A synthetic Listeriolysin-O peptide-based indirect ELISA for detection of L. monocytogenes infection in ruminants

Abstract

The present invention discloses A synthetic peptide Listeriolysin O (LLO-3 and LLO-4) as an antigen, comprising Seq. ID 1 and Seq. ID 2 derived from Listeriolysin O protein of L. monocytogenes strain at 18-29 amino acid position for LLO-3 and at 25-36 amino acid position for LLO-4 for sero-screening of listeriosis in ruminants. The synthetic peptides identified from the Listeriolysin-O protein of L. monocytogenes strains have been standardized and compared with results of existing Listeriolysin O (LLO-2) in i-ELISA test format with screening of the field ‘sera’ samples using Listeriolysin O (LLO-3 and LLO-4) peptides as antigens for serodiagnosis of Listeriosis in bovines and caprine

Description

FIELD OF THE INVENTIONThe present invention relates to the development of a category of serodiagnosisfor Listeriosis. More particularly, the present invention relates to the ListeriolysinO (LLO) peptide-based indirect enzyme linked immunosorbent assay techniquefor the detection of Listeria monocytogenes infection.BACKGROUND OF THE INVENTIONListeriosis is known as a circling disease and / or silage disease which is highlyfatal in ruminants such as sheep, goats, cattle, buffalo, camel, including nonruminants like horses, pigs, canines, rodents, wild animals, birds, and humans.According to the Centre for Disease Control and Prevention (CDC), the diseaseListeriosis was included in the list of nationally notifiable diseases in the year,2001. It is the second most common cause of fatal food-borne illness afterSalmonella infections. The fatality rate associated with Listeriosis is as high as30%, especially in high-risk population groups such as immune-compromisedindividuals, the elderly, and the newborns with an overall hospitalization rate of >95%. The annual global burden of Listeriosis in the year 2010 was estimated to bemore than 23,000 illnesses, almost 5500 deaths, and 172,823 disability-adjustedlife-years (DALYs). Globally, the majority of Listeriosis cases have been reportedeither in sporadic or epidemic form, and most often the infection in animals issubclinical but in many cases a severe form can also occur. Listeriosis ischaracterized by neural, visceral, and reproductive clinical entities usuallymanifested with septicaemia, encephalitis, meningitis, meningoencephalitis,rhomb encephalitis, abortion, stillbirth, perinatal infections, and gastroenteritis inhumans and animals. Moreover, "Meningitis" due to Listeria has been ranked atthird position among the bacterial causes of meningitis in humans.In the true sense, the epidemiological data available on Listeriosis in India to dateis not adequate for assessing the extent of infection in human beings and animals.The disease largely remains undiagnosed and underreported due to the lack ofmandatory notification and awareness on Listeriosis and also due to the lack ofreliable, rapid, and simple diagnostic tests.Conventionally, the most authentic diagnosis of Listeriosis is made by culturebased method which is also regarded as the "Gold standard test". However, itrequires 2 to 3 days to provide presumptive positive results and an additional 2 to4 days to confirm the suspected colonies by biochemical tests. Besides beingtime-consuming and laborious, conventional testing fails to discriminate betweenpathogenic and non-pathogenic strains. The pathogenicity testing of Listeriaisolates is performed either by in vitro pathogenicity tests namely, hemolysis onsheep blood agar (SBA); phosphatidylinositol-specific phospholipase C (PI-PLC)activity on Agar Listeria according to Ottaviani and Agosti (ALOA) medium orPhosphoinositide-specific phospholipase (PI-PLC) substrate medium and by the invivo methods like chick embryo and mouse inoculation tests. Hence, it becomesmandatory to correlate these isolates with the cases of Listeriosis.In the last two decades, nucleic acid amplification-based methods such aspolymerase chain reaction (PCR) have been employed to detect L. monocytogenesisolates. Many gene amplification procedures such as polymerase chain reaction(PCR), multiplex PCR (m PCR), and Real-Time PCR have been developed for theidentification, differentiation, and quantification of Listeria species, however,such processes also have limitations like to perform these tests, sophisticatedlaboratories including costly instruments are required. Besides this, they are noteconomical, and highly trained personnel are required to perform and analyze theresults.Many serodiagnostic assays employing the somatic (O), flagellar (H), coldextracted or sonicated antigens, and outer membrane protein (OMP) of Listeriaspp. have been evaluated for screening animal and human Listeriosis cases.However, these conventional assays cannot be relied upon because of their poorspecificity and sensitivity. In the recent past, indirect ELISA aimed at identifyinganti-Listeriolysin O antibody (ALLO) which has been extensively used for theSerodiagnosis of Listeria infections in humans, goats, sheep, buffaloes, and cattle.However, the cross-reactivity of antibodies against anti-Listeriolysin O antibody(ALLO) with those produced against the streptolysin O (SLO) which is ahaemolysin produced by Streptococcus spp., remains a major limitation of thisassay which calls for the adsorption of test sera with streptolysin O (SLO) beforeits testing.Currently, synthetic peptide-based diagnostic assays have been devised as novelapproaches for the rapid and reliable diagnosis of infectious diseases. The,synthetic peptides are quite stable, economical to scale up, have high specificityand antigenicity, greater reproducibility, and have no variation in results betweenthe batches. Therefore, in recent years specific synthetic peptide(s) have been usedas specific antigens in developing rapid immunodiagnostic tests for reliablescreening of important infectious agents including zoonotic pathogens, namely,Mycobacterium tuberculosis, Listeria monocytogenes, and Coxiella burnetii.The present invention is based on immunoassays that aim at identifying antigenswith the advantage of screening a large number of samples, are highlyeconomical, easy to perform, and can be interpreted easily. Ideally, such assaysmust have sufficient diagnostic sensitivity and specificity to detect the causativeagent or their antibodies. In the present invention, two specific synthetic peptidesof Listeriolysin O (LLO-3, LLO-4) have been identified which can be used as anantigen in indirect enzyme-linked immunosorbent assay (ELISA) for seroscreening of Listeriosis in bovines and caprine.OBJECTIVE OF THE INVENTIONThe principle object of the present invention is to identify specific antigens i.e.,synthetic peptides of Listeriolysin O (LLO) for the development of aserodiagnostic assay for the detection of Listeriosis infection in bovines andcaprine.Another object of the present invention is to develop an indirect enzyme-linkedimmunosorbent assay (ELISA) method employing identified synthetic peptides ofListeriolysin O (LLO-3 and LLO-4).Another object of the present invention is to evaluate the performance of thedeveloped indirect enzyme-linked immunosorbent assay (ELISA) method fordiagnostic sensitivity by comparing the results with the existing syntheticListeriolysin O (LLO-2) peptide-based indirect enzyme-linked immunosorbentassay (ELISA) results.Yet another object of the present invention is to evaluate the performance of thedeveloped enzyme-linked immunosorbent assay (ELISA) method for diagnosticsensitivity by comparing the results with the gold standard cultural-based method.Other objects, features, and advantages will become apparent from the detaileddescription and appended claims to those skilled in the art.SUMMARY OF THE INVENTIONThe present invention discloses a synthetic peptide of Listeriolysin O (LLO-3, andLLO-4) as an antigen comprising Seq. ID 1 and Seq. ID 2 derived fromListeriolysin O protein of the L. monocytogenes strain at (18-29) amino acid (aa)position for LLO-3 and at (25-36) aa position for LLO-4 for sero-screening ofListeriosis in ruminants.Seq. ID 1 is AQQTEAKDASAFSeq. ID 2 is DASAFNKENSIS.In brief, protein sequences of Listeriolysin O (LLO) protein (NCBI protein ID:NP_463733) of the L. monocytogenes strain were retrieved from the NCBIdatabase. These retrieved sequences from the NCBI database were subjected toidentify trans-membrane regions using TOPCON software (http: / / topcons.net)and / or IEDB online analysis resources. These identified regions were furtherscreened for their antigenicity (B-cell epitopes), surface probability,hydrophobicity plot, and flexibility scores using the protean module ofLASERGENE software. The highly specific peptide sequences identified afterNCBI protein BLAST were shortlisted for the detection of L. monocytogenes(Table 1). The identified peptides matched with Listeria monocytogenes (LLO)while no other bacterial toxins matched the query. Further, the peptide of theregion between amino acid (18 to 36) matched with "NCBI BLASTP" search dataand the peptide sequence matched with Listeria monocytogenes (Listeriolysin O(LLO).The identified Listeriolysin O (LLO-3 and LLO-4) peptides were synthesizedfrom Sigma Aldrich, USA. The purity of the synthetic peptides found to be morethan 80% by High-performance liquid chromatography (HPLC) technique.Another embodiment of the present invention is related to the protocol followedfor the synthesis of the identified peptides on "Wang" resin by solid phasechemistry.BRIEF DESCRIPTION OF THE DRAWINGSThe present invention will be described in detail herein with the detaileddescription and related drawing that is provided only for illustrative purposes andrelate well with the preferred embodiments of the invention and are not intendedto limit the scope of the present disclosure. The invention is best understood byreference to the detailed description which follows in conjunction with the belowmentioned accompanying drawing provided herein:Figure 1 shows schematic presentation of Listeriolysin O (LLO) protein (NCBIProtein ID: NP_463733) sequence of L. monocytogenes strain and the location ofthe identified LLO-3, LLO-4 and LLO-2 peptides.Figure 2 shows the invitrogen peptide design tool analysis.Figure 3 shows the Karplus & Schulz Flexibility Prediction of the identifiedLLO-3 and LLO-4 peptides.Figure 4 shows the Parker Hydrophobicity Prediction of the identified LLO-3 andLLO-4 peptides.Figure 5 shows the Emini Surface Accessibility Prediction of the identified LLO3 and LLO-4 peptides.Figure 6 shows the standardization of synthetic Listeriolysin O (LLO-3 and LLO4) peptides and comparison with Listeriolysin O (LLO-2) in indirect enzymelinked immunosorbent assay (i-ELISA) test format.Figure 7 shows the screening of field "sera" samples (representative figure) usingListeriolysin O (LLO-3 and LLO-4) peptides as antigens for serodiagnosis ofListeriosis.DETAILED DESCRIPTION OF THE INVENTIONThe preferred embodiments of the present invention are provided so that thedisclosure will be thorough and will fully convey the scope to those who areskilled in the art. Various specific details are set as specific components to providean overall understanding of the preferred embodiments of the present disclosure.It will be apparent to those skilled in the art that the specific details need not beemployed and the preferred embodiments may be embodied in many differentforms and the steps followed do not limit the scope of the disclosure. It is also tobe understood that in the embodiments, the best known technology adopted forthe development of the present invention is described in detail.In any embodiment described herein, the open-ended terms "comprising,""comprises", and the like (which are synonymous with "including," "having" and"characterized by") may be replaced by the respective partially closed phrases"consisting essentially of," consists essentially of," and the like or the respectiveclosed phrases "consisting of," "consists of", and the like.As used herein, the singular forms "a", "an" and "the" designate both the singularand the plural, unless expressly stated to designate the singular only.An embodiment of the present invention involves identification of peptides ofListeriolysin O (LLO-3 and LLO-4) and its evaluation as an antigen in indirectenzyme-linked immunosorbent assay (ELISA) format technique for serologicalscreening of Listeriosis cases in bovines and caprine. The protein sequences ofListeriolysin O (LLO) protein (NCBI protein ID: NP_463733) of L.monocytogenes strain were retrieved from the NCBI database. These retrievedsequences from NCBI database subjected to identify trans-membrane regionsusing TOPCON software (http: / / topcons.net) and / or IEDB online analysisresources. These identified regions were further screened for their antigenicity (Bcell epitopes), surface probability, hydrophobicity plot and flexibility scores usingprotean module of LASERGENE software. The highly specific peptide sequencesidentified after NCBI protein BLAST were shortlisted for the detection of L.monocytogenes infection in bovines and caprines (Table 1).Table 1. Sequence of synthetic peptides of Listeriolysin O (LLO)With reference to Fig. 1 a schematic presentation of Listeriolysin O (LLO) protein(NCBI Protein ID: NP_463733) sequence of L. monocytogenes strain and thelocation of the identified LLO-3, LLO-4 and LLO-2 peptides is disclosed.Synthesis of the identified peptides by solid phase chemistryIdentified linear peptide synthesis of Listeriolysin O (LLO-3, LLO-4) was done onWang resin. Following are the steps involved in the chemical synthesis ofpeptides:(1) Loading of first N-protected amino acid on solid resin (Wang resin, a modifiedMerrifield resin);(2) Checking loading efficiency of the first amino acid and capping of open endson Wang resin;(3) Removal of N-terminal protection from amino acid onto resin;(4) Coupling of next incoming amino acids;(5) Repeating steps 2, 3, and 4 till the desired length of peptide was synthesized;(6) Deprotection and cleavage of the peptide from the resin; and(7) Desalting of the peptide and purification.Detailed Procedure for linear peptide synthesis on Wang resin1. Linear peptide synthesis was done on 50mg of Wang resin which was firstswelled with dimethyl formamide (DMF) overnight.2. For the addition of the first amino acids, N, N'-Diisopropylcarbodiimide(DIPC, 20mg) and 4-Dimethylaminopyridine (DMAP, 0.37 mg) per coupling isadded on the first amino acid dissolved in small amount of DMF and shaken for15 minutes on ice and then added on Wang resin.3. Calculated amount of the first amino acid along with Hobt, HBTU and DIEAwere added in a small amount of DMF and shaken for 5 minutes contained inMerrifield"s vessels and the vessels are then put in a shaker adjusted at C for aminimum of 2 hours.4. Excess and unattached amino acid is drained from the vessel and washed onceeach with DMF followed by DCM and DMF.5. De-blocking of the side chain is done with 20% Piperidine at °C for aminimum of 25 minutes and later the excess of Piperidine is drained and washedonce with DMF, twice with DCM and DMF respectively.6. All the next incoming amino acids are added sequentially and finally cleavageof the peptide from the resin using cleavage mixture (500 μl of mixture to 100 mgbeads) is done with shaking for 3-5 hours and after centrifugation at high speed10,000 rpm to collect the supernatant liquid.7. The cleavage step is repeated and the supernatant collected is washed andprecipitated with diethyl-ether five times.8. The precipitated peptides are then dried and concentrated in a vacuum drier.9. The purity of the synthesized peptide was checked by employing HighPerformance Liquid Chromatography (HPLC) and Mass Spectroscopy (MS).10. The isolated sequences of the synthetic LLO peptides are then stored indesiccators at room temperature.Fig. 2, Fig. 3, Fig. 4 and Fig. 5 show the results of invitrogen peptide design toolbased independent flexibility, hydrophobicity and surface accessibilitypredictions. The study shows that a peptide from the region between amino acidof (18-36) would have the best chances of binding the host antibodies.Another embodiment of the present invention relates with the positive andnegative control studies based on indirect enzyme-linked immunosorbent assay(ELISA).Positive and negative controlThe indirect enzyme-linked immunosorbent assay (ELISA) was standardizedusing positive serum samples of bovines and caprine confirmed by culture andpolymerase chain reaction (PCR) method while the serum samples of bovines andcaprine found to be negative by culture and polymerase chain reaction (PCR)method was employed as a negative control. Synthetic peptides of Listeriolysin O(LLO-3 and LLO-4) employed as coating antigens.Another embodiment of the present invention relates to the standardization ofsynthetic Listeriolysin O (LLO-3 and LLO-4) peptide based on enzyme-linkedimmunosorbent assay (i-ELISA) technique.Standardization of synthetic LLO (LLO-3 and LLO-4) peptide based indirectenzyme-linked immunosorbent assay (i-ELISA)The synthetic Listeriolysin O (LLO-3 and LLO-4) peptide based indirect enzymelinked immunosorbent assay (ELISA) was standardized by checkerboard analysisas per the method described in Low et al., (1992) literature with certainmodifications.In the process, 96-well flat bottom polyvinyl plates (Greiner, Germany) coatedwith synthetic Listeriolysin O (LLO-3 and LLO-4) peptides as antigens usingcoating buffer solutions. Different concentrations viz., 4.0, 0.4, 0.04 and 0.004μg / ml for each of the Listeriolysin O (LLO-3 and LLO-4) peptides were coated onELISA plate followed by the addition of the antigens to the wells atconcentrations of 100 μl / well and the coated plates kept at 37ºC for 2 hoursfollowed by incubation at temperature of 4 ºC overnight. The coated plates furtherwashed thrice with phosphate-buffered saline (PBS) with Tween 20 (PBS-T) ofpH 7.2 to remove the unbound antigens. The unsaturated sites of the plates wereblocked by adding a buffer of concentration of 200 μl / well (4.0 % of skimmedmilk powder and 2.0 % of bovine serum albumen in phosphate-buffered saline(PBS, pH 7.2). The plates then incubated at a temperature of 37 ºC for 2 hours andsubsequently washed thrice with phosphate-buffered saline (PBS-T). The knownpositive and negative sera samples diluted at various ratio concentrations of 1:25,1:50, 1:100, 1:200, 1:400 and 1:800 in phosphate-buffered saline (PBS) werethen added to the plates at a concentration rate of 100μl / well and then incubatedat 37 ºC for 2 hours. The plates then washed thrice with phosphate buffered saline(PBS-T) to which the mouse anti-goat antibody conjugated to horseradishperoxidase (HRP) enzyme conjugate for goat sera (IgG-HRPO conjugate) and therabbit anti-bovine antibody conjugated to horseradish peroxidase (HRP) enzyme(IgG-HRPO conjugate) for bovine sera added at concentration of 100μl / well alongwith dilutions to concentration ratios of (1:1000), (1:2000), (1:5000) and(1:10000). The plates then incubated at room temperature for 1 hour and washedthrice with phosphate saline buffer (PBS-T). Finally, the substrate solutioncontaining 10 mg of O-phenylene-diamine dihydrochloride (OPD) per 10 mL ofcitrate buffer containing 30% hydrogen peroxide (6μl / 10 ml of citrate buffer)were added at a concentration of 100μl / well and then incubated for 15 minutes inthe dark. The obtained plates were then analysed by an indirect enzyme-linkedimmunosorbent assay (ELISA) reader (Thermo Scientific Multiskan Ex, USA) at492 nm wavelength.Based on the checkerboard analysis, the serum sample at a dilution of (1:200)with a positive to negative ratio of more than 2.5 is considered positive forListeriosis in enzyme-linked immunosorbent assay (ELISA) employing eithersynthetic Listeriolysin O (LLO-3 and LLO-4) peptides (0.4μg / ml) as an antigenand (1:2000) dilution of mouse anti-goat IgG-HRPO conjugate for goat sera andrabbit anti-bovine IgG-HRPO conjugate for bovine sera. The standardizedELISAs employed for detecting the anti-listeria antibodies in bovine and caprinesera by employing the synthetic Listeriolysin O LLO (LLO-3 and LLO-4)peptides as a Serodiagnostic antigen.Fig. 6 shows the standardization of synthetic Listeriolysin O (LLO-3 and LLO-4)peptides and comparison with Listeriolysin O (LLO-2) in indirect enzyme-linkedimmunosorbent assay (i-ELISA) test format.Adsorption of test sera with Streptolysin O (SLO)To detect specific anti-Listeriolysin Oantibodies (ALLO) in bovines and caprine asan indicator for Listeria infection, their sera require prior adsorption withStreptolysin O (SLO) to remove cross-reacting anti-streptolysin O (ASLO)antibodies because Streptolysin O (SLO) and Listeriolysin O (LLO) areantigenically related. The adsorption of sera is carried out as per the methoddescribed in Berche et al. (1990) with certain modifications.The purified SLO (obtained from Sigma-Aldrich, USA) was coated onto 96 wellflat bottom enzyme-linked immunosorbent assay (ELISA) plates at aconcentration of 60 μg / ml which was further added to the wells at a concentrationof 200 μl / well and incubated for 3 hours at 37 ºC. Each serum (of 0.5 mL volume)and further diluted to (1 / 100) and (1 / 200) concentration ratios, respectively inphosphate-buffered saline (PBS) and added to the wells for further incubation at 4ºC overnight on a rocker. Streptolysin O (SLO) adsorbed sera was used in indirectplate ELISA's to screen the sera for ALLO antibodies against syntheticListeriolysin O (LLO-3 and LLO-4) peptides as per the method described above.Interpretation of indirect enzyme-linked immunosorbent assay (ELISA)resultsFor results interpretation, a comparison of positive (P) and negative (N) valueswas done wherein "P" stands for optical density (OD) of the unknown sample and"N" stands for optical density (OD) of the negative samples. The value of "N" wascalculated by taking the average of optical density values (ODs) of 2 knownListeriosis negative serum samples. The samples showing a P / N ratio of ≥ . havebeen considered seropositive tests for Listeriosis.Fig. 7 shows the screening of field "sera" samples (representative figure) usingListeriolysin O (LLO-3 and LLO-4) peptides as antigens for serodiagnosis ofListeriosis.Evaluation of synthetic Listeriolysin O (LLO-3 and LLO-4) peptide-basedindirect-ELISA with available synthetic LLO-2 peptide-based i-ELISA usingfield sera samples of bovines and caprineA total of 1096, serum samples collected from bovine (n=442) and caprine(n=654) were screened with LLO-3 and LLO-4 peptides in enzyme-linkedimmunosorbent assay (i-ELISA). All serum samples were also screened withavailable synthetic LLO-2 peptide-based i-ELISA to identify suitable syntheticpeptides of Listeriolysin O (LLO) for future sero surveillance of Listeriosis inbovines and caprine.On testing of 442 bovine serum samples with the three synthetic peptidesListeriolysin O (LLO-2, LLO-3, and LLO-4) based i-ELISA technique, theseropositivity observed against the unabsorbed caprine sera was 24.88%, 26.24%,and 22.39% which after adsorption with Streptolysin O (SLO) marginally reducedby (P>0.05) to 22.85%, 23.52% and 22.39%, respectively as shown in Table 2.Similarly, testing of 654 caprine serum samples with three synthetic ListeriolysinO peptide (LLO-2, LLO-3 and LLO-4) based i-ELISA, the seropositivityobserved against the unabsorbed caprine sera samples found were 9.17%, 8.56%and 8.71% which after adsorption with SLO slightly reduced (P>0.05) to 7.03%,7.28% and 6.88%, respectively (Table 2).Table 2. Consolidated results of serum samples tested by using syntheticpeptides of Listeriolysin O (LLO-3 and LLO-4) and its comparison withLLO-2 in indirect enzyme-linked immunosorbent assay (ELISAs)The difference in the number of positive and negative samples detected by syntheticListeriolysin O (LLO) peptides (LLO-3 and LLO-4) before and after Streptolysin O(SLO) adsorption was statistically non-significant (as P>0.05) in both bovines andcaprine. No significant difference was observed in the number of samples detectedby synthetic peptides of LLO (LLO-3 and LLO-4) when compared with LLO-2peptide results before and after adsorption with SLO on i-ELISA format (P>0.05) inbovines and caprine.Table 3 to Table 6 shows the contingency results of the samples screened by thesynthetic Listeriolysin O (LLO-3 and LLO-4) peptides in comparison withListeriolysin O (LLO-2) peptide-based results using indirect enzyme-linkedimmunosorbent assay (i-ELISA) before and after SLO adsorption.Table 3. Contingency table of bovine sera screened by Listeriolysin O peptides(LLO-2) and (LLO-3) synthetic peptide-based enzyme-linkedimmunosorbent assay (i-ELISA) format before and after Streptolysin O(SLO) adsorption.Table 4. Contingency table of bovine sera screened by Listeriolysin O (LLO2) and (LLO-4) synthetic peptide-based enzyme-linked immunosorbent assay(i-ELISA) format before and after Streptolysin O (SLO) adsorption.Table 5. Contingency table of caprine sera screened by Listeriolysin O (LLO2) and (LLO-3) synthetic peptide-based indirect enzyme-linkedimmunosorbent assay (i-ELISA) format before and after Streptolysin O(SLO) adsorption.Table 6. Contingency table of caprine sera screened by Listeriolysin O (LLO2) and (LLO-4) synthetic peptide-based indirect enzyme-linkedimmunosorbent assay (i-ELISA) format before and after Streptolysin O(SLO) adsorption.Table 7 and Table 8 show the data of relative sensitivity and specificity of theListeriolysin O (LLO-3 and LLO-4) peptides in comparison with the results ofalready existing LLO-2 peptides for both bovine and caprine samples which clearlysignifies the high efficacy of the LLO (LLO-3 and LLO-4) peptides over theexisting LLO-2 peptide for the detection of L. monocytogenes infection inruminants.Table 7. Relative sensitivity and specificity of synthetic peptides ofListeriolysin O (LLO-3 and LLO-4) peptides when compared with LLO-2peptide for bovine sera samples.Table 8. Relative sensitivity and specificity of synthetic peptides ofListeriolysin O peptide (LLO-3 and LLO-4) when compared with LLO-2peptide for caprine sera samplesADVANTAGES OF THE PRESENT INVENTION1. An advantage of the present invention is that the developed Listeriolysin-Opeptide-based indirect ELISA for detection of L. monocytogenes infection is acost-effective technique which is extremely stable, scalable and reproduciblewith the same purity.2. Another advantage of the present invention is the identified synthetic peptidesof Listeriolysin-O (LLO-3 and LLO-4) have excellent sensitivity andspecificity.3. Another advantage of the present invention is the good reproducibility ofindirect enzyme-linked immunosorbent assay (i-ELISA) by employing thesynthetic peptides of Listeriolysin-O (LLO-3 and LLO-4).4. Another advantage of the present invention is that the developed method doesnot require the prior adsorption of serum samples with Streptolysin O (SLO) andcross-reaction with other closely related toxins (Streptolysin O, Pneumolysin Oand Perfringolysin O) is eliminated.5. Another advantage of the present invention is that the Listeriolysin-O (LLO)peptides identified in this innovation (LLO-3 and LLO-4) or their derivedantibodies can be used as user-friendly methods in various field applicabilitytest formats for rapid and onsite detection of L. monocytogenes or its infectionin ruminants.6. Yet another advantage of the present invention is that there is no requirementfor higher infrastructure, sophisticated types of equipment, trained manpower,and most importantly time can be saved for the detection of L. monocytogenesor its infection in ruminants.

Claims

1. A synthetic Listeriolysin O peptide (LLO-3 and LLO-4) as an antigen, comprising Seq. ID 1 and Seq. ID 2 derived from Listeriolysin O protein of L. monocytogenes strain at 18-29 amino acid position for LLO-3 and at 25-36 amino acid position for LLO-4 for detection of L. monocytogenes infection in ruminants, wherein, Seq. ID 1 is AQQTEAKDASAF; and Seq. ID 2 is DASAFNKENSIS.

2. The synthetic Listeriolysin O peptide (LLO-3 and LLO-4) as claimed in claim 1, wherein the LLO-3 and LLO-4 peptides comprise of 12 amino acids.

3. A method for preparation of the synthetic Listeriolysin O peptide (LLO-3 and LLO-4) as claimed in claim 1, comprising the steps of: a) loading of the first N-protected amino acid on solid Wang resin which was first swelled with DMF overnight, b) checking the loading efficiency of the first amino acid and capping of open ends on Wang resin, c) removal of N-terminal protection from amino acid onto resin, d) coupling of the next incoming amino acids, e) repeating steps b, c, and d till the desired length of the peptide was synthesized, f) deprotection and cleavage of the peptide from the resin, g) desalting of the peptide, and h) purity check of peptide by employing HPLC and Mass Spectroscopy.

4. The synthetic Listeriolysin O peptide (LLO-3 and LLO-4) based indirect ELISA for serodiagnosis of L. monocytogenes infection in ruminants as claimed in claim 1, comprising the steps of: a) coating the ELISA plate with 100 μl of synthetic LLO-3 and LLO-4 peptides as antigens, b) incubating the plates at 37ºC for 2 h thereafter keeping the plate at 4ºC overnight, c) washing the plate thrice, d) blocking the plate with PBS and incubated at 37ºC for 2 h, e) washing the plate thrice, f) diluting the positive and negative sera in PBS added to the plates and then incubating the plate at 37ºC for 2 h, g) washing the plate thrice, h) adding anti-goat IgG-HRPO and anti-bovine IgG-HRPO into the plate, i) incubated the plate at room temperature for 1 h and washed thrice with PBS-T, j) adding the substrate O-phenylene-diamine dihydrochloride (OPD) and incubating the plates for 15 min. in the dark, and k) stopping the colour reaction by adding 2N sulphuric acid @ 50μl and reading the plates by an ELISA at 492 nm wavelength.