CN113845589B - SmcL monoclonal antibody, hybridoma cell strain secreting antibody, application of monoclonal antibody and competitive ELISA detection method - Google Patents

Abstract

The invention provides a SmcL monoclonal antibody, a hybridoma cell strain secreting the antibody, application of the hybridoma cell strain and a competitive ELISA detection method, wherein the monoclonal antibody is secreted by the hybridoma cell strain or a subculture cell strain thereof preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2021175. The monoclonal antibody secreted by the hybridoma cell strain has the advantages of high titer, good specificity and strong affinity with natural antigen. The Listeria monocytogenes SmcL monoclonal antibody competition ELISA detection method established based on the antibody has high sensitivity, good stability and no cross reaction with other pathogenic bacteria, can effectively detect the level of the SmcL antibody in clinical serum of sheep, and can be used for epidemiological investigation and clinical immune monitoring of Listeria monocytogenes.

Description

SmcL monoclonal antibody, hybridoma cell strain secreting antibody, application of monoclonal antibody and competitive ELISA detection method

Technical Field

The invention relates to the technical field of biotechnology detection, in particular to a SmcL monoclonal antibody, a hybridoma cell strain secreting the antibody, application of the antibody and a competitive ELISA detection method.

Background

Listeria monocytogenes (Lm) and Listeria monocytogenes (Listeria ivanovii) are the main pathogenic bacteria of the genus Listeria, causing listeriosis in humans and animals. Wherein, the listeria monocytogenes mainly infects ruminants, which causes the fulminant abortion of cattle and sheep and brings huge economic loss to the breeding industry. In recent years, human gastroenteritis and septicemia caused by listeria monocytogenes have been reported, and high attention needs to be paid.

The monitoring to the listeriosis in the breeding link is strengthened, infected animals can be found in the farm stage, and measures can be taken in time, so that the diseased livestock can be prevented from entering the food industry chain, and the method has important significance to public health and safety. The "gold standard" for listeriosis diagnosis is the isolation of pathogenic bacteria. The method is long in time consumption, and due to the reasons of short existence time of viable bacteria, irregular bacterium elimination and the like, the detection result is often inaccurate. Serological detection methods are commonly used for the auxiliary diagnosis of infectious diseases, and are a supplement and support to traditional bacterial isolation and culture methods. The ELISA method has the advantages of simple and convenient operation, high speed and capability of screening large batches of samples at high flux, and is widely used in animal epidemiological investigation and clinical immune monitoring. Currently, a commercial diagnostic kit for detecting listeriosis is an indirect ELISA method based on listeriolysin O (LLO), and the kit cannot distinguish infection caused by listeriosis monocytogenes and listeriosis. To date, there have been no reports of serological detection methods for listeria monocytogenes infection.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art. Therefore, the invention provides a SmcL monoclonal antibody, a hybridoma cell strain secreting the antibody, application of the SmcL monoclonal antibody and a competitive ELISA detection method, and aims to realize effective detection of Listeria monocytogenes, and the monoclonal antibody secreted by the hybridoma cell strain has the advantages of high titer, good specificity, high affinity and strong affinity with natural antigens.

Based on the above purposes, the invention provides a hybridoma cell strain secreting listeria monocytogenes SmcL monoclonal antibody, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021175 and the preservation address of China, wuhan university, the preservation time is 2021 year, 7 month and 8 days, and the hybridoma cell strain is classified and named as: hybridoma cell line 6E10.

The invention also provides a SmcL monoclonal antibody of Listeria monocytogenes, which is secreted and generated by the hybridoma cell strain or the subculture cell strain thereof. The SmcL monoclonal antibody is used for detecting Listeria monocytogenes.

The monoclonal antibody comprises a heavy chain and a light chain, wherein the light chain comprises three complementarity determining regions of which the amino acid sequences are respectively shown as SEQ ID NO. 1-3; the heavy chain comprises three complementarity determining regions of which the amino acid sequences are respectively shown as SEQ ID NO. 4-6.

Preferably, the full-length amino acid sequence of the light chain is shown as SEQ ID NO.7, and the full-length amino acid sequence of the heavy chain is shown as SEQ ID NO. 9.

The invention also provides a polynucleotide encoding the monoclonal antibody.

Preferably, the nucleotide sequence of the light chain encoded by the polynucleotide is shown in SEQ ID NO.8, and the nucleotide sequence of the heavy chain encoded by the polynucleotide is shown in SEQ ID NO. 10.

The invention also provides application of the monoclonal antibody in a detection reagent or a kit for detecting listeria monocytogenes.

The invention also provides a kit for detecting listeria monocytogenes, which comprises an enzyme-labeled antibody, wherein the enzyme-labeled antibody is a monoclonal antibody of listeria monocytogenes SmcL which is not limited to horse radish peroxidase labeling.

Preferably, the kit further comprises coating antigen (prokaryotically expressed rHis-SmcL protein), listeria monocytogenes positive serum/negative serum, blank control, coating liquid, sealing liquid, diluent, washing liquid, 3,3',5,5' -Tetramethylbenzidine (TMB) substrate liquid, reaction and other common experimental materials which are detected by persons in the field by adopting competitive ELISA.

Furthermore, the coating antigen can be coated on the ELISA plate in advance, a blank ELISA plate and a detection antigen can also be provided, and the detection antigen is coated on the ELISA plate by an operator by a conventional method before detection.

Further, the coating solution may be a Carbonate Buffer Solution (CBS) at PH 9.6; the washing solution is a PBST solution with pH of 7.2, and concentrated or unconcentrated washing solution can be selected according to requirements; the confining liquid is PBS solution containing 2% skimmed milk powder; the blank control is PBS solution with pH 7.2; the diluent was a PBS solution containing 1% bsa; the reaction termination solution is 2mol/L H ₂ SO ₄ And (3) solution.

In general, in the kit of the present invention, each reagent is separately stored.

The invention also provides a competitive ELISA detection method of the Listeria monocytogenes SmcL monoclonal antibody, and the detection method comprises the steps of detecting Listeria monocytogenes by using the SmcL monoclonal antibody; the enzyme-labeled antibody and a natural antibody in serum compete to coat a binding site of an antigen SmcL, and then incubation and detection are carried out.

The specific steps for detecting the SmcL antibody in the serum by using the detection method provided by the invention are as follows:

1) Detecting an antigen-coated ELISA plate;

(1) diluting the antigen to be detected with a coating solution, and adding the diluted antigen to each hole of an ELISA plate;

(2) washing the ELISA plate with washing solution for 3 times, each time for 2min;

(3) adding 300 mu L of confining liquid into each hole, and incubating for 3h at 37 ℃;

(4) washing the ELISA plate with washing solution for 3 times, each time for 2min;

2) Sample incubation and detection;

(1) mixing Listeria monocytogenes negative serum and positive serum which are diluted by diluent, blank control and the enzyme-labeled antibody according to the volume ratio of 1:1, adding the mixture into each hole, incubating for 3h at 37 ℃ at 100 mu L/hole;

(2) discarding the sample reaction solution, and washing the ELISA plate with a washing solution for 6 times, each time for 2min;

(3) adding substrate solution, 100 μ L/hole, and incubating at 37 deg.C for 5min;

(4) adding stop solution, 50 μ L/well, and labeling with enzymeTo the OD _450nm Measuring the light absorption value under the wavelength; calculation of inhibition PI = (1-OD of serum sample) _450nm OD of value/blank control _450nm ) X 100%, PI > 28.61% is positive, PI ≤ 28.61% is negative.

The invention has the beneficial effects that: the monoclonal antibody secreted by the hybridoma cell strain has the advantages of high potency, good specificity and strong affinity with natural antigen. The Listeria monocytogenes SmcL monoclonal antibody competition ELISA detection method established based on the antibody has high sensitivity and good stability, has no cross reaction with other pathogenic bacteria, and can effectively detect the level of the SmcL antibody in clinical serum of sheep. The establishment of the method fills the blank of the listeria monocytogenes serological detection method, and can be used for listeria monocytogenes epidemiological investigation and clinical immune monitoring.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a ROC curve diagram of the SmcL monoclonal antibody competition ELISA method of the present invention;

FIG. 2 is a sensitivity curve diagram of the SmcL monoclonal antibody competition ELISA method of the present invention;

FIG. 3 shows the specific results of the SmcL monoclonal antibody competition ELISA method of the present invention;

FIG. 4 is a graph showing the determination of SmcL antibody titer in a clinically positive serum sample according to the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.

It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have a general meaning as understood by one having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined.

Example 1

Obtaining of hybridoma cell line 6E10

The preservation number is CCTCC NO: C2021175.

1.1 animal immunization

The specific immunization steps are as follows: female BALB/c mice 6-8 weeks old were immunized with rHis-SmcL protein. For the first immunization, rHis-SmcL protein and Freund complete adjuvant are mixed and fully emulsified according to the volume ratio of 1:1, the immunization dose is 50 mu g/mouse, and subcutaneous multi-point injection is carried out. After 14 days, the second immunization was carried out, the adjuvant used in the second immunization was Freund's incomplete adjuvant, and the rest of the procedure was identical to that of the first immunization. 7 days after the second immunization, mouse serum was collected, and the antibody titer of SmcL in the serum was measured by indirect ELISA, and the mouse with the highest antibody level was selected for boosting. Booster immunizations were performed 14 days after the second immunization, and rHis-SmcL protein was intraperitoneally injected into mice at an immunization dose of 100 μ g/mouse. On day 3 after the boost, mouse spleen lymphocytes were taken for cell fusion.

1.2 cell fusion

The method comprises the following specific steps: killing immunized mice according to biosafety method, soaking and sterilizing with 75% alcohol for 5min, aseptically taking splenocytes, fusing with myeloma cell SP2/0 in logarithmic growth phase under the action of PEG (MW 1450), using ICR mouse abdominal cavity macrophage as feeder cell, suspending the fused cells and feeder cell with HAT culture medium, subpackaging with 96-well plate, setting at 37 deg.C, 5% CO ₂ Culturing in an incubator. Adding fresh HAT culture medium after 5 days, culturing with HT culture medium after 10 days, periodically observing, changing liquid and detecting.

1.3 establishment of Indirect ELISA detection method

Positive cell clones were screened by indirect ELISA. The matrix test determines the coating concentration of the detection antigen rMBP-SmcL protein. Detecting transverse gradient dilution of antigen-coated buffer solution, coating an enzyme label plate with 100 mu L of each hole, and standing overnight at 4 ℃; PBST washing 3 times, each hole add 300 u L of confining liquid, 4 degrees C overnight; diluting immune mouse serum longitudinally by multiple, wherein each hole is 100 mu L, diluting SPF mouse serum by the same multiple to be used as a negative control, and incubating for 2h at 37 ℃; washing with PBST for the third timeAdding a working concentration of HRP-labeled goat anti-mouse IgG antibody, incubating at 37 ℃ for 1h, wherein each well is 100 mu L; after PBST washing, 100 mu L of TMB is added into each hole for developing for 5min; after addition of stop solution, the enzyme reader was used at OD _450nm And measuring the absorbance value at the wavelength, and judging the optimal coating concentration of the detection antigen.

1.4 screening Positive clones

And detecting the antibody condition secreted by the hybridoma cells by adopting a well-established indirect ELISA method. The specific method comprises the following steps: adding culture supernatant of hybridoma cells into a pre-coated enzyme label plate, performing 100 mu L/hole treatment, taking the supernatant of SP2/0 cells as negative control, taking immune multi-antiserum as positive control, and performing water bath at 37 ℃ for 2h; PBST wash 3 times; adding a working concentration of HRP-labeled goat anti-mouse IgG antibody, 100 mu L/hole, and performing water bath at 37 ℃ for 1h; after washing, TMB color development for 5min, enzyme labeling instrument for OD determination after color development termination _450nm And (6) reading. Measured hole OD _450nm More than two times of reading of the negative control is judged as positive. The selected 1 positive clone was designated as positive cell clone 6E10.

1.5 cloning of Positive hybridoma cells

The selected positive cell clone 6E10 was subcloned by limiting dilution method for 3 times and preserved. The positive cell clone 6E10 corresponds to a hybridoma cell strain with the preservation number of CCTCC NO: C2021175.

Preparation of Listeria monocytogenes SmcL monoclonal antibody

2.1 preparation of ascites

When ascites is prepared, BALB/c female mice of about 10 weeks old are selected. Liquid paraffin as an inducer is injected into the abdominal cavity of the mouse, and 500 mu L of the liquid paraffin is injected into the mouse. One week later, each mouse was injected with 5 × 10 ⁵ And (3) hybridoma cells. When the abdomen of the mouse is obviously enlarged, the ascites can be collected. Centrifuging the collected ascites at room temperature to remove precipitate, collecting supernatant, and storing at-70 deg.C. The monoclonal antibody secreted by the hybridoma cell strain or the passage cell strain (corresponding to the hybridoma cell 6E 10) with the preservation number of CCTCC NO: C2021175 is marked as the monoclonal antibody 6E10.

2.2 determination of ascites titer

Adding times into an ELISA plate coated with a detection antigenSpecific diluted monoclonal antibody ascites, each hole is 100 mu L; diluting SP2/0 ascites at the same multiple ratio as a negative control, and incubating for 2h at 37 ℃; washing with PBST for 3 times, adding HRP-labeled goat anti-mouse IgG antibody with working concentration, 100 mu L per well, and incubating at 37 ℃ for 1h; after PBST washing, TMB was added for color development, and OD was read after termination _450nm Absorbance values were measured and the experimental results were analyzed. And (3) analyzing the ascites titer of the monoclonal antibody by taking the P/N value of more than or equal to 2.1 as a judgment standard.

The results show that the ascites titer of the monoclonal antibody 6E10 reached 1.

2.3 purification and labeling of monoclonal antibodies

The prepared 6E10 ascites fluid was purified using Protein G affinity chromatography method.

And (4) carrying out horseradish peroxidase labeling on the purified 6E10 monoclonal antibody.

2.4 sequencing of hybridoma cells

The sequencing result of the hybridoma cell shows that the amino acid sequence of the complementarity determining region 1 (CDR 1) of the monoclonal antibody 6E10 light chain variable region is shown as SEQ ID NO.1, and specifically comprises the following steps: KASQDINSNLS.

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: RTNRLLD.

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 components: LQYAEFPPT.

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: the DAWMD.

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: EIRSKAKNHATYYAESVKG.

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: GFAY.

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

MRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTITCKASQDINSNLSWFQQKPGKSPKTLMYRTNRLLDGVPSRFSGSGSGQDYSLTISSLEYEEMGMYYCLQYAEFPPTFGGGTKLEIK

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

ATGAGGACCCCTGCTCAGTTTCTTGGAATCTTGTTGCTCTGGTTTCCAGGTATCAAATGTGACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGGGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCAATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATGTATCGTACAAACAGACTCTTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGAAATGGGAATGTATTATTGTCTACAGTATGCTGAGTTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

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

MYLGLNYVFIVFLLNGVQSEVKLEESGGGLVQPGGSKKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKAKNHATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCNGGFAYWGQGTLVTVSA

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

ATGTACTTGGGACTGAACTATGTATTCATAGTTTTTCTCTTAAATGGTGTCCAGAGTGAAGTAAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCAAGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGAAGCAAAGCTAAAAATCATGCAACATACTATGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAGCTTAAGAGCTGAAGACACTGGCATTTATTACTGTAACGGGGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

characterization of monoclonal antibodies

3.1 subclass identification of monoclonal antibodies

And (3) identifying the monoclonal antibody by using a commercial murine monoclonal antibody subclass kit. Adding hybridoma cell culture supernatant into the enzyme label plate coated with the detection protoprotein, and incubating for 2h at 37 ℃. Goat anti-mouse IgG1, igG2a, igG2b, igG3, igM, igA were diluted at 1. HRP-rabbit anti-sheep IgG was diluted to 1 in 5000, 100. Mu.L/well, incubated at 37 ℃ for 15min. Adding TMB to develop color, reading OD after termination _450nm Absorbance values were measured and the experimental results were analyzed.

The results showed that mAb 6E10 was of the IgG1 subclass.

3.2 specificity identification of monoclonal antibodies

The specificity of the antigen recognized by the monoclonal antibody 6E10 is identified by Western blot. And respectively taking purified rHis-SmcL, rMBP-SmcL, his tag protein and MBP tag protein to perform SDS-PAGE electrophoresis. After electrophoresis was completed, proteins were transferred onto NC membranes using a rapid transfer machine. The blocking conditions were 5% skim milk in PBST incubated for 2h at room temperature. Diluting the purified 6E10 monoclonal antibody to working concentration with 5% skimmed milk PBST, shaking and incubating for 3h at room temperature, and washing with PBST for 5 times, each for 2min. HRP-goat anti-mouse IgG1 was diluted at 8000, incubated at room temperature for 1h with shaking, and washed 6 times for 2min with PBST after completion. After ECL color development for 2min, the image was taken with a GE Amersham Imager 600 super-sensitive multifunctional Imager.

The results show that the monoclonal antibody 6E10 reacts specifically with the purified rHis-SmcL and rMBP-SmcL proteins, the band size is correct, and the monoclonal antibody does not react with both the His tag protein and the MBP tag protein.

3.3 Cross-reactivity characterization of monoclonal antibodies

Respectively extracting natural secretory proteins of Listeria monocytogenes, lm, listeria innocua, listeria selaginella, salmonella, escherichia coli and Staphylococcus aureus. Western blot was used to identify the cross-reactivity of mAb 6E10 to different strains.

The results show that the monoclonal antibody 6E10 only reacts with the natural secretory protein of Listeria monocytogenes, and has no cross reaction with other common pathogenic bacteria.

3.4 affinity assay of monoclonal antibodies

The detection original protein is respectively diluted to different concentrations and added into an enzyme label plate, and each well is 100 mu L. The purified monoclonal antibody 6E10 was diluted in a gradient of 100. Mu.L per well and incubated at 37 ℃ for 2h. HRP-goat anti-mouse IgG (1 diluted 5000) was added, 100. Mu.L per well, and incubated at 37 ℃ for 1h. And after the completion, color development is performed. The calculation formula of the antibody affinity constant (Kaff) was Kaff = (n-1)/2 (n [ Ab']－[Ab] _t ). Wherein, [ Ab']And [ Ab ]] _t Respectively represents an antigen concentration of [ Ag']And [ Ag] _t OD =1/2OD _max The corresponding antibody concentration; n is antigen [ Ag']And [ Ag] _t Dilution factor in between.

The affinity constant of the monoclonal antibody 6E10 is calculated to be 1.97 multiplied by 10 according to the formula ¹⁰ M ^-1 And belongs to high affinity antibodies.

Establishment of SmcL monoclonal antibody competition ELISA method

4.1 determination of working concentration of coating antigen and enzyme-labeled antibody

The optimal coating concentration of the antigen and the optimal dilution multiple of the enzyme-labeled antibody are determined by an ELISA chessboard method. The rHis-SmcL protein was diluted to different concentrations (1.5. Mu.g/mL, 1.25. Mu.g/mL, 1.0. Mu.g/mL and 0.75. Mu.g/mL), 100. Mu.L/well using CBS, and incubated overnight at 4 ℃. The following day 300. Mu.L of PBS containing 2% BSA was added per well and incubated at 37 ℃ for 3h. Serum samples were diluted according to 1: 10000. 1. And (3) simultaneously adding the diluted serum sample and the enzyme-labeled antibody into an enzyme-labeled plate, wherein each well contains 50 mu L of the diluted serum sample and the enzyme-labeled antibody, placing the mixture on a shaking table, uniformly mixing the mixture at room temperature for 5min, and then incubating the mixture at 37 ℃ for 2h. Adding TMB to develop color, reading OD after termination _450nm And (4) processing the absorbance value. The formula for calculating the serum inhibition (PI) is: inhibition rate = (1-detection of serum OD) _450nm Blank control OD _450nm ) X100%. The condition with the maximum positive inhibition rate/negative inhibition rate (P/N) value is selected as the optimal working concentration of the antigen and the enzyme-labeled antibody.

Comparing the inhibition rate of the negative serum and the positive serum of each group and the P/N value, and determining that the optimal coating antigen concentration of the rHis-SmcL protein is 1.25 mu g/mL, and the optimal dilution multiple of the enzyme-labeled antibody is 1 10000 (72 ng/mL).

4.2 determination of serum dilution factor

The elisa plate was coated with optimal antigen concentration. Serum samples were diluted according to 1. And calculating PI values and P/N values under each dilution factor, and selecting the condition with the maximum P/N value as the optimal dilution factor of the serum sample.

Considering the P/N value and the inhibition rate of negative and positive, 1.

4.3 determination of confining liquid

The blocking effect of different blocking solutions was investigated by setting 2% BSA, 5% BSA, 2% skim milk and 5% skim milk as blocking solutions, respectively. And comparing the PI value and the P/N value under each condition, and selecting the confining liquid under the condition of the maximum P/N value as the optimal confining liquid.

The results show that 2% skim milk is the best confining liquid.

4.4 determination of the seal time

On the basis of the optimal conditions, different closing times (2 h, 2.5h, 3h and 3.5 h) are set, and the rest of the operation is unchanged. And respectively calculating PI values and P/N values of all time points, and selecting the time point with the maximum P/N value as the optimal closing time.

The P/N value is larger and the stability is better when the material is sealed for 3 hours. The most ideal closing time is therefore 3h.

4.5 determination of incubation time of enzyme-labeled antibody

Based on the optimal conditions determined by the experiment, different enzyme-labeled antibody incubation times (0.5 h, 1.0h, 1.5h, 2.0h, 2.5h and 3 h) are set, and the rest operations are unchanged. Respectively calculating PI values and P/N values of different time points, and selecting the time point with the maximum P/N value as the optimal incubation time of the enzyme-labeled antibody.

3.0h was chosen as the optimal incubation time for the enzyme-labeled antibody.

4.6 determination of the action time of the substrate

On the basis of the determined optimal conditions, different color development times (5 min, 7min, 9min and 11 min) are set respectively, and the rest operations are unchanged. And calculating PI values and P/N values under different color development time, and selecting the time point with the maximum P/N value as the optimal action time of the substrate.

When the substrate action time is 5min, the P/N value is the largest, so 5min is selected as the optimal action time of the substrate.

4.7 determination of Cut off value

62 positive sera and 40 negative sera were tested according to the competitive ELISA reaction conditions determined in the above experiment, and PI value was calculated for each serum. The data were analyzed using the SPSS Statistics 17.0 software and receiver operating characteristic curves (ROC curves) were plotted. The value at which the you-den index (you-den index = sensitivity + specificity-1) is the largest was taken as the Cut off value for the competition ELISA.

The data were analyzed using the SPASS software and ROC curves were plotted (FIG. 1), with the diagonal being the reference line. The area under the ROC curve was examined and the results showed that the area under the curve was 0.989 and P were woven to be 0.001, with statistically significant differences, indicating that the method can be used for the detection of SmcL antibodies in serum. The optimal cut-off for the competitive ELISA was 28.61% when the Ewing index was maximal, where sensitivity and specificity were 88.7% and 100%, respectively. This indicates that the sample was judged to be positive when the inhibition rate was greater than 28.61% and negative otherwise.

Characterization of the SmcL monoclonal antibody competition ELISA method

5.1 detection Limit determination of competitive ELISA

Based on the principle of the self-inhibition rate experiment, the purified SmcL monoclonal antibody 6E10 is diluted to different concentrations (5 mu g/mL, 2.5 mu g/mL, 1.25 mu g/mL, 0.63 mu g/mL, 0.32 mu g/mL, 0.16 mu g/mL, 0.08 mu g/mL and 0.04 mu g/mL) in a gradient way and is respectively mixed with the enzyme-labeled antibody with the optimal working concentration in equal volume. And detecting samples with different concentrations according to the established competitive ELISA method, and calculating the PI value. The sensitivity curve of the competition ELISA was plotted with the sample concentration as abscissa and the PI value as ordinate as shown in FIG. 2. The lowest antibody concentration when the PI value is larger than Cut off is the detection limit of the method.

From the above results, the limit of detection of the SmcL monoclonal antibody competition ELISA method was preliminarily determined to be 0.16. Mu.g/mL.

5.2 specific assay for competitive ELISA

According to the established competitive ELISA method, positive serum of Lm, escherichia coli, campylobacter, mycobacterium tuberculosis, salmonella enteritidis, salmonella typhimurium and influenza A virus H5N1 is respectively detected, and positive serum and negative serum contrast are established at the same time. Calculating the PI value of each serum sample, comparing with the Cut off of the method, and judging whether cross reaction exists.

As shown in FIG. 3, the competitive ELISA method showed good specificity and no cross-reaction with other common pathogenic bacteria or virus-positive sera.

5.3 competitive ELISA reproducibility test

The reproducibility of the SmcL monoclonal antibody competition ELISA method was examined. For the in-batch repeatability experiment, the same batch of antigen-coated elisa plates are used, 8 sheep serum samples are detected at three time points respectively, experimental data are counted, and the Coefficient of Variation (CV) of each sample in three experiments is analyzed. For the batch-to-batch repeatability experiment, three batches of antigens are respectively prepared, 8 sheep serum samples are detected by using enzyme label plates coated with different batches of antigens under the identical experiment conditions, the experiment data is counted, and the CV value is analyzed.

8 sheep serum samples with different antibody levels were randomly selected for the in-and between-batch reproducibility experiments. The experimental data were statistically analyzed and the results are shown in table 1. The competition ELISA method has the intra-batch variation coefficient of 2.99-13.49 percent and the inter-batch variation coefficient of 3.31-11.30 percent which are all lower than 15 percent. The results show that the SmcL monoclonal antibody competition ELISA has small variation degree and good repeatability.

TABLE 1 competitive ELISA repeatability test results

Detection of sheep serum samples

A competitive ELISA method established by the research is utilized to detect 73 serum samples of 5 sheep farms separating Listeria monocytogenes in epidemiological investigation, and the antibody level of SmcL protein in the sheep serum is analyzed. Referring to the experimental conditions of the indirect ELISA method in example 1, 7 clinical serum samples positive in detection result were randomly selected and their SmcL antibody titers were measured.

73 serum samples from 5 sheep farms are detected by the method, and the result shows that SmcL antibodies in 30 serum samples are positive, 43 serum antibodies are negative, and the positive rate reaches 41.10%. 7 positive samples are randomly selected for titer analysis, the result is shown in figure 4, and the SmcL antibody titer in serum is more than 1.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Sequence listing

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Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp

35 40 45

Ile Asn Ser Asn Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro

50 55 60

Lys Thr Leu Met Tyr Arg Thr Asn Arg Leu Leu Asp Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser

85 90 95

Ser Leu Glu Tyr Glu Glu Met Gly Met Tyr Tyr Cys Leu Gln Tyr Ala

100 105 110

Glu Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 8

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgaggaccc ctgctcagtt tcttggaatc ttgttgctct ggtttccagg tatcaaatgt 60

gacatcaaga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagggtcact 120

atcacttgca aggcgagtca ggacattaat agcaatttaa gctggttcca gcagaaacca 180

gggaaatctc ctaagaccct gatgtatcgt acaaacagac tcttagatgg ggtcccatca 240

aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 300

gaagaaatgg gaatgtatta ttgtctacag tatgctgagt ttcctccgac gttcggtgga 360

ggcaccaagc tggaaatcaa a 381

<210> 9

<211> 134

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly

1 5 10 15

Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Lys Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Lys Asn His Ala Thr Tyr

65 70 75 80

Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Gly Ile Tyr Tyr Cys Asn Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ala

130

<210> 10

<211> 402

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgtacttgg gactgaacta tgtattcata gtttttctct taaatggtgt ccagagtgaa 60

gtaaagcttg aggagtctgg aggaggcttg gtgcaacctg gaggatccaa gaaactctct 120

tgtgctgcct ctggattcac ttttagtgac gcctggatgg actgggtccg ccagtctcca 180

gagaaggggc ttgagtgggt tgctgaaatt agaagcaaag ctaaaaatca tgcaacatac 240

tatgctgagt ctgtgaaagg gaggttcacc atctcaagag atgattccaa aagtagtgtc 300

tacctgcaaa tgaacagctt aagagctgaa gacactggca tttattactg taacgggggg 360

tttgcttact ggggccaagg gactctggtc actgtctctg ca 402

Claims (8)

1. A hybridoma cell strain secreting Listeria monocytogenes SmcL monoclonal antibody is characterized in that the preservation number of the hybridoma cell strain is CCTCC NO: C2021175.

2. A Listeria monocytogenes SmcL monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. The listeria monocytogenes SmcL monoclonal antibody of claim 2, wherein the monoclonal antibody comprises a heavy chain and a light chain comprising three complementarity determining regions having amino acid sequences set forth in SEQ ID nos. 1-3, respectively; the heavy chain comprises three complementarity determining regions of which the amino acid sequences are respectively shown as SEQ ID NO. 4-6.

4. The listeria monocytogenes SmcL monoclonal antibody of claim 3, wherein the full-length amino acid sequence of the light chain is set forth as SEQ ID No.7 and the full-length amino acid sequence of the heavy chain is set forth as SEQ ID No. 9.

5. A polynucleotide encoding the monoclonal antibody of any one of claims 2-4.

6. The polynucleotide of claim 5, wherein the nucleotide sequence encoding the light chain of the polynucleotide is represented by SEQ ID No.8 and the nucleotide sequence encoding the heavy chain of the polynucleotide is represented by SEQ ID No. 10.

7. A kit for detecting Listeria monocytogenes, which is characterized by comprising an enzyme-labeled antibody, wherein the enzyme-labeled antibody is a horseradish peroxidase-labeled Listeria monocytogenes SmcL monoclonal antibody, and the Listeria monocytogenes SmcL monoclonal antibody is secreted and generated by the hybridoma cell strain of claim 1.

8. The kit for detecting listeria monocytogenes of claim 7, further comprising one or more of a coating antigen, listeria monocytogenes positive and/or negative serum, a coating solution, a blocking solution, a diluent, a wash solution, a substrate solution, or a reaction stop solution.

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