Antigen for preparing salmonella monoclonal antibody, monoclonal antibody, polyclonal antibody and application

Abstract

A specific protein fragment capable of representing salmonella as an antigen is obtained through screening and experimental verification, a mouse is immunized with the specific protein fragment as the antigen, and hybridoma cells capable of stably secreting monoclonal antibodies are obtained through a cell fusion technology. And finally, the anti-salmonella monoclonal antibody with high titer and good specificity is prepared. Similarly, the specific protein fragment is used for immunizing a rabbit to obtain the anti-salmonella polyclonal antibody. On the basis of a mouse monoclonal antibody and a rabbit polyclonal antibody, a salmonella colloidal gold immunochromatography detection method is established, the detection method is good in specificity, and a rapid, simple and convenient detection means is provided for detection of salmonella in food.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering and immunoassay, and specifically relates to antigens for preparing Salmonella antibodies, anti-Salmonella monoclonal antibodies, anti-Salmonella polyclonal antibodies, and the applications of the antibodies. Background Art

[0002] Salmonella belongs to the Enterobacteriaceae family and is a type of Gram-negative enterobacterium mainly parasitizing in the intestines of humans and animals. It has no spores, generally no capsules, and is aerobic or facultatively anaerobic. The optimal growth temperature of Salmonella is 35 - 37 °C, and the optimal growth pH is 6.8 - 7.8. The surface antigen structure of Salmonella is divided into three types, namely somatic (O) antigen, flagellar (H) antigen, and surface envelope (K) antigen, among which O antigen and H antigen are the most important antigens in Salmonella.

[0003] As a type of foodborne pathogen with strong harmfulness and wide influence, the diseases caused by Salmonella rank among the top in global foodborne diseases. Animal-derived foods such as poultry meat, eggs, dairy products, etc. are easily contaminated with Salmonella, and there are no obvious changes in the characteristics of the contaminated food. After humans are infected with Salmonella, poisoning symptoms such as abdominal pain, diarrhea, vomiting, fever, etc. will occur, and in severe cases, septicemia may even endanger life. It is estimated that there are up to 16 million Salmonella infection cases globally every year, including 600,000 death cases. Although there are many kinds of antibiotics that can be used to combat Salmonella infection, with the abuse of antibiotics and the emergence of drug-resistant genes due to the mutation of Salmonella, this line of defense of antibiotics is also in jeopardy. Therefore, it is very important to establish a on-site, rapid, efficient, and sensitive Salmonella detection method to timely handle contaminated foods, ensure the safe entry of foods into the circulation field, and minimize the risk of endangering the lives and safety of consumers.

[0004] There are more than 2,500 identified Salmonella serotypes worldwide. Approximately 300 serotypes have been discovered in China. The serotypes related to human diseases are mainly concentrated in groups A - E, among which Salmonella typhimurium, Salmonella enteritidis, and Salmonella choleraesuis are the most common. Common Salmonella detection methods include biochemical culture methods, immunoassay methods, molecular detection methods, etc. Biochemical culture methods are the standard methods for Salmonella detection, with high accuracy and authority. However, the detection cycle is long (usually 5 - 10 days), the operation is cumbersome, more relying on subjective judgment, and there is cross - reactivity in the biochemical identification of Enterobacteriaceae. Therefore, biochemical culture methods have certain limitations in terms of detection speed, specificity, sensitivity, etc., and are not suitable for on - site rapid detection. Molecular detection methods mainly include polymerase chain reaction (PCR), real - time fluorescence quantitative PCR (RT - PCR), loop - mediated isothermal amplification (LAMP), etc. based on Salmonella 16S rRNA or specific genes. These methods have the characteristics of high sensitivity, short detection time, and high detection efficiency compared with biochemical culture methods. However, they also have disadvantages such as easy contamination, being easily affected by sample matrix and primers, and having many false negatives. Moreover, the detection threshold of this detection method is relatively high, requiring precise instrument equipment and professional operators, and is not suitable for on - site rapid detection of Salmonella.

[0005] Immunoassay methods are based on the principle of specific binding of antigen and antibody, with advantages such as convenience, speed, stability, reliability, strong specificity, and high sensitivity. Currently, the main techniques include enzyme - linked immunosorbent assay (ELISA), immunochromatographic strip method (ICA), immunomagnetic bead separation method, etc.

[0006] Colloidal gold immunochromatography is to fix the antibody in a certain area of the nitrocellulose membrane, then immerse the sample at one end of the dried nitrocellulose membrane. The sample moves along the membrane under the action of capillary action. When it reaches the area with colloidal gold - labeled antibody, the antigen in the sample will specifically bind to the gold - labeled antibody, and the colloidal gold is used to make the area where the immune reaction occurs show color for specific detection of pathogens. Colloidal gold immunochromatography is a solid - phase membrane immunoassay method that combines colloidal gold labeling technology and immunochromatography method. The rapid detection test strips developed from it have received extensive attention in the domestic and foreign research fields in recent years. This method has the advantages of being fast, simple, highly specific, visible to the naked eye, and not requiring special instrument equipment, and can be used for on - site rapid qualitative detection of samples.

[0007] Through retrieval, no patent publication documents related to this invention patent application have been found. Summary of the Invention

[0008] The innovation of the present invention lies in overcoming and improving the deficiencies of the prior art. By analyzing and sequencing the outer membrane protein sequences of Salmonella, outer membrane amino acid sequences with high coverage within the genus Salmonella and strong specificity outside the genus are screened out, corresponding them into nucleotide sequences, and then using genetic engineering techniques to express the specific protein. Using the specific protein as an antigen to immunize animals, the obtained antibodies can specifically recognize Salmonella, and at the same time, the direct use of Salmonella pathogens during the experiment can be avoided, which greatly reduces the risk of Salmonella contamination and ensures public health and the safety of operators.

[0009] The object of the present invention is to provide an antigen for preparing monoclonal antibodies against Salmonella, monoclonal antibodies, and polyclonal antibodies, and to establish a colloidal gold immunochromatographic assay that is rapid, simple, convenient, and can be used for on-site real-time detection by using the antibodies, for the on-site rapid detection of Salmonella in food.

[0010] The technical solution of the present invention is as follows:

[0011] An antigen for preparing monoclonal antibodies against Salmonella, and the protein amino acid sequence of the antigen is SEQ ID NO.1:

[0012] MTSTIASLMFVAGAAVAADPTPVSVSGGTIHFEGKLVNAACAVSTKSADQTVT

[0013] LGQYRTASFTAIGNTTAQVPFSIVLNDCDPKVAANAAVAFSGQADNTNPNLLA

[0014] VSSADNSTTATGVGIEILDNTSSPLKPDGATFSAKQSLVEGTNTLRFTARYKATAAATTPGQANADATFIMKYE.

[0015] Monoclonal antibodies prepared using the antigen as described above

[0016] Polyclonal antibodies prepared using the antigen as described above

[0017] Moreover, the polyclonal antibodies are prepared by immunizing animals with the antigen.

[0018] Application of the monoclonal antibodies as described above in detection products for detecting or assisting in the detection of Salmonella. For example, application in kits, test strips, and other detection products for Salmonella.

[0019] Application of the polyclonal antibodies as described above in detection products for detecting or assisting in the detection of Salmonella. For example, application in kits, test strips, and other detection products for Salmonella.

[0020] A method for detecting or assisting in the detection of Salmonella using the monoclonal antibody described above, wherein the monoclonal antibody is used in the method.

[0021] A method for detecting or assisting in the detection of Salmonella using the monoclonal antibody described above, wherein the polyclonal antibody is used in the method.

[0022] An immunoassay method for Salmonella using the monoclonal antibody described above, wherein the monoclonal antibody serves as a capture antibody.

[0023] An immunoassay method for Salmonella using the polyclonal antibody described above, wherein the polyclonal antibody serves as a detection antibody.

[0024] The advantages and positive effects achieved by the present invention are as follows:

[0025] 1. The present invention uses a specific protein of Salmonella instead of Salmonella as an antigen to immunize mice, and hybridoma cells are screened through cell fusion technology. The monoclonal antibody secreted by the hybridoma cells can specifically recognize Salmonella, has no cross-reaction with other foodborne pathogenic bacteria, and the titer can reach 10 7 . The present invention avoids the use of Salmonella pathogens and obtains Salmonella monoclonal antibodies with high sensitivity, strong specificity, and high titer.

[0026] 2. The present invention simultaneously immunizes New Zealand white rabbits with this protein to prepare polyclonal antibodies. Based on the prepared rabbit-derived polyclonal antibodies and mouse-derived monoclonal antibodies, the present invention has established a rapid, simple, and highly specific colloidal gold immunochromatographic assay for Salmonella. The mouse monoclonal antibody is conjugated with colloidal gold to form a gold-labeled antibody, and the rabbit polyclonal antibody serves as a detection antibody. The detection limit is 10 6 CFU / mL. Five strains within the genus Salmonella and six strains outside the genus Salmonella are selected for specificity testing, and the results show good specificity. It meets the requirements of rapid, simple, and real-time detection in the food industry and has the value of popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the assembly of the test strip

[0028] Figure 2 It is a purification diagram of the antigen after recombinant expression in Escherichia coli in the present invention, wherein, M: protein Marker, 1: bacteria before induction, 2: bacteria after induction, 3, 4, 5: purified protein.

[0029] Figure 3 It is an optimization diagram of the loading buffer of the colloidal gold immunochromatographic test strip in the present invention.

[0030] Figure 4 It is the optimized graph of the usage amount of the gold-labeled antibody in the colloidal gold immunochromatographic test strip of the present invention.

[0031] Figure 5 It is the graph for determining the detection limit of the colloidal gold immunochromatographic test strip of the present invention.

[0032] Figure 6 It is the graph for determining the specificity of the colloidal gold immunochromatographic test strip of the present invention. Among them, 1-7 are Salmonella enteritidis (CMCC 50041), Escherichia coli O157:H7 (ATCC 32250), Staphylococcus aureus (ATCC 26003), Cronobacter sakazakii (ATCC 29544), Listeria monocytogenes (ATCC 19115), Pseudomonas aeruginosa (CICC 35150), Shigella flexneri (CICC 10865) respectively, and 8-13 are Salmonella newport (ATCC 6962), Salmonella typhimurium (CMCC 50115), Salmonella agona (ATCC 51957), Salmonella enteritidis (CMCC 50041), Salmonella choleraesuis (CICC 21494), Salmonella enteritidis (CICC 10467) respectively. Detailed implementation manners

[0033] The embodiments of the present invention will be described in detail below. It should be noted that this embodiment is narrative and not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0034] The raw materials used in the present invention are all conventional commercially available products without special instructions; the methods used in the present invention are all conventional methods in the art without special instructions.

[0035] An antigen for preparing Salmonella monoclonal antibody, and the protein amino acid sequence of the antigen is SEQ ID NO.1:

[0036] MTSTIASLMFVAGAAVAADPTPVSVSGGTIHFEGKLVNAACAVSTKSADQTVT

[0037] LGQYRTASFTAIGNTTAQVPFSIVLNDCDPKVAANAAVAFSGQADNTNPNLLA

[0038] VSSADNSTTATGVGIEILDNTSSPLKPDGATFSAKQSLVEGTNTLRFTARYKATAAATTPGQANADATFIMKYE.

[0039] The monoclonal antibody prepared by using the antigen as described above

[0040] Polyclonal antibodies prepared using the antigen as described above

[0041] Moreover, the polyclonal antibodies are prepared by immunizing an animal with the antigen.

[0042] Application of the monoclonal antibody as described above in detection products for detecting or assisting in the detection of Salmonella. For example, application in kits, test strips and other detection products for Salmonella.

[0043] Application of the polyclonal antibody as described above in detection products for detecting or assisting in the detection of Salmonella. For example, application in kits, test strips and other detection products for Salmonella.

[0044] A method for detecting or assisting in the detection of Salmonella using the monoclonal antibody as described above, wherein the monoclonal antibody is used in the method.

[0045] A method for detecting or assisting in the detection of Salmonella using the polyclonal antibody as described above, wherein the polyclonal antibody is used in the method.

[0046] An immunoassay method for Salmonella using the monoclonal antibody as described above, wherein the monoclonal antibody is used as a capture antibody.

[0047] An immunoassay method for Salmonella using the polyclonal antibody as described above, wherein the polyclonal antibody is used as a detection antibody.

[0048] Specifically:

[0049] 1. In the present invention, a specific protein fragment representing Salmonella as an antigen is obtained through screening and sequence alignment, and the protein is recombinantly expressed by genetic engineering techniques. The recombinant protein is used as an antigen to immunize mice, and the antibody titer in the mouse blood can reach 1:243000 after three booster immunizations. The spleen cells of the immunized mice are fused with sp2 / 0 myeloma cells, and HAT selective medium is used for cell screening. Only the successfully fused hybridoma cells can survive. Subcloning is continued by the limiting dilution method, and after 4 rounds of subcloning and screening, hybridoma cells with strong growth ability, high titer and capable of secreting monoclonal antibodies are obtained. Intraperitoneal injection is performed at a dose of 10 6 cells per mouse, and the mouse ascites is taken and purified 7 - 10 days later to obtain the monoclonal antibody.

[0050] 2. Immunize New Zealand white rabbits with the same antigen to obtain polyclonal antibodies, and establish a colloidal gold strip immunochromatographic detection method based on monoclonal antibodies and polyclonal antibodies. Connect the monoclonal antibody with colloidal gold to prepare a gold-labeled antibody. Through experiments, it is obtained that the optimal pH for the connection between the monoclonal antibody and colloidal gold is 8.0, and the optimal antibody amount connected to each mL of colloidal gold is 15 μg. Coat the polyclonal antibody and goat anti-mouse IgG on the T line and C line of the test strip respectively, and assemble the test strip.

[0051] Optimize the performance of the test strip, and the obtained results are as follows: the optimal concentration of the detection antibody on the T line is 1.6 mg / mL, the optimal dilution factor of goat anti-mouse IgG on the C line is 100 times, the optimal usage amount of the gold-labeled antibody is 6 μL, the optimal sample loading buffer is PBS, and the detection limit of the test strip for Salmonella is 10 6 CFU / mL. Select 6 foodborne pathogenic bacteria outside the genus Salmonella, Escherichia coli O157:H7 (ATCC 32250), Staphylococcus aureus (ATCC 26003), Cronobacter sakazakii (ATCC29544), Listeria monocytogenes (ATCC 19115), Pseudomonas aeruginosa (CICC 35150), Shigella flexneri (CICC10865), and 5 different serotypes of Salmonella within the genus Salmonella to verify the specificity of the test strip, and the results show good specificity.

[0052] More specifically, the related preparation and verification are as follows:

[0053] Example 1 Preparation of Antigen

[0054] 1.1 Construction of recombinant plasmid

[0055] Obtain the target gene by PCR, and perform double digestion on the purified PCR product and pET-30a plasmid. After the target gene is ligated to the plasmid, it is transformed into Escherichia coli DH5α competent cells, positive clones are screened, and the strain is expanded and cultured and preserved.

[0056] 1.2 Expression of recombinant protein

[0057] 1.2.1 Transformation. Extract the recombinant plasmid and transform it into the expression strain Escherichia coli BL21(DE3) competent cells, and spread it on an LB solid culture plate containing Kana antibiotic, and culture it upside down overnight.

[0058] 1.2.2 Strain expansion culture. Pick colonies with good growth status and inoculate them into 10 ml of LB liquid medium containing Kana, and culture them at 37 °C overnight

[0059] 1.2.3 Induced expression. Take 100 μL of overnight culture and inoculate it into 10 mL of LB liquid medium containing Kana at a ratio of 1:1000. Incubate for about 2 h until the logarithmic phase. Take 200 μL of the bacterial solution as the control before induction, and add IPTG inducer with a final concentration of 0.1 mM to the remaining bacterial solution. Continue to incubate for 5 - 6 h.

[0060] 1.2.4 Detection of protein expression form. Centrifuge the induced bacterial solution to remove the LB medium, wash it twice with PBS, and resuspend the bacteria with PBS at 1 / 10 of the original volume. Use a high-pressure cell disruptor to break the bacterial solution, centrifuge at 12,000 rpm for 10 min at 4°C to separate the supernatant and precipitate, and resuspend the precipitate with the same volume of PBS. Take 10 μL of the supernatant and precipitate respectively for SDS-PAGE, observe the electrophoresis results. If the protein content in the supernatant is significantly higher than that in the precipitate, it indicates that the protein expression form is soluble expression; if the protein content in the precipitate is significantly higher than that in the supernatant, it indicates that the protein expression form is inclusion body.

[0061] 1.2.5 Optimization of induction conditions. Select an IPTG inducer concentration of 0.1 mM and induce at different temperatures (37°C, 30°C, 20°C). Detect the induced bacterial solution by SDS-PAGE, and select the induction temperature with the highest protein expression as the optimal induction temperature; use different concentrations of inducer (0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM) to induce protein expression at the optimal induction temperature, and select the inducer concentration with the highest protein expression detected by SDS-PAGE as the optimal concentration.

[0062] 1.2.6 Large-scale expression. Express the protein (1 L) on a large scale according to the optimal induction conditions, centrifuge to remove the LB medium, and wash it twice with PBS. After repeated freezing and thawing on ice for 2 - 3 times, break the bacteria by high pressure. Centrifuge the broken bacterial solution at 12,000 rpm for 10 min at 4°C. If the protein is soluble expressed, directly purify it with Ni-NTA·His·Bind affinity column; if the protein is inclusion body, it needs to be denatured and renatured first and then purified.

[0063] 1.3 Protein purification

[0064] 1.3.1 Denaturation and renaturation. Resuspend the precipitate with 1×PBS, centrifuge at 4℃, 12000rpm for 10min, remove the supernatant and keep the precipitate. Suspend the precipitate with 50mL Buffer A, mix well, centrifuge at 4℃, 12000rpm for 10min, discard the supernatant, and repeat this operation once. Suspend the precipitate with 50mL Buffer B, mix well, centrifuge at 4℃, 12000rpm for 10min, discard the supernatant, and repeat this operation once. Suspend the precipitate with 50mL Buffer C, mix well, and shake rapidly in a 37℃ shaker for 1h. After the precipitate in the solution is completely dissolved, centrifuge at 4℃, 12000rpm for 10min, and keep the supernatant. Carefully pour the supernatant into the pre-treated dialysis bag, use 50 times the volume of protein refolding dialysis fluid, dialyze at 4°C for 12 hours, and use gradient dialysis fluid containing different concentrations of urea (2M urine 2 times, 1M urea 1 time, 0.5M urea 1 time, no urea 2 times). The protein after dialysis is the refolded protein, centrifuge the refolding solution at 4°C, 12000rpm for 10 minutes, and retain the supernatant.

[0065] 1.3.2 Protein purification using Ni-NTA·His·Bind affinity column.

[0066] a. Refrigerate the Binding Buffer, Washing Buffer, and Elution Buffer used for purification at 4°C in advance.

[0067] b. Take 10 mL of Ni-NTA·His·Bind resin stored in 20% ethanol and add it to the empty chromatography column with the lower end sealed.

[0068] c. Wash the column with 5 column volumes of sterile ultrapure water to rinse out the ethanol in the resin.

[0069] d. Wash the column with 3 column volumes of Binding Buffer to equilibrate the column conditions.

[0070] e. Add the protein solution to be purified, and take 50 μL of the protein solution as a control before adding. Collect the protein solution flowing out of the chromatography column, pass it through the column again, and then place the inlet and outlet in the protein solution, circulate through the column on ice for 1 to 2 hours to allow the protein and resin to fully bind. Take 50 μL of the bound protein solution and save it for SDS-PAGE electrophoresis detection.

[0071] f. Wash the column with 10 column volumes of Binding Buffer.

[0072] g. Wash the column with 6 column volumes of Washing Buffer to remove impurities.

[0073] h. Wash the column with 6 column volumes of Elution Buffer. First, allow the Elution Buffer to react fully with the resin for 10 min before starting the column run. Collect the eluate, which should contain the purified protein at this time. Collect one tube every 2 mL and take 20 μL from each tube for SDS-PAGE electrophoresis to detect the purification effect.

[0074] The results are shown in Figure 2 , there is a single band at around 30 kDa, indicating that the target protein is correctly expressed and has a relatively high purity, and can be used for subsequent immunization.

[0075] Example 2 Preparation of Polyclonal Antibodies

[0076] 2.1 Immunization of Rabbits

[0077] Select 2 male New Zealand white rabbits weighing about 1.5 kg as immunized animals. After purchasing, first keep them in a standard experimental animal room for 1 week. Adjust the protein concentration to 2 mg / ml with physiological saline, take 1 mL and mix it with an equal volume of complete Freund's adjuvant for emulsification. Take blood before immunization as a negative control. The first immunization method is subcutaneous multiple-point injection and thigh muscle injection, and the immunization dose is 2 mg. Boost the immunization every 2 weeks, with an immunization dose of 1 mg, and the immunization method is subcutaneous multiple-point injection, for a total of 4 immunizations. Take blood after the third immunization and measure the antibody titer by indirect ELISA. Seven days after the last immunization, take whole blood and centrifuge to obtain serum.

[0078] 2.2 Detection of Antiserum Titer by Indirect ELISA

[0079] Take blood from the rabbit ear vein, place it at room temperature for 2 h, and then at 4℃ overnight. Centrifuge at 4℃ and 3000 rpm for 10 min, and take the supernatant as serum.

[0080] (1) Coating the plate: Dilute the antigen protein to 1 mg / mL with coating buffer, and add it to the 96-well ELISA plate at a coating amount of 100 μL (1 μg) / well, incubate overnight at 4℃ or for 2 h at 37℃.

[0081] (2) Washing: Discard the liquid in the ELISA plate wells, wash the plate 3 times with 200 μL / well of PBST, and let it stand for 2 min each time. After the last washing, discard the liquid in the wells and pat dry the residual liquid in the wells on the filter paper.

[0082] (3) Blocking: Add blocking solution (5% skim milk powder - PBST), 200 μL / well, incubate at 37℃ for 1 h, discard the blocking solution, wash the plate 3 times with PBST, and finally pat dry the residual liquid in the wells on the filter paper.

[0083] (4) Adding serum: Dilute the serum with PBST in gradients, diluting it 1000, 3000, 9000, 27000, 81000, and 243000 times respectively. Add 100 μL per well, and add PBST to the last row as a blank control. Incubate at 37 °C for 1 h, wash the plate 4 times with PBST, and finally pat dry the residual liquid in the wells on filter paper.

[0084] (5) Adding secondary antibody: Dilute the goat anti-rabbit secondary antibody labeled with HRP 5000 times, add 100 μL per well, incubate at 37 °C for 30 min, discard the liquid, wash the plate more than 5 times with PBST, and finally pat dry the wells on filter paper.

[0085] (6) Color development: Add the color developing solution, 100 μL per well, and react at 37 °C in the dark for 15 min.

[0086] (7) Termination: Add the termination solution, 50 μL per well, to terminate the color development reaction.

[0087] (8) Reading: Measure the absorbance value of each well with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. Wells with an absorbance above 0.8 are positive wells, and the maximum dilution factor corresponding to the positive wells is the antibody titer in the rabbit blood.

[0088] The results showed that the antibody titers in the sera of Rabbit 1 and Rabbit 2 both met the requirements for subsequent experiments, and the titers increased with the increase in the number of immunizations.

[0089] 2.3 Purification of polyclonal antibody

[0090] In this experiment, Protein A affinity chromatography medium was used for the purification of polyclonal antibody. The specific purification steps are as follows:

[0091] (1) Take 2 mL of rabbit serum and thaw it at 4 °C. Dilute the serum with the equilibration buffer (Binding Buffer) at a ratio of 1:2.

[0092] (2) Shake the packing material well, aspirate 3 mL of the slurry (actually 1.5 mL of the packing material) into the chromatography column, and pre-add 3 mL of the equilibration buffer to the chromatography column.

[0093] (3) Let the resin settle naturally, add 15 mL of the equilibration buffer, and flow out the equilibration liquid at a flow rate of 2 mL / min.

[0094] (4) Load the sample at a flow rate of 0.5 mL / min, load the effluent again, and cycle 3 times to ensure that the antibody is completely bound to the column material. Collect the final effluent, and subsequently use SDS-PAGE to measure the binding ability of the column material.

[0095] (5) Wash the column material with 90 mL of the equilibration buffer until the A280 absorbance of the effluent reaches a stable state.

[0096] (6) Elute the antibody with 10 - 15 mL of Elution Buffer at a flow rate maintained at 0.5 mL / min, and collect the effluent. Measure the absorbance of the effluent. 280 Collect the eluate with an absorbance > 0.2 and immediately add neutralization buffer to adjust the pH to 7.4.

[0097] (7) Column material regeneration: First, wash with 30 mL of elution buffer, then wash with 15 mL of equilibration buffer until the pH of the effluent is neutral, and finally add 20% ethanol for sealed storage.

[0098] 2.4 Determination of polyclonal antibody concentration

[0099] At 4°C, dialyze the above - purified antibody in PB buffer for 3 days. Take 10 μL of the dialyzed antibody, dilute it 20 - fold with PBS, and measure its absorbance at 280 nm using a UV - visible spectrophotometer. Use PBS as the blank control and substitute into formula (2 - 1) to obtain the antibody concentration.

[0100] Antibody concentration (mg / ml) = (A – A 空白 ) / 1.35 × 20 Formula (2 - 1)

[0101] Where, A: Absorbance of the antibody at 280 nm; A 空白 : Absorbance of PBS without antibody at 280 nm; 1.35: Protein coefficient; 20: Dilution factor.

[0102] The results showed that the antibody titer produced by rabbit 2 was higher, so the polyclonal antibody from rabbit 2 was selected for subsequent experiments.

[0103] Example 3 Preparation of monoclonal antibody

[0104] 3.1 Immunization of mice

[0105] Newly - bought mice are immunized after 7 days of adaptation in the standard animal house. The first immunization is carried out by multiple subcutaneous injections on the back. Take an appropriate amount of protein (100 μg / mouse) and mix it with an equal volume of complete Freund's adjuvant, and emulsify it completely. After that, incomplete Freund's adjuvant is used for immunization, and the immunization interval is 2 weeks. A boost immunization is carried out 3 days before cell fusion. The boost immunization does not use adjuvant, only pure protein, and the immunization dose is 200 μg / mouse.

[0106] 3.2 Detection of antiserum titer by indirect ELISA

[0107] 7 - 10 days after the third immunization, detect the titer of mouse antiserum. The steps are the same as those for detecting the titer of rabbit antiserum above.

[0108] The results are shown in Table 1. The antibody titer in the serum of mouse No. 3 is relatively high, and the next step of cell fusion can be carried out.

[0109] Table 1 Antibody titers in the sera of mice after four immunizations

[0110]

[0111] 3.3 Cell fusion

[0112] Resuscitate sp2 / 0 myeloma cells two weeks before cell fusion. When the cell quantity is sufficient, inject the cells subcutaneously into the back of the mice, 300 μL (0.5 - 1×10 6 cells) for each mouse. Observe the growth of tumors on the backs of the mice 10 days later.

[0113] (1) Give a boost immunization to the mice three days before cell fusion, and sterilize the scissors and forceps required for dissection in advance.

[0114] (2) Isolation and collection of sp2 / 0 myeloma cells

[0115] a. Decapitate the mice with tumors and soak them in 75% alcohol for 3 - 5 minutes.

[0116] b. Fix the mice on the dissection board with their backs up in a sterile laminar flow hood. Peel off the fur under sterile conditions and put the removed tumor pieces into a homogenizer.

[0117] c. Add 5 mL of 1640 basal medium, grind thoroughly, supplement with 10 mL of basal medium, mix well and let stand for 2 min. Gently aspirate 5 mL of the supernatant into a 50 mL sterile centrifuge tube, then supplement with 10 mL of basal medium twice. After each supplementation, mix well and let stand for 2 min. Aspirate 10 mL for the first time and all for the second time. When aspirating for the last time, appropriately discard about 3 mL of the liquid at the bottom to avoid sucking large tissue pieces.

[0118] d. Centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the cells with 1640 basal medium, and control the volume at about 30 mL.

[0119] f. Pour 15 mL of lymphocyte separation medium into another 50 mL centrifuge tube, and slowly add the cell suspension from the previous step along the wall above the lymphocyte separation medium (the volume ratio of the cell suspension to the lymphocyte separation medium is 2:1 - 1:1)

[0120] g. Centrifuge at 1200 rpm for 15 min, aspirate the white and dense cell layer outside the liquid interface, wash the cells twice with 1640 basal medium, and then resuspend the cells with 10 mL of basal medium.

[0121] h. Aspirate 10 μL of the cell suspension for 10 - fold dilution and counting, and use a Thoma hemocytometer for counting.

[0122] (3) Preparation of feeder cells

[0123] a. Sacrifice an unimmunized BALB / c mouse by taking blood from its eyeballs, soak it in 75% alcohol for 3 - 5 minutes, and collect the serum as negative serum.

[0124] b. Take out the mouse carcass, wipe off the excess alcohol, and fix the mouse in a supine position on the dissection board in a sterile laminar flow hood. Take a set of sterile scissors and forceps, cut through the fur layer between the two hind legs, and carefully cut through the fur layer from bottom to top to expose the abdominal cavity.

[0125] c. Replace with a new set of scissors and forceps, make an opening in the abdominal cavity from the lower left part, cut upward and to the right from the opening, and then invert the cut peritoneum completely from the lower left to the upper right to expose the peritoneum.

[0126] d. Replace with another new set of scissors and forceps, take out the spleen, carefully cut the tissues adhered around the spleen, and place the spleen in the grinder.

[0127] f. The grinding step is the same as above

[0128] g. Centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the cells with 10 mL of HAT medium, and place it in a 37°C incubator for standby.

[0129] (4) Preparation of immune spleen cells

[0130] The preparation of immune spleen cells is the same as that of feeder spleen cells.

[0131] (5) Cell fusion

[0132] a. Mix 1 - 2×10 7 myeloma cells and 10 8 spleen cells in a ratio of 1:10 or 1:15, and then centrifuge at 1000 rpm for 10 min.

[0133] b. Pour out the supernatant, dry the residual liquid on the tube wall with a sterile filter paper, and gently tap the bottom of the tube to slightly loosen the cell clumps (to facilitate the full action of PEG on the cells).

[0134] c. Place the centrifuge tube containing the cell mixture in a 37°C water bath, and slowly add 800 μL of 50% PEG preheated at 37°C within 1 min 4000 , while adding, shake the centrifuge tube.

[0135] d. After adding, continue to shake for 30 s and let it stand for 30 s.

[0136] e. Slowly add 15 mL of the termination solution (1640 basal medium) preheated to 37°C to terminate the fusion reaction. The specific addition steps are as follows: add 1 mL in the 1st minute, 2 mL in the 2nd minute, 3 mL in the 3rd minute, 4 mL in the 4th minute, and 5 mL in the 5th minute. When adding, it must be added slowly and the centrifuge tube should be gently shaken.

[0137] f. Place the centrifuge tube in an incubator at 37°C for 10 min, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0138] g. Mix the feeder cells and the fused cells, and supplement the HAT medium according to the seeding amount (the force when resuspending the cells must be small because the newly fused cells are easily dispersed if the force is large). Seed the cell suspension into a 96-well cell culture plate, 200 μL per well. Generally, 4 - 8 96-well plates are inoculated for one fusion.

[0139] h. Incubate the 96-well plate in an incubator at 37°C.

[0140] i. Do not move the cells within three days after fusion. On the 4th day, perform a half-medium change with HAT medium. On the 6th day, start performing a half-medium change with HT medium. Continuously change the medium 4 times in the following days. When the fused cell colonies grow to 1 / 4 of the well, take the supernatant of the culture medium for antibody detection.

[0141] 3.4 Screening of hybridoma cells

[0142] (1) First screening: Seven days after cell fusion, observe the cell wells and mark the wells with hybridoma cells, and calculate the fusion rate. When the hybridoma cells grow to cover 1 / 10 of the bottom, take a small amount of the supernatant of the culture medium and use the indirect ELISA method to detect whether it is positive, and calculate the positive rate. The wells with positive results in three consecutive detections are determined as positive wells, and the positive wells are expanded and cultured in a 24-well plate.

[0143] The results of the first screening showed that the fusion success rate was 61.8%, and the positive cell rate was 8.4%. The results are shown in Table 2:

[0144] Table 2 Results of the first cell screening

[0145]

[0146] (2) Second screening: When the hybridoma cells in the 24-well plate grow to cover 1 / 10 of the bottom, coat the ELISA plate with various serotypes of Salmonella, Escherichia coli, Cronobacter sakazakii, Staphylococcus aureus and other foodborne pathogenic bacteria, and take the supernatant for indirect ELISA determination. Select the wells that show positive reactions with all Salmonella and no cross-reactions with other foodborne pathogenic bacteria for subcloning.

[0147] 3.5 Subcloning

[0148] In the uncloned wells, there are positive cells secreting antibodies and negative cells not secreting antibodies. The negative cells will compete with the positive cells for growth, causing the positive cells to gradually turn negative. At the same time, to ensure that the cells in each well are derived from the division of a single hybridoma cell, the cells in the positive wells should be cloned in a timely manner.

[0149] (1) Prepare feeder cells before cloning. The preparation steps are the same as above. Suspend the prepared feeder cells in HT medium and keep them at 37 °C for later use.

[0150] (2) Pick positive cell wells, gently pipette the cells with a cut pipette tip, and perform a 10-fold dilution before counting.

[0151] (3) For the first subcloning, dilute the cells to 30 cells / mL with HT medium and add 100 μL / well to a 96-well cell plate, i.e., 3 cells / well. For the second subcloning, dilute according to 1 cell / well, and for the third subcloning, dilute according to 0.5 - 1 cell / well. Inoculate the remaining cells into a 24-well plate for expansion culture and preservation.

[0152] (4) Add the previously prepared feeder cells to the wells, 50 μL / well. The concentration of the feeder cells should be appropriate to cover the bottom of the well.

[0153] (5) Place the cell plate in the cell culture incubator. After three days, add 50 μL of HT medium to each well without moving the plate. At the same time, observe whether there are single cell clusters formed in the cell wells and make marks.

[0154] (6) On the 7th day of culture, take the culture supernatant for indirect ELISA detection to screen the cells. Use Salmonella and other foodborne pathogenic bacteria as the coating antigen, and the coating concentration is 0.5 - 1×10 8 CFU / mL. Select the cells in the wells that show a positive reaction only to Salmonella, have no cross-reaction with other bacteria, have a high antibody titer, show monoclonal growth, and have a good growth state for the next subcloning.

[0155] (7) After three rounds of subcloning, when the positive rate of the cell wells is 100%, amplify this strain of hybridoma cells in large quantities, freeze more than 4 tubes of cells, and make marks.

[0156] Finally, 5 strains of hybridoma cells that can stably secrete monoclonal antibodies were obtained, named: 5E10, 5G8, 3E4, 2C10, 6G11.

[0157] 3.6 Large-scale preparation and purification of monoclonal antibodies

[0158] Select female BALB / c mice at 8 - 10 weeks old and inject sterile paraffin oil intraperitoneally at a dose of 500 μL per mouse. One week later, inject hybridoma cells intraperitoneally, with 0.5 - 1×10 6 cells per mouse, and the cell suspension is 400 μL per mouse. After 7 - 10 days, it can be observed that the abdomen of the mice bulges significantly, and the ascites is collected in a timely manner. Centrifuge the ascites at 4℃ and 3000 rpm for 10 min, discard the upper white fat and the lower cell precipitate, aspirate the middle light yellow liquid, and store it at -20℃ for purification.

[0159] Purify the antibody by the caprylic acid - saturated ammonium sulfate method.

[0160] (1) Take out the ascites from the -20℃ refrigerator, thaw it at 4℃, centrifuge at 12000 rpm for 10 min, and take the supernatant.

[0161] (2) Add 3 volumes of acetate buffer to the supernatant of the centrifuged ascites.

[0162] (3) According to the amount of adding 33 μL of caprylic acid per milliliter of ascites, under ice - bath conditions, slowly drip - add caprylic acid while stirring, stir for 30 min, and let it stand at 4℃ for 2 h.

[0163] (4) After the precipitation of miscellaneous proteins is complete, centrifuge at 4℃ and 12000 rpm for 15 min, collect the supernatant, and filter it through a 0.45 - μm filter membrane.

[0164] (5) Add 1 / 10 volume of 0.1M PBS buffer with pH 7.4 to the supernatant, and adjust the pH to 7.4 with 2M NaOH.

[0165] (6) Under stirring conditions, slowly drip - add saturated ammonium sulfate solution to make the final concentration of ammonium sulfate reach 45%, stir in an ice - bath for 30 min, and let it stand at 4℃ overnight.

[0166] (7) Centrifuge the above solution at 4℃ and 12000 rpm for 15 min, discard the supernatant, add 2 mL of 0.01M PBS to resuspend the precipitate, dialyze it in 0.01M PBS, change the solution every 4 h, and change the solution 3 times.

[0167] (8) Centrifuge the dialyzed antibody at 4℃ and 12000 rpm for 15 min to remove insoluble precipitates, aliquot it into 2 - mL sterile centrifuge tubes, add an equal volume of glycerol, store it at -20℃, and measure the antibody concentration.

[0168] Example 4 Establishment of the detection method for colloidal gold immunochromatographic test strips

[0169] The double antibody sandwich ELISA method was used to select the most suitable pair combinations of 5 monoclonal antibodies and polyclonal antibodies, and finally a group of optimal combinations was screened out. The results are shown in Table 3. The P / N value of the paired combination of monoclonal antibody 6G11 and polyclonal antibody was the highest. Therefore, monoclonal antibody 6G11 and polyclonal antibody were selected for paired use.

[0170] Table 3 Results of paired monoclonal and polyclonal antibodies

[0171]

[0172] 4.1 Connection of monoclonal antibody and colloidal gold

[0173] It was known from experiments that the optimal pH for the connection of monoclonal antibody and colloidal gold was 8.0, and the optimal antibody amount for connection per milliliter of colloidal gold was 15 μg. After the connection of colloidal gold and monoclonal antibody, a blocking agent and a stabilizer were added. After centrifugation, the gold-labeled antibody was resuspended with the working solution and stored at 4 °C for later use.

[0174] 4.2 Assembly of test strip

[0175] The assembly of the test strip is as Figure 1 shown. The test strip consists of a nitrocellulose membrane (NC membrane), a PVC backplate, a sample pad, and an absorbent pad. First, the NC membrane was cut and pasted on the PVC backplate, and the absorbent pad (10 mm × 2.7 mm) and the sample pad (10 mm × 2.5 mm) were pasted in sequence. On a three-dimensional flat membrane spraying and gold-spraying instrument, polyclonal antibody and goat anti-mouse secondary antibody were respectively coated on the NC membrane as the C line and the T line, and dried at 37 °C for 6 h or overnight. After drying, the test strip was cut into small strips with a width of 3.7 mm using a microcomputer automatic cutting machine and stored dry in a sealed bag.

[0176] 4.3 Optimization of loading buffer

[0177] PBS, PB, Tris, and borate solution were selected as different loading buffers to resuspend Salmonella, and detected by loading on the test strip. The results showed that the colors of the T line and the C line of the test strip using PBS and PB were significantly uniform, while the colors of the Tirs and borate buffer were lighter and uneven. The results are shown in Figure 3 .

[0178] Subsequently, blank experiments were carried out using PBS and PB. PB would produce false positive phenomena. Therefore, PBS was finally selected as the optimal loading buffer.

[0179] 4.4 Optimization of the concentrations of detection antibody and goat anti-mouse secondary antibody

[0180] The detection antibody was respectively diluted to 0.8, 1.2, 1.6, 2.0 mg / mL, and the goat anti-mouse secondary antibody was respectively diluted 50, 100, 200, 400 times to obtain 16 combinations, and detected by loading at the same time.

[0181] The results showed that when the concentration of the detection antibody for the T line was 1.6 mg / mL and the secondary antibody for the C line was diluted 100-fold, the colors of the T line and the C line were appropriate and uniform. Therefore, the optimal concentration of the detection antibody was 1.6 mg / mL, and the optimal dilution factor of the goat anti-mouse secondary antibody was 100-fold. The results are shown in Table 4.

[0182] Table 4 Optimization of the concentrations of the detection antibody and goat anti-mouse IgG

[0183]

[0184] Note: C represents the control line; T represents the test line; + indicates the presence of color, and the more +, the darker the color; - indicates the absence of color

[0185] 4.5 Optimization of the amount of gold-labeled antibody used

[0186] 2, 4, 6, 8, and 10 μL of the gold-labeled antibody were separately pipetted and added to 100 μL of Salmonella for sample loading and detection.

[0187] The results showed that when the amount of the gold-labeled antibody used was 6 μL, the colors of the T line and the C line were bright and uniform. Therefore, 6 μL of the gold-labeled antibody was selected for subsequent experiments. The results are shown in Figure 4 .

[0188] 4.6 Determination of the detection limit of the test strip

[0189] Salmonella in the logarithmic phase was taken, and the bacterial liquid concentration was controlled at 10 8 CFU / mL with PBS, and then the bacterial liquid was diluted to 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, and 10 4 CFU / mL with PBS respectively. Bacterial liquids with different concentrations were separately taken for detection to determine the detection limit of the bacterial liquid of the test strip.

[0190] The results showed that Salmonella with a concentration of 10 6 CFU / mL could be detected by the test strip, while the test strip could not detect Salmonella at 10 5 CFU / mL. Therefore, the detection limit of the test strip was 10 6 CFU / mL. The results are shown in Figure 5 .

[0191] 4.7 Verification of the specificity of the test strip

[0192] Different strains outside the genus Salmonella and strains within the genus Salmonella were used respectively. Escherichia coli O157:H7 (ATCC 32250), Staphylococcus aureus (ATCC 26003), Cronobacter sakazakii (ATCC 29544), Listeria monocytogenes (ATCC 19115), Pseudomonas aeruginosa (CICC 35150), Shigella flexneri (CICC 10865), several common foodborne pathogenic bacteria were selected for specificity verification. Different serotypes of Salmonella preserved in this laboratory were selected to verify the coverage within the genus Salmonella of the test strip.

[0193] The results are as Figure 6 shown. 1 - 7 are Salmonella enteritidis (CMCC 50041), Escherichia coli O157:H7 (ATCC32250), Staphylococcus aureus (ATCC 26003), Cronobacter sakazakii (ATCC 29544), Listeria monocytogenes (ATCC 19115), Pseudomonas aeruginosa (CICC 35150), Shigella flexneri (CICC 10865) respectively, and 1 - 7 are all negative reactions. 8 - 13 are Salmonella newport (ATCC 6962), Salmonella typhimurium (CMCC 50115), Salmonella agona (ATCC 51957), Salmonella enteritidis (CMCC 50041), Salmonella choleraesuis (CICC 21494), Salmonella enteritidis (CICC 10467) respectively, and 8 - 13 are all positive reactions, indicating that the test strip has good specificity for other foodborne pathogenic bacteria outside the genus Salmonella.

[0194] Example 5 Sequencing of the Monoclonal Antibody

[0195] The screened monoclonal antibody 6G11 was sequenced. The total RNA of the hybridoma cell line secreting the 6G11 monoclonal antibody was extracted using an RNA extraction kit. The RNA was reverse transcribed into cDNA using specific reverse primers or universal primers. Subsequently, the VH and VL antibody fragments of the monoclonal antibody 6G11 were amplified using the GenScript rapid amplification of cDNA ends (RACE) technology. The amplified antibody fragments were cloned into a sequencing vector, and clones with the correct - sized inserted fragments were screened by colony PCR. For each fragment bacterium, no less than 5 colonies with the correct - sized inserted fragments were selected for sequencing, and the sequencing results of each clone were compared and analyzed. Finally, the sequence information of antibody 6G11 is shown in Table 5. For convenience of description, the light - chain complementarity - determining regions (CDR) 1 - 3 are represented by LCDR1, LCDR2, and LCDR3 respectively, and the heavy - chain complementarity - determining regions (CDR) 1 - 3 are represented by HCDR1, HCDR2, and HCDR3 respectively.

[0196] Table 5 Sequence Information of the Monoclonal Antibody 6G11 in this Example

[0197]

[0198]

[0199] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that: various substitutions, changes, and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments and the drawings.

Claims

1. An antigen for preparing a Salmonella monoclonal antibody, characterized in that: The amino acid sequence of the antigen is shown in SEQ ID NO.

1.

2. A monoclonal antibody prepared using the antigen as claimed in claim 1, characterized in that The amino acid sequences of HCDR1-HCDR3 of the monoclonal antibody are shown in SEQ ID NOs. 4-6, and the amino acid sequences of LCDR1-LCDR3 are shown in SEQ ID NOs. 7-9.

3. A polyclonal antibody prepared using the antigen as claimed in claim 1.

4. A method for preparing anti-Salmonella monoclonal antibodies using the antigen according to claim 1, characterized in that: The following steps are involved: The antigen described in claim 1 is used to immunize mice, hybridoma cells are screened by cell fusion technology, and the anti-Salmonella monoclonal antibody is secreted by the hybridoma cells.

5. A method for preparing anti-Salmonella polyclonal antibodies using the antigen according to claim 1, characterized in that it comprises the following steps: The anti-Salmonella polyclonal antibody is prepared by immunizing New Zealand white rabbits with the antigen described in claim 1.

6. Use of the monoclonal antibody according to claim 2 in preparing a detection product for detecting or assisting in the detection of Salmonella.

7. Use of the polyclonal antibody according to claim 3 in preparing a detection product for detecting or assisting in the detection of Salmonella.

8. A method for detecting or assisting in detecting Salmonella using the monoclonal antibody according to claim 2, characterized in that: The monoclonal antibody of claim 2 is used in the method, and the method is not intended for diagnosis or treatment of a disease.

9. A method for detecting or assisting in detecting Salmonella using the polyclonal antibody according to claim 3, characterized in that: The method uses the polyclonal antibody according to claim 3, and the method is not intended for diagnosis or treatment of a disease.

10. Use of the antigen as claimed in claim 1 to prepare monoclonal antibodies or polyclonal antibodies against Salmonella.

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