity.

25. The method as claimed in claim 24, wherein the diluted bacterial stock yields 50 to 250 colony forming units (cfus).

Images