CN106093409A - The preparation method of the colloidal gold strip that detection Salmonella in Food based on Salmonella core polysaccharide monoclonal antibody belongs to - Google Patents

Abstract

The invention discloses a method for preparing a colloidal gold test strip for detecting Salmonella in food based on the Salmonella core polysaccharide monoclonal antibody SQX6D8, which belongs to the field of immunoanalysis. In the present invention, the conjugate of Salmonella typhimurium lipopolysaccharide (LPS) and bovine serum albumin (BSA) synthesized by sodium periodate method is used as the coating source of the colloidal gold test strip test line (T line), and the core of Salmonella The polysaccharide monoclonal antibody SQX6D8 was used as a gold-labeled antibody. The present invention is different from the principle of the colloidal gold test strip sandwich method of conventional pathogenic bacteria. The colloidal gold test strip method for Salmonella adopts the principle of indirect competition for detection, and the Salmonella core polysaccharide-specific monoclonal antibody ensures that the method is effective against Salmonella within the genus. Both have cross-reaction and have no cross-reaction to bacteria outside the genus, which provides an analytical method for the comprehensive, simple and rapid detection of Salmonella in food.

Description

Detection of Salmonella in food based on monoclonal antibody to Salmonella core polysaccharide The preparation method of the colloidal gold test strip

technical field

The invention relates to a preparation method of a colloidal gold test strip for detecting Salmonella in food based on the Salmonella core polysaccharide monoclonal antibody SQX6D8, belonging to the field of immune analysis.

Background technique

Salmonella is a global foodborne pathogen. Biologically, Salmonella is a kind of Gram-negative bacteria with blunt ends, no spores, and generally no capsule. The main antigens are O antigen, H antigen, and Vi antigen. Animal foods such as poultry, eggs, and dairy products are easily contaminated with Salmonella. After the human body ingests food containing bacteria, it will cause acute gastroenteritis, typhoid fever, children with low immunity and even sepsis and other symptoms.

There are more than 2,000 serotypes of Salmonella, and the common serotypes in clinical practice are mainly Salmonella enteritidis, Salmonella typhimurium, and Salmonella paratyphi A. The application of well-regulated production operations and management systems such as hazard analysis and critical point control (HACCP) can largely reduce the occurrence of foodborne pathogens. However, the quality monitoring of raw materials, production process and products is also an important means to ensure food biosafety.

At present, the methods for detecting Salmonella mainly include biochemical culture method, immunological detection method, and molecular detection method. The traditional biochemical culture method is the national standard method for detecting Salmonella. Although it is authoritative and reliable, it generally takes 5-10 days to get the result, and the operation process is cumbersome, which cannot meet the requirements of rapid detection; the molecular detection method is based on Salmonella deoxyribonucleic acid (DNA) The polymerase chain reaction (PCR) was established. At present, it has been developed into traditional PCR, real-time fluorescent quantitative PCR (RT-PCR), and loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP). Compared with traditional PCR, RT-PCR has the characteristics of realizing quantitative detection of target DNA, stronger specificity, effectively solving the problem of PCR pollution, and high degree of automation. The LAMP method has the characteristics of simplicity, rapidity and strong specificity. This technology is not inferior to conventional PCR technology in terms of sensitivity, specificity, and detection range. It does not rely on special instruments and equipment, and can perform high-throughput rapid detection on site, and the detection cost is much lower than real-time fluorescent quantitative PCR. However, it has been reported in the literature that false negatives may occur when the LAMP method detects Salmonella in milk. This may be caused by the primer being affected by the sample matrix. Similarly, conventional PCR and real-time fluorescent PCR also face the problems of high detection cost and high technical requirements for operators.

Colloidal gold test strip method for detection of pathogenic microorganisms has the advantages of simplicity, rapidity and low cost. At present, there is no report on the colloidal gold test strips for the detection of Salmonella at the genus level. The main difficulties focus on the preparation of monoclonal antibodies with uniform crossover and high affinity, and the acquisition of paired antibodies for double-antibody sandwiches. In our previous patents, we disclosed the synthesis of cross-linked immunogens (patent application number: 201410314040.3), and the establishment of a cross-type double-antibody sandwich ELISA method (201510183851.9). However, when using paired antibodies to establish a colloidal gold test strip method, there is a problem that the T line does not develop color. The reasons may be: (1) the affinity of the screened monoclonal antibody meets the ELISA but is not enough to meet the requirements of the colloidal gold test strip; (2) It is difficult for the core polysaccharide to be exposed on the surface of the bacteria. When the reaction time of the colloidal gold test strip method is very short (10 min), it is difficult for the antibody immobilized on the T line to effectively capture the Salmonella in the sample. Therefore, we used the newly prepared monoclonal antibody SQX6D8 with higher affinity, and synthesized heterologous LPS conjugates as the T-line coating agent, and used the principle of competition method for detection, which overcomes the difficulty of capturing Salmonella with T-line antibodies. problem. The detection sensitivity of this method to the 12 strains of Salmonella tested was 10 ⁵ - 5×10 ⁶ CFU/mL, and the crossover was relatively uniform. Campylobacter, Campylobacter coli, Vibrio parahaemolyticus, Staphylococcus aureus, and Listeria monocytogenes had no cross-reactivity.

Contents of the invention

The purpose of the present invention is to establish a colloidal gold test strip for detecting Salmonella at the genus level, which is used for rapid detection of Salmonella in food with high specificity and high accuracy.

The technical scheme of the present invention, in order to achieve the above object, the present invention establishes a colloidal gold test strip method for detecting Salmonella in food based on the competition method principle of Salmonella core polysaccharide monoclonal antibody, and the method also includes an optimization process.

Among them, the monoclonal antibody SQX6D8 is obtained by immunizing 8-week-old BALB/c mice with the EDC method LPS-BSA artificial antigen as the immunogen, and is fused and screened by hybridoma technology. It has the characteristics of high affinity and good inhibitory effect.

Among them, the LPS-BSA conjugate synthesized by the _NaIO4 method, which was originally coated with T line, has a better inhibitory effect on LPS-BSA than the homologous EDC method.

The principle of detection and analysis of the method of the present invention: the principle of indirect competition is used for detection, the test line (T line) is sprayed with a synthetic LPS-BSA conjugate as the coating source, the Salmonella core polysaccharide-specific monoclonal antibody SQX6D8 and the red gold nanometer Particles were conjugated as gold-labeled antibodies. During detection, the Salmonella in the sample first binds to the gold-labeled antibody to inhibit the binding of the gold-labeled antibody to the T-line coating source, and the color intensity of the T-line is inversely proportional to the number of Salmonella in the sample. The monoclonal antibody specific to the core polysaccharide of Salmonella ensures that the method has cross-reaction to Salmonella within the genus and no cross-reaction to bacteria outside the genus.

A monoclonal cell line SQX6D8 based on the core polysaccharide of Salmonella has been deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms, referred to as CGMCC, and the preservation number is CGMCC No.12019.

The monoclonal antibody SQX6D8 based on the core polysaccharide of Salmonella is secreted and produced by the monoclonal cell line SQX6D8 with the deposit number CGMCC No. 12019.

A method for preparing a colloidal gold test strip for detecting Salmonella in food based on the Salmonella core polysaccharide monoclonal antibody SQX6D8, the specific steps are:

(1) Preparation of Salmonella core polysaccharide monoclonal antibody SQX6D8: the monoclonal antibody was obtained by immunizing the strain with the preservation number CGMCC No.12019;

(2) Synthesis of T-line coated original LPS-BSA conjugate:

a. Activation: take mutant Salmonella typhimurium lipopolysaccharide Ra-LPS 10mg and dissolve it in ultrapure water according to the reaction mass ratio NaIO ₄ : Ra-LPS 5: 1, add 47 μL of NaIO ₄ solution with a concentration of 200mM/L to the mutant In the Salmonella typhimurium lipopolysaccharide Ra-LPS solution, react at 25°C for 2h; then take 10μL of 1M/L ethylene glycol solution into the reaction solution, and react at 25°C for 2h;

b. Coupling: Add the mutant Salmonella typhimurium lipopolysaccharide Ra-LPS reaction solution activated in step a dropwise to the BSA solution according to the reaction molar ratio Ra-LPS:BSA ratio of 5:1, and use 0.01M, pH9 .6 carbonate buffer solution CB to adjust the pH of the reaction solution to 8.5; react overnight at room temperature, and obtain the synthesized LPS-BSA conjugate after dialysis;

(3) Synthesis of gold nanoparticles: Synthesize 30nm gold nanoparticles by citric acid reduction method, add 300mL of chloroauric acid solution with a mass concentration of 0.01% in a cleaned flask, heat to complete boiling under magnetic stirring, and pour into the solution Quickly add 4.8mL of trisodium citrate solution with a mass concentration of 1% to the solution, boil the solution for 10min until the color turns bright wine red, cool the flask at room temperature, and store it at 4°C;

(4) Coupling of gold-labeled antibody: adjust the pH of 1mL colloidal gold solution to 8 with 0.1M potassium carbonate solution; add 10 μg of Salmonella core polysaccharide monoclonal antibody SQX6D8 and react at room temperature for 2 hours; add 50 μL with a mass-volume ratio of 10 % BSA solution to block and react at room temperature for 2 hours; centrifuge the colloidal gold solution at 4°C, 6000g, and 20min to remove uncoupled colloidal gold and monoclonal antibodies; use 0.01M phosphoric acid containing 0.2% Tween and 0.2% sucrose Wash the gold-labeled antibody with salt buffer;

(5) Preparation of gold standard test strips: fix the nitrocellulose membrane on the PVC bottom plate, attach the water-absorbing pad to the end of the nitrocellulose membrane and the PVC bottom plate close to the quality control line, and stick the sample pad to the nitrocellulose The other end of the plain film and the PVC bottom plate close to the test line; the distance between the quality control line and the test line is 6mm; the LPS-BSA conjugate is sprayed to the test line with a film sprayer, and 0.5mg/mL goat anti-mouse IgG The secondary antibody was sprayed to the quality control line; dried at 37°C for 2 hours and cut into strips for later use to obtain colloidal gold test strips based on the Salmonella core polysaccharide monoclonal antibody SQX6D8 for the detection of Salmonella in food.

The Salmonella includes 12 strains, followed by Paratyphoid A (Group A), Salmonella Agona (Group B), Salmonella Typhimurium (Group B), Paratyphoid B (Group B), Salmonella Thompson (C1 Salmonella), Blockley Salmonella (Group C2), Salmonella Kentucky (Group C3), Salmonella Enteritidis (Group D), Salmonella Typhoid (Group D), Salmonella Dublin (Group D), Salmonella duck (Group E), Salmonella Arizona.

Beneficial effects of the present invention: the Salmonella-specific colloidal gold test strip provided by the present invention is different from the principle of the double-antibody sandwich method of the conventional macromolecular colloidal gold test strip, but adopts a competition method for detection.

The monoclonal antibody SQX6D8 specific to the core polysaccharide of Salmonella has higher affinity and more uniform crossover. The synthetic LPS-BSA conjugate is used as the T-line coating agent to compete with Salmonella in the sample. The gold-labeled antibody of SQX6D8 coupled to gold nanoparticles was established The colloidal gold test strip can detect Salmonella at the genus level and has no cross-reaction with other test bacteria. The method has the characteristics of simple and fast operation, good stability and low cost, and has the value of popularization and application.

Preservation of biological material samples: the monoclonal cell line SQX6D8 has been preserved in the General Microbiology Center of the Chinese Microbiological Culture Collection Management Committee, referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation Date: January 20, 2016, deposit number CGMCC No.12019.

Description of drawings

Fig. 1 Schematic diagram of the principle of the Salmonella-specific colloidal gold test strip.

Figure 2 Detection of mutant lipopolysaccharide (RaLPS) by Salmonella-specific colloidal gold test strips.

Figure 3 Salmonella specific colloidal gold test strips for the detection of Salmonella.

Figure 4 Salmonella-specific colloidal gold test strip cross-reactivity.

detailed description

Example 1

The concrete steps of this colloidal gold test strip development are:

(1) Preparation of Salmonella core polysaccharide monoclonal antibody SQX6D8:

Using the immunogen synthesis method disclosed in Patent Application No.: 201410314040.3, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide ( NHS) method to synthesize mutant Salmonella lipopolysaccharide (Ra-LPS) and keyhole limpet hemocyanin (KLH) complete antigens, and as immunogens to immunize mice, through routine cell fusion and Salmonella cells with LPS and different O antigens as package The positive cells were screened by the original, and finally the genus-specific monoclonal antibody SQX6D8 with high affinity and uniform crossover to Salmonella was prepared.

(2) Synthesis of T-line-coated original LPS-BSA conjugate: Synthesize the conjugate using the synthesis method disclosed in Patent Application No.: 2014103140403, and the specific steps are as follows:

a. Activation: Dissolve 10 mg of mutant Salmonella typhimurium lipopolysaccharide Ra-LPS in ultrapure water, and add 47 μL of NaIO ₄ solution with a concentration of 200 mM/L dropwise according to the reaction mass ratio NaIO ₄ : Ra-LPS 5:1 to the mutant Salmonella typhimurium lipopolysaccharide Ra-LPS solution, react at 25°C for 2h; then take 10μL of 1M/L ethylene glycol solution into the reaction solution, react at 25°C for 2h;

b. Coupling: Add the activated mutant Salmonella typhimurium lipopolysaccharide Ra-LPS reaction liquid dropwise to the BSA solution according to the molar ratio Ra-LPS:BSA ratio of 5:1, and use 0.01M, pH9.6 The carbonate buffer solution CB adjusted the pH of the reaction solution to 8.5; reacted overnight at room temperature, and obtained the synthesized LPS-BSA conjugate after dialysis;

(3) Synthesis of gold nanoparticles: Synthesize 30nm gold nanoparticles by citric acid reduction method, add 300mL of chloroauric acid solution with a mass concentration of 0.01% in a cleaned flask, heat to complete boiling under magnetic stirring, and pour into the solution Quickly add 4.8mL of trisodium citrate solution with a mass concentration of 1% to the solution, boil the solution for 10min until the color turns bright wine red, cool the flask at room temperature, and store it at 4°C;

(4) Coupling of gold-labeled antibody: adjust the pH of 1 mL of colloidal gold solution to 8 with 0.1 M potassium carbonate solution; add 10 μg of Salmonella core polysaccharide monoclonal antibody SQX6D8 and react at room temperature for 2 hours; add 50 μL of 10% ( M/V) BSA solution was blocked, and reacted at room temperature for 2h. Centrifuge the colloidal gold solution at 4°C, 6000g, and 20min to remove unconjugated colloidal gold and monoclonal antibody; wash the gold-labeled antibody with 0.01M phosphate buffer containing 0.2% Tween and 0.2% sucrose;

(5) Preparation of gold standard test strips: Fix the absorbent pad 1 and the sample pad 5 on the polyvinyl chloride PVC bottom plate 6 and the nitrocellulose membrane 2 according to the positions shown in the figure, and mark the test line 4 and the quality control line 3 in turn. position, with an interval of 6mm; spray LPS-BSA conjugate and goat anti-mouse IgG secondary antibody (0.5mg/mL) to test line 4 and quality control line 3 respectively with a film sprayer BioJet Quanti3000; dry at 37°C for 2 hours and dry Cut into strips and set aside.

Embodiment 2 adopts this colloidal gold test strip to detect Salmonella Ra LPS:

First, Ra LPS (1mg/mL) was diluted with 0.01M phosphate buffer to 100 ng/mL, 50 ng/mL, 25 ng/mL, 10 ng/mL and 5 ng/mL, blank 0.01M phosphoric acid Salt buffer as a control;

Then use the wet method to detect Ra LPS, take 7 μL of the gold-labeled antibody prepared in step (4) of Example 1 and 47 μL of the resuspension (0.1% Tween, 0.2% sucrose, 1% BSA in 0.01M phosphate buffer ) into the microplate and mix with a pipette. Add 150 μL of different concentrations of Ra LPS to different microwell plates, mix well with a pipette gun, and react at room temperature for 5 minutes; insert the prepared colloidal gold test strip into the microwell plate, and react at room temperature for 10 minutes to read. Interpretation basis: blank sample, quality control line and test line have color at the same time, and the test line has a darker color, positive sample. The quality control line has color, the color of the test line is very light or no color compared with the blank sample, and the negative sample. The specific test results are shown in Figure 2.

Embodiment 3 Adopt this colloidal gold test strip to detect 12 strains of Salmonella:

The 12 strains were Paratyphoid A (Group A) CMCC 50093, Salmonella Agona (Group B) CICC 21586, Paratyphoid B (Group B) CMCC 50094, Salmonella Typhimurium (Group B) ATCC 13311, Tompu Salmonella genus (group C1) CICC21480, Salmonella Brockley (group C2) CICC 21489, Salmonella Kentucky (group C3) CICC 21488, Salmonella enteritidis (group D) ATCC13076, Salmonella typhi (group D) CMCC 50071, Salmonella Dublin (group D) ) CICC 21497, Duck Salmonella (Group E) CICC 21498, Arizona Salmonella ATCC 13314.

The specific detection process is as follows: the pure culture of Salmonella tested is diluted with 0.01M phosphate buffer to 10 ⁷ CFU/mL, 10 ⁶ CFU/Ml, 10 ⁵ CFU/mL and 10 ⁴ CFU/mL, and the diluent Make a blank control, and other processes are the same as in Example 2. The specific test results are shown in Figure 3.

Example 4 Testing the cross-reactivity of Salmonella-specific colloidal gold test strips:

The other 8 strains tested were: Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157, Escherichia coli vulgaris, Enterobacter cronobacter, Vibrio parahaemolyticus, Campylobacter jejuni, Campylobacter coli. The test concentrations were all 5×10 ⁸ CFU/mL, the test process was the same as in Example 2, and the results are shown in FIG. 4 .

Claims (4)

1. A monoclonal cell line SQX6D8 based on the core polysaccharide of Salmonella has been deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, referred to as CGMCC, and the preservation number is CGMCC No.12019. 2. The monoclonal antibody SQX6D8 based on the core polysaccharide of Salmonella, characterized in that it is secreted and produced by the monoclonal cell line SQX6D8 with the deposit number CGMCC No. 12019. 3. a kind of preparation method of the colloidal gold test strip of Salmonella in the detection food based on Salmonella core polysaccharide monoclonal antibody SQX6D8, it is characterized in that concrete steps are: (1) Preparation of Salmonella core polysaccharide monoclonal antibody SQX6D8: the monoclonal antibody was obtained by immunizing the strain with the preservation number CGMCC No.12019; (2) Synthesis of T-line coated original LPS-BSA conjugate: a. Activation: Dissolve 10 mg of mutant Salmonella typhimurium lipopolysaccharide Ra-LPS in ultrapure water, and add 47 μL of NaIO ₄ solution with a concentration of 200 mM/L dropwise according to the reaction mass ratio NaIO ₄ : Ra-LPS 5:1 to the mutant Salmonella typhimurium lipopolysaccharide Ra-LPS solution, react at 25°C for 2h; then take 10μL of 1M/L ethylene glycol solution into the reaction solution, react at 25°C for 2h; b. Coupling: Add the mutant Salmonella typhimurium lipopolysaccharide Ra-LPS reaction solution activated in step a dropwise to the BSA solution according to the reaction molar ratio Ra-LPS:BSA ratio of 5:1, and use 0.01M, pH9 .6 carbonate buffer solution CB to adjust the pH of the reaction solution to 8.5; react overnight at room temperature, and obtain the synthesized LPS-BSA conjugate after dialysis; (3) Synthesis of gold nanoparticles: Synthesize 30nm gold nanoparticles by citric acid reduction method, add 300mL of chloroauric acid solution with a mass concentration of 0.01% in a cleaned flask, heat to complete boiling under magnetic stirring, and pour into the solution Quickly add 4.8mL of trisodium citrate solution with a mass concentration of 1% to the solution, boil the solution for 10min until the color turns bright wine red, cool the flask at room temperature, and store it at 4°C; (4) Coupling of gold-labeled antibody: adjust the pH of 1mL colloidal gold solution to 8 with 0.1M potassium carbonate solution; add 10 μg of Salmonella core polysaccharide monoclonal antibody SQX6D8 and react at room temperature for 2 hours; add 50 μL with a mass-volume ratio of 10 % BSA solution to block and react at room temperature for 2 hours; centrifuge the colloidal gold solution at 4°C, 6000g, and 20min to remove uncoupled colloidal gold and monoclonal antibodies; use 0.01M phosphoric acid containing 0.2% Tween and 0.2% sucrose Wash the gold-labeled antibody with salt buffer; (5) Preparation of gold standard test strips: Fix the nitrocellulose membrane (2) on the PVC bottom plate (6), and attach the water-absorbing pad (1) to the nitrocellulose membrane (2) and the PVC bottom plate (6). ) close to the end of the quality control line (3), adhere the sample pad (5) to the other end of the nitrocellulose membrane (2) and PVC bottom plate (6) close to the test line (4); the quality control line (3) The distance from the test line (4) is 6mm; spray the LPS-BSA conjugate to the test line (4) with a film sprayer, and spray 0.5mg/mL goat anti-mouse IgG secondary antibody to the quality control line (3) place; dried at 37°C for 2 hours and cut into strips for later use, to obtain colloidal gold test strips based on the Salmonella core polysaccharide monoclonal antibody SQX6D8 for the detection of Salmonella in food. 4. according to the preparation method of the colloidal gold test strip of Salmonella in the detection food based on the Salmonella core polysaccharide monoclonal antibody SQX6D8 of claim 3, it is characterized in that: described Salmonella comprises 12 strains, is successively paratyphoid fever ( Group A), Salmonella Agona (Group B), Salmonella Typhimurium (Group B), Paratyphoid B (Group B), Salmonella Thompson (Group C1), Salmonella Blockley (Group C2), Salmonella Kentucky (C3 group), Enteritis Salmonella (D group), Typhoid Salmonella (D group), Dublin Salmonella (D group), Duck Salmonella (E group), Arizona Salmonella.