CN113563465B

CN113563465B - Salmonella PhoN protein antibody and detection method and application thereof

Info

Publication number: CN113563465B
Application number: CN202110656656.9A
Authority: CN
Inventors: 焦新安; 高杨; 孟闯; 潘志明; 丁睿清; 徐双媛; 曹李妍; 康喜龙; 顾丹
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2023-02-03
Anticipated expiration: 2041-06-11
Also published as: CN113563465A

Abstract

The invention belongs to the technical field of biology and provides a salmonella PhoN protein antibody and a detection method and application thereof. The invention also provides a preparation method and application of the monoclonal antibody. The monoclonal antibody provided by the invention has the advantages of high titer, strong specificity and wide reaction spectrum. The invention adopts the immunomagnetic bead enrichment method based on the PhoN antigen monoclonal antibody of the outer membrane protein of the salmonella, can effectively enrich the salmonella typhimurium and the salmonella infantis, compared with the prior art, the immunomagnetic bead enrichment method has better specificity and broad spectrum, can simultaneously enrich at least two kinds of salmonella, simplifies the detection steps, shortens the detection time, and provides a reliable sample pretreatment technology for rapidly and efficiently detecting various types of salmonella serotypes.

Description

Salmonella PhoN protein antibody and detection method and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a hybridoma lymphocyte technology and an immunomagnetic bead technology, and further relates to an anti-salmonellae PhoN protein detection antibody prepared by the two technologies, a detection method and application.

Background

The Salmonella (Salmonella) is an important food-borne zoonosis pathogen, causes salmonellosis by infecting animals and humans, seriously harms the development of livestock industry and human health, and has important public health significance. Up to now, as many as 2600 serotypes of salmonella have been found. According to relevant statistics, the food poisoning caused by salmonella is top-ranked among the bacterial food poisoning worldwide. There are 16 million people infected with salmonella each year in europe and about 140 million people infected with salmonella each year in the united states, accounting for 30% of total food-borne diseases. Therefore, high-throughput detection of salmonella is of high interest in various countries. The search for a rapid detection technology for salmonella is particularly important.

The monoclonal antibody (McAb) has the advantages of strong specificity, high sensitivity, high purity and the like, and the application of the technology improves the specificity, sensitivity and stability of the serological method of salmonella. The McAb-based detection and diagnosis application, such as immune enrichment magnetic beads, serological diagnosis kit and the like, can be used for rapidly detecting and diagnosing salmonella. However, the existing salmonella detection method has the problems of long detection time consumption, low specificity, complex steps and the like.

The currently legal salmonella detection method is a microbial culture method, mainly comprises the processes of pre-enrichment, isolated culture, genus and species identification and the like, has strong repeatability, high stability and high accuracy, and is the most reliable detection method at present. However, the whole detection process has long period, complicated operation and low sensitivity, and the treatment of the early selective enrichment culture needs to consume 3 days, so that the time cost is greatly increased, and the timely and effective monitoring cannot be realized. Immunomagnetic separation (IMS) based on magnetic nanomaterials has been widely used for the separation of food-borne pathogenic bacteria. The biologically modified and functionalized magnetic nano material can specifically identify food-borne pathogenic bacteria, and an external magnetic field is used for separating bacteria, so that the specific rapid enrichment and separation of a small amount of pathogenic bacteria in food can be realized. Therefore, the development of a sample pretreatment technology such as immunomagnetic beads for specifically and rapidly enriching salmonella has great significance for improving the salmonella detection efficiency.

Disclosure of Invention

In view of the above disadvantages of the prior art, the present invention aims to provide a PhoN protein detection antibody for salmonella and a detection method thereof, which are used for solving the problems of long time consumption and low specificity of the detection of salmonella in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a salmonella PhoN protein antibody, comprising a heavy chain and a light chain, wherein the light chain comprises three complementarity determining regions with amino acid sequences shown as SEQ ID nos. 1-3, and the heavy chain comprises three complementarity determining regions with amino acid sequences shown as SEQ ID nos. 4-6.

Further, the amino acid sequence of the light chain is shown as SEQ ID NO. 7; the amino acid sequence of the heavy chain is shown as SEQ ID NO. 9.

In another aspect of the present invention, there is provided a polynucleotide encoding the antibody as described above.

Further, the nucleotide sequence of the polynucleotide for coding the light chain is shown in SEQ ID NO. 8; the nucleotide sequence of the heavy chain encoded by the polynucleotide is shown in SEQ ID NO. 10.

In another aspect of the present invention, there is provided a recombinant expression vector comprising the polynucleotide described above.

Another aspect of the present invention provides a host cell comprising or incorporating a recombinant expression vector as described above.

In another aspect, the invention provides the use of the above antibodies, polynucleotides, recombinant expression vectors and host cells for detecting at least two serotypes of salmonella (salmonella typhimurium and salmonella infantis).

In another aspect of the present invention, there is provided a salmonella immunomagnetic bead detection composition comprising the antibody described above.

Furthermore, the composition also comprises magnetic beads, a washing solution, an activating solution and a sealing solution.

Further, the antibody is a purified IgG antibody against a salmonella PhoN protein.

The magnetic bead is used for preparing immunomagnetic beads, and the preparation method of the immunomagnetic beads comprises the following steps: after washing the magnetic beads with a washing solution, activating the magnetic beads with an activating solution; coupling the IgG antibody of the PhoN protein of the purified salmonella with the magnetic beads, and then sealing by using sealing liquid; then washing with a washing solution to obtain the immunomagnetic beads coupled with the salmonella PhoN protein antibodies.

Further, the washing solution was 0.01mol/L morpholine ethanesulfonic acid Tween solution (MEST, pH6.0;0.05% Tween-20); the activating solution is 5mg/mL carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution which are respectively prepared from 0.01mol/L morpholine ethanesulfonic acid monohydrate solution (MES, PH 6.0); the blocking solution was PBST (pH 7.4, 1% BSA).

In another aspect of the invention, a method for detecting salmonella antibody immunomagnetic beads is provided, and the method comprises the step of detecting different serotypes of salmonella by using the antibody.

Further, the purified IgG antibodies to salmonella PhoN protein.

Further, the method specifically comprises the following steps:

(1) Washing and activating the magnetic beads, coupling antibodies of the salmonella PhoN protein, and sealing;

(2) Adding the sealed immunomagnetic beads into the quantitative bacterial liquid, and incubating;

(3) Magnetically separating the supernatant and the magnetic beads, and washing the magnetic beads with a washing solution;

(4) And respectively coating plates for counting and analyzing the results.

Further, the method specifically comprises the steps of washing the magnetic beads for 3 times by using a washing solution, activating by using an activating solution, and coupling the antibody of the salmonella PhoN protein; adding confining liquid after magnetic separation, and washing for 3 times; mixing the sealed immunomagnetic beads with quantitative bacteria, and incubating; magnetically separating the incubated mixed liquid into a supernatant and immunomagnetic beads, and washing the immunomagnetic beads for 2 times by using a washing solution; and respectively diluting the supernatant and the immunomagnetic beads, and then coating the diluted supernatant and the diluted immunomagnetic beads on a plate, and calculating the enrichment efficiency.

Further, the washing solution was a morpholine ethanesulfonic acid Tween solution monohydrate (MEST, pH6.0;0.05% Tween-20) at pH 0.01 mol/L; the activating liquid is a 5mg/mL carbodiimide (EDC) solution and an N-hydroxysuccinimide (NHS) solution which are respectively prepared from a 0.01mol/L morpholine ethanesulfonic acid monohydrate solution (MES, PH 6.0); the blocking solution was PBST (pH 7.4, 1% BSA).

Further, the dosage of the magnetic beads and the dosage of the antibodies are respectively 2mg and 200 mu g; the activation time is 0.5h, the coupling time is 3h, and the sealing time is 1h.

Further, the immunomagnetic bead-enriched bacteria were incubated at 37 ℃ and 80rpm for 1 hour.

Further, the analysis result means that the enrichment efficiency = (total number of colonies after magnetic separation/total number of colonies before magnetic separation) × 100% was calculated.

The detection antibody and the detection method for salmonella provided by the invention have the following beneficial effects:

the Outer Membrane Protein (OMP) of Salmonella is one of the important components of the Salmonella antigens, and has many important functions as flagella and pili. The salmonella outer membrane protein is an antigen which can stimulate an organism to generate stronger humoral immune response and higher antibody level, and plays an important role as a virulence factor in the pathogenic process of bacteria. The PhoN protein is an outer membrane protein of salmonella, is a non-specific acid phosphatase, can stimulate an organism to generate humoral immunity and cellular immunity, and has good immunogenicity. More importantly, phoN exists in most serotypes of Salmonella, but does not exist in bacteria of non-Salmonella enterobacteriaceae such as Escherichia coli, and shows application potential as a Salmonella detection target.

The invention relates to an immunomagnetic bead detection method based on a PhoN monoclonal antibody of salmonella. Compared with the prior art, the salmonella PhoN protein antibody and the immunomagnetic bead detection method thereof have better broad-spectrum property and specificity, shorten the detection time, enrich at least two kinds of salmonella simultaneously, simplify the detection steps, shorten the detection time and provide a reliable sample pretreatment technology for quickly and efficiently detecting various salmonella serotypes. Therefore, the invention provides a sensitive and rapid detection method for detecting the salmonella antibody and an important detection means for the prevention, control and purification of the salmonella.

Drawings

FIG. 1 shows the identification of the reactivity of monoclonal antibody 4B2 with antigenic protein;

FIG. 2 shows the identification of the reactivity of monoclonal antibody 4B2 with different proteins;

FIG. 3 shows the specific identification of monoclonal antibody 4B2 and Salmonella;

FIG. 4 shows the result of enrichment of Salmonella by monoclonal antibody-coupled magnetic beads;

FIG. 5 shows the enrichment of Salmonella and other bacteria with immunomagnetic beads.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention.

Example 1: obtaining of hybridoma cell lines

1.1 animal immunization

The specific immunization program was as follows: fully emulsifying the salmonella PhoN protein and Freund's complete adjuvant, and then carrying out primary immunization on mice, wherein each mouse is immunized at 50 mu g, and abdominal subcutaneous multipoint injection is adopted; after 14 days, a second immunization, 50. Mu.g/mouse, was performed by abdominal subcutaneous multiple injections after sufficient emulsification of PhoN protein with Freund's incomplete adjuvant. After 7 days of secondary immunization, the mice were bled to detect serum antibody titers. After 14 days of secondary immunization, the mice were injected intraperitoneally with PhoN protein without adjuvant for boosting.

1.3 cell fusion

After 3 days of boosting, the mice were bled and serum was collected and stored at-20 ℃ for positive control for subsequent screening. Mice were sacrificed according to biosafety method, alcohol soaked for sterilization, splenocytes taken and myeloma cells SP2/0 in logarithmic growth phase were fused under the action of polyethylene glycol PEG 50% in the proportion of 1:5 cells. ICR mouse abdominal cavity macrophage is used as a feeder cell, the fused cell and the feeder cell are suspended, mixed evenly and paved in a 96-well plate by using HAT culture medium, and the 96-well plate is placed in a 37 ℃ cell culture box for culture. HAT medium was added after 5 days, and HT medium was used after 9 days.

1.4 establishment of Indirect ELISA detection method and screening of Positive clones

Positive cell clones were screened by indirect ELISA. The specific method comprises the following steps: coating an ELISA plate with the optimal antigen coating concentration determined according to a square matrix test, wherein the concentration is 100 mu L/hole, and the temperature is kept overnight at 4 ℃; PBST was washed 3 times, added with 1% BSA in PBS blocking solution, 200. Mu.L per well, and incubated at 37 ℃ for 2h; after the sealing is finished, PBST is washed for 3 times, hybridoma cell supernatant is added, SP2/0 cell supernatant is used as negative control, immune mouse polyclonal antiserum is used as positive control, 100 mu L/hole is carried out, and water bath at 37 ℃ is carried out for 2h; PBST is washed for 5 times, horse radish peroxidase HRP marked goat anti-mouse IgG with working concentration is added, 100 mu L/hole is formed, and water bath is carried out for 1h at 37 ℃; washing for 7 times, adding TMB for developing for 5min, and detecting OD with enzyme-labeling instrument after the development is terminated ₄₅₀ The values, the experimental results were determined according to the following formula: OD ₄₅₀ Cell pore/OD ₄₅₀ A negative Kong 2.1.1 was judged as a positive well. The positive clone selected was named 4B2.

1.6 cloning of Positive hybridoma cells

The selected positive cell clone 4B2 was subcloned 3 times by limiting dilution and stored.

Example 2: preparation of salmonella PhoN protein monoclonal antibody

2.1 preparation and purification of ascites

Adopts a method of inducing ascites in vivo. 9-12 week-old healthy BALB/c mice are injected with 0.5 mL/mouse and 7-1 mouse of liquid paraffin in abdominal cavityAfter 0 days, hybridoma cells 4B2,2 × 10, diluted with PBS and cultured to logarithmic phase growth, were each inoculated intraperitoneally ⁵ One cell/one; after 7-10 days, ascites were collected, the supernatant was collected by centrifugation, and the cells were stored at-70 ℃.

The prepared ascites fluid was purified by Protein A affinity chromatography and stored at-70 ℃.

Example 3: detection of monoclonal antibody characteristics

3.1 characterization of monoclonal antibody subclasses

And (4) identifying the monoclonal antibody subclasses by using a monoclonal antibody subclass kit. Respectively adding hybridoma cell culture supernatant into an ELISA (enzyme-Linked immuno sorbent assay) ELISA plate coated with an antigen in advance, incubating at the temperature of 37 ℃ for 2 hours at a concentration of 100 mu L/hole; PBST washing 3 times; adding 100 μ L/well of goat anti-mouse IgA, igG1, igG2a, igG2b, igG3 and IgM diluted with PBS at 1; adding HRP-rabbit anti-sheep IgG enzyme-labeled antibody diluted by PBS 1; PBST washing 5 times; adding TMB color development solution at a concentration of 100 μ L/hole, and standing at room temperature for 5min; 2M H was added ₂ S0 ₄ The reaction was stopped, 50. Mu.L/well, OD was measured with a microplate reader ₄₅₀ According to OD ₄₅₀ And determining the subclass of the monoclonal antibody.

The results showed that the subclass of mAb 4B2 was IgG2a.

The identification result shows that the amino acid sequence of the complementarity determining region 1 (CDR 1) of the monoclonal antibody light chain variable region is shown in SEQ ID NO.1, and specifically comprises the following steps: KASDHIHNWLA.

The amino acid sequence of the complementarity determining region 2 (CDR 2) of the light chain variable region is shown in SEQ ID NO.2, and specifically comprises the following steps: GATSLET.

The amino acid sequence of the complementarity determining region 3 (CDR 3) of the light chain variable region is shown in SEQ ID NO.3, and specifically comprises the following steps: QQYWTYT.

The amino acid sequence of the complementarity determining region 1 (CDR 1) of the heavy chain variable region is shown in SEQ ID NO.4, and specifically comprises the following steps: SYWIH.

The amino acid sequence of the complementarity determining region 2 (CDR 2) of the heavy chain variable region is shown as SEQ ID NO.5, and specifically comprises the following steps: SIYPGNSETSYNQKFKG.

The amino acid sequence of the complementarity determining region 3 (CDR 3) of the heavy chain variable region is shown as SEQ ID NO.6, and specifically comprises the following steps: GGYGNYLSPFDY.

The full-length amino acid sequence of the light chain is shown as SEQ ID NO. 7:

MKFPSQLLLFLLFRITGIICDIQMTQSSSYLSVSLGGRVTITCKASDHIHNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWTTYTFGGGTKLEIK。

the nucleotide sequence of the coding light chain is shown as SEQ ID NO. 8:

ATGAAGTTTCCTTCTCAACTTCTGCTCTTCCTGCTGTTCAGAATCACAGGCATAATATGTGACATCCAGATGACACAATCTTCATCCTACTTGTCTGTATCTCTAGGAGGCAGAGTCACCATTACTTGCAAGGCAAGTGACCACATTCATAATTGGTTAGCCTGGTATCAGCAGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAACCAGTTTGGAAACTGGGGTTCCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGACTACGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。

the full-length amino acid sequence of the heavy chain is shown as SEQ ID NO. 9:

MECNWILPFILSVISGVYSDVQLQQSGTVLARPGASVKMSCKASGYSFTSYWIHWVKQRPGQGLEWVGSIYPGNSETSYNQKFKGKAKLTAVTSASTAYMELSSLTNEDSAVYYCTRGGYGNYLSPFDYWGQGTTLTVSS。

the nucleotide sequence of the coding heavy chain is shown as SEQ ID NO. 10:

ATGGAATGTAACTGGATACTTCCTTTTATTCTGTCGGTAATTTCAGGGGTCTACTCAGATGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCCGTGAAGATGTCCTGCAAGGCTTCTGGCTACAGCTTTACCAGCTACTGGATACACTGGGTAAAACAGAGGCCTGGACAGGGTCTAGAGTGGGTTGGTTCTATTTATCCTGGAAATAGTGAGACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAGTCACATCCGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGAGGGGGGTATGGTAACTACCTCTCCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。

3.2 measurement of ascites titer of monoclonal antibody

Coating the PhoN protein on 96-well ELISA plates at the optimal coating concentration using coating buffer overnight at 4 ℃; washing for 3 times, adding 200 mu L of confining liquid into each hole, and incubating for 2h at 37 ℃; washing for 3 times by using PBST, adding diluted monoclonal antibody ascites in a multiple ratio, diluting SP2/0 ascites in the same multiple as a negative control, and incubating for 2h at 37 ℃; PBST washing 5 times; adding HRP-goat anti-mouse IgG with working concentration, 100 mu L/hole, and incubating for 1h at 37 ℃; PBST WashAfter washing, adding TMB color development liquid for color development, 100 mu L/hole, and incubating for 5min at 37 ℃; 2M H was added ₂ SO ₄ Stopping the reaction, and detecting OD by an enzyme-linked immunosorbent assay ₄₅₀ And (4) measuring the titer of the monoclonal antibody ascites by taking the P/N more than or equal to 2.1 as a judgment standard.

The results show that the potency of mab 4B2 is 1. The monoclonal antibody 4B2 has high titer, which indicates that the established detection method can generate high sensitivity.

3.3 monoclonal antibody immunoreactivity identification

And analyzing the specificity of the monoclonal antibody by adopting an established indirect ELISA method. Coating the purified His-PhoN, GST-PhoN, his-PdgL, his-OmpC, his-OmpF recombinant protein and pCold I empty carrier lysate with the same concentration to an ELISA plate, performing PBS control, and refrigerating at 4 ℃ overnight; on the next day, PBST is washed for 3 times, 3-5 min/time, and residual liquid is patted dry as much as possible; blocking with 200. Mu.L of 1% BSA in PBST per well, 2h at 37 ℃; diluting ascites, adding the diluted ascites into an ELISA plate which is sealed, carrying out water bath at 37 ℃ for 2h, wherein each well is 100 mu L; PBST wash 5 times; adding HRP-goat anti-mouse IgG antibody with working concentration, 10 mu L/hole, and water bath at 37 ℃ for 1h; PBST is washed for 7 times, TMB color development liquid is added in the dark for color development, each hole is 100 mu L, and the action is carried out for 10min at 37 ℃; after the color development is finished, adding sulfuric acid stop solution (2M H) ₂ SO ₄ ) The termination was performed at 50. Mu.L per well, OD was performed with a microplate reader ₄₅₀ Reading the value and analyzing the reactivity of the monoclonal antibody.

The results (FIG. 2) show that the monoclonal antibody 4B2 can specifically recognize His-PhoN and GST-PhoN recombinant proteins, and has no reaction with other proteins.

3.4 monoclonal antibody specificity assay

Adding appropriate amount of 5 XProtein Loading buffer into the purified His-phoN, GST-phoN recombinant Protein and BL21 (DE 3) -pCold I and BL21 (DE 3) -pGEX-6P-1 recombinant bacteria contrast respectively to carry out boiling water bath for 10min, and carrying out SDS-PAGE gel electrophoresis at 120V; salmonella typhimurium (S.typhimurium), salmonella enteritidis (S.enteritidis), salmonella delavata (S.derby), salmonella infantis (S.infantis), salmonella london (S.london), salmonella indiana (S.indiana), salmonella lavalis (S.corvallis), salmonella reyneana (S.rissen), salmonella typhimurium 8978 (S.Fairfield) Escherichia coli (E.coli), shigella sonnei (Sh.sonnei), listeria monocytogenes (L.monocytogenes), campylobacter jejuni (C.jejuni) and the like are cultured, treated by ultrasonic disruption, and subjected to SDS-10min in water bath electrophoresis by adding a proper amount of 5 × Protein Loading buffer, respectively.

Using Pyxis ^TM Gel Processor rapid transfer printing instrument and matched Pyxis thereof ^TM Protein Transfer Stack kit, transferred for 8min, blocked with 5% BSA in PBST for 1h at room temperature; PBST was washed 5 times, and ascites from mice diluted with 5% BSA in PBST solution 1; PBST washing 5 times, adding containing 5% BSA PBST solution 1 diluted with HRP-goat anti mouse IgG antibody, room temperature shaking table incubation for 1h; and (3) washing the PBST for 5 times, uniformly mixing the solution A and the solution B1:1 of the TMB two-component developing solution, developing for 1min in a dark place, stopping the developing reaction, and scanning and taking a picture by using an ultrasensitive multifunctional imager.

The results (FIG. 2) show that the monoclonal antibody 4B2 reacts well with His-PhoN and GST-PhoN recombinant proteins in the Western Blot assay.

The results (FIG. 3) show that monoclonal antibody 4B2 and 9 strains of Salmonella all reacted in the Western Blot test, and did not react with other species of bacteria, indicating that the monoclonal antibody secreted by the hybridoma cell line has good specificity.

Example 4: preparation and application of salmonella immune enrichment magnetic bead

4.1 activated PM3-050 Carboxylic beads

The vial was shaken up and 200uL of PM3-050 magnetic carboxyl beads (2 mg) were transferred to a 1.5mL centrifuge tube. Each of the cells was washed 3 times with 500. Mu.L of 0.01mol/L morpholine ethanesulfonic acid Tween solution (MEST, pH6.0;0.05% Tween-20), and the supernatant was separated by a magnetic holder and aspirated. Respectively preparing 5mg/mL carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution by MES stored at 4 ℃, respectively adding 200 mu L newly prepared EDC and NHS solution into magnetic beads, uniformly mixing by vortex to fully suspend the magnetic beads, placing the magnetic beads on a mixing instrument to keep the magnetic beads suspended, activating for 30min at 37 ℃, placing the centrifuge tube on a magnetic rack to carry out magnetic separation, discarding the supernatant, adding 500 mu L MEST, washing once, and discarding the supernatant; the beads were transferred to a new 1.5mL centrifuge tube, 500. Mu.L MEST was added, and the tube was washed 3 times to remove the supernatant as clean as possible. After the steps, the carboxyl on the surface of the PM3-050 magnetic bead is activated, and covalent coupling can be carried out on the biological ligand with primary amino.

4.2 coupling of activated magnetic beads to antibodies

Add 100. Mu.g, 200. Mu.g, 300. Mu.g of antibody to 5.1 activated beads, adjust the total volume to 500. Mu.L with MES solution, mix the beads and bioligands gently. And (3) placing the mixed magnetic beads on a rotary mixer, reversing and uniformly mixing, coupling at 37 ℃ for 3h, and fully and uniformly mixing the activated magnetic beads and the antibody suspension, wherein the activated magnetic beads are ensured to be in a suspension state in the period. The tube was placed on a magnetic separation rack for magnetic separation, the supernatant removed, 1mL of PBST (pH 7.4, 1% BSA) was added to resuspend the beads, and the mixture was blocked at 37 ℃ for 1h, during which time the beads were kept in suspension by mixing on a rotary mixer by inversion. The centrifuge tubes were placed on a magnetic separation rack for magnetic separation, the supernatant was removed and the beads were washed 3 times with 500 μ L PBST. Magnetic separation, supernatant removal, magnetic beads with PBST heavy suspension, stored at 4 degrees C.

4.3 enrichment of Salmonella by immunomagnetic beads

Adding the sealed magnetic beads into 1mL of the bacterial liquid of salmonella typhimurium, salmonella infantis, escherichia coli and shigella, fully and uniformly mixing, and then acting at 37 ℃ and 80rpm for 60min. Placing the centrifugal tube on a magnetic frame, turning for 2min, standing for 1min, respectively transferring the supernatants of the groups into new finger-shaped tubes when the magnetic beads are fully adsorbed on the tube wall, and coating 100 μ L of diluted supernatant on an LB plate; respectively diluting the bacterial liquid before and after the action of the immunomagnetic beads in a proper gradient, and coating 100 mu L of the diluted bacterial liquid on an LB (Langmuir-Blodgett) flat plate; all plates were incubated at 37 ℃ for 24h. The concentration of the bacterial suspension was calculated by a plate counting method, and the capture rate was calculated by immunomagnetic bead capture rate (%) = (total number of colonies after magnetic separation/total number of colonies before magnetic separation) × 100%.

The results (FIG. 4) show that the capture efficiency of Salmonella typhimurium and Salmonella infantis was the highest in the 200. Mu.g antibody-coupled magnetic bead assay, 79% and 77%, respectively, and therefore 200. Mu.g coupled magnetic beads were used for enrichment of Salmonella and other bacteria.

The results show (fig. 5) that in the immunomagnetic bead enrichment test of salmonella and other genus bacteria, the prepared salmonella immunomagnetic beads have 77% and 77% capture efficiency on salmonella typhimurium and salmonella infantis respectively, and 6.1% and 3.5% capture efficiency on escherichia coli and shigella, which indicates that the prepared salmonella immunomagnetic beads can specifically bind to salmonella.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

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130 135 140

<210> 10

<211> 420

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggaatgta actggatact tccttttatt ctgtcggtaa tttcaggggt ctactcagat 60

gttcagctcc agcagtctgg gactgtgctg gcaaggcctg gggcttccgt gaagatgtcc 120

tgcaaggctt ctggctacag ctttaccagc tactggatac actgggtaaa acagaggcct 180

ggacagggtc tagagtgggt tggttctatt tatcctggaa atagtgagac tagctacaac 240

cagaagttca agggcaaggc caaactgact gcagtcacat ccgccagcac tgcctacatg 300

gagctcagca gcctgacaaa tgaggactct gcggtctatt actgtacaag aggggggtat 360

ggtaactacc tctccccctt tgactactgg ggccaaggca ccactctcac agtctcctca 420

Claims

1. A salmonella PhoN protein antibody, comprising a heavy chain and a light chain, wherein the light chain comprises three complementarity determining regions comprising a light chain variable region complementarity determining region 1, a light chain variable region complementarity determining region 2 and a light chain variable region complementarity determining region 3, the amino acid sequence of the light chain variable region complementarity determining region 1 is shown in SEQ ID No.1, the amino acid sequence of the light chain variable region complementarity determining region 2 is shown in SEQ ID No.2, and the amino acid sequence of the light chain variable region complementarity determining region 3 is shown in SEQ ID No. 3; the heavy chain comprises three complementarity determining regions, including a heavy chain variable region complementarity determining region 1, a heavy chain variable region complementarity determining region 2 and a heavy chain variable region complementarity determining region 3, wherein the amino acid sequence of the heavy chain variable region complementarity determining region 1 is shown in SEQ ID NO.4, the amino acid sequence of the heavy chain variable region complementarity determining region 2 is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region complementarity determining region 3 is shown in SEQ ID NO. 6.

2. The antibody of claim 1, wherein the amino acid sequence of said light chain is set forth in SEQ ID No.7 and the amino acid sequence of said heavy chain is set forth in SEQ ID No. 9.

3. A polynucleotide encoding the antibody of any one of claims 1-2.

4. The polynucleotide of claim 3, wherein the nucleotide sequence encoding said light chain in said polynucleotide is represented by SEQ ID No.8 and the nucleotide sequence encoding said heavy chain in said polynucleotide is represented by SEQ ID No. 10.

5. A recombinant expression vector comprising the polynucleotide of claim 3 or 4.

6. A host cell comprising or incorporating the recombinant expression vector of claim 5.

7. An immunomagnetic bead assay composition prepared based on a salmonella PhoN protein, wherein the composition comprises the antibody of claim 1 or 2.

8. The composition of claim 7, wherein the test composition further comprises magnetic beads, a wash solution, an activation solution, a blocking solution; the antibody is a purified IgG antibody against the PhoN protein of Salmonella.

9. A method for immunomagnetic bead detection of antibodies to salmonella, comprising detecting salmonella using the antibody of claim 1 or 2, for non-disease diagnostic and therapeutic purposes.