CN112250766A

CN112250766A - Anti-vancomycin monoclonal antibody and application thereof

Info

Publication number: CN112250766A
Application number: CN202010896337.0A
Authority: CN
Inventors: 李春生; 李玉静; 刘鹏茹; 贾春旭; 刘静静; 张静; 吴萌
Original assignee: Institute of Biology of Hebei Academy of Sciences
Current assignee: Institute of Biology of Hebei Academy of Sciences
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2021-01-22

Abstract

The invention relates to an anti-vancomycin monoclonal antibody and application thereof, and belongs to the technical field of immunology and veterinary drug residue analysis. The anti-vancomycin monoclonal antibody is secreted and generated by a hybridoma cell strain with the preservation number of CCTCC NO: C2020117, the titer of the antibody is 1:256000, and the subtype is IgG₁Affinity constant Ka 4.23X 10⁸L/mol, inhibition ratio IC to vancomycin₅₀1.14. mu.g/L, no cross-reaction to other structural analogues. The monoclonal antibody can be used for preparing an enzyme linked immunosorbent assay kit and a colloidal gold test chromatography test strip for detecting vancomycin so as to achieve the aim of quickly and sensitively detecting vancomycin drug residues in animal derived food.

Description

Anti-vancomycin monoclonal antibody and application thereof

Technical Field

The invention relates to an anti-vancomycin monoclonal antibody, a hybridoma cell strain for generating the monoclonal antibody and application of the antibody in detection of vancomycin. Belongs to the technical field of immunology and veterinary drug residue analysis.

Background

Vancomycin (VCM) belongs to glycopeptide antibiotics, and the molecular formula is C₆₆H₇₅C_l2N₉O₂₄The product has weak alkalinity, is easy to dissolve in water, is slightly soluble in methanol, and is insoluble in organic reagents such as acetone, butanol, diethyl ether and the like. Vancomycin can inhibit the growth and reproduction of bacteria, has strong antibacterial effect on various gram-positive cocci and bacilli, has good effect on treating super bacteria such as super germs, and is the best known antibiotic at present, namely the so-called first-line medicament. The use of the drugs is strictly regulated in China, but some lawbreakers use the strong antibacterial action of vancomycin and begin to abuse in the medical field and the animal husbandry, vancomycin drugs are excessively used to enable the bacteria capable of resisting the vancomycin to grow continuously, bacteria capable of resisting the vancomycin, such as vancomycin drug-resistant enterococcus (VRE), are generated at present, the appearance of super bacteria and the hidden danger of infectious disease prevention and treatment are caused, and the residual drugs in animal bodies can generate secondary harm after being eaten by human beings. In view of this, vancomycin has been banned from 1997 in livestock breeding in the united states. Vancomycin is specified as a forbidden veterinary drug in a publication No. 560 of veterinary drug local standard disuse catalog of Ministry of agriculture of China in 2005 and 10 months, production and operation are forbidden, and a method for detecting vancomycin is proposed to be established. Vancomycin was listed in list of non-edible substances that may be illegally added to food and food additives that are easily abusable in food (fourth lot) by the expert group of the national food safety work offices in 2010. Aiming at the current situation, the establishment of a method for rapidly detecting the residual quantity of glycopeptide antibiotics such as vancomycin in animal derived food is urgently needed

At home and abroad, the research on the content determination of the glycopeptide antibiotics such as vancomycin and the like is mostly concentrated in the field of medicine and health, research objects relate to serum, plasma, urine and pharmaceutical preparations, and the detection method mainly comprises instrument methods such as High Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS) and the like and an immunological rapid detection method, but the reports on the analysis method of the vancomycin in animal-derived food are less. The instrument and the method are sensitive and accurate, but the operation is complicated, the requirements on experimental equipment and technology are high, and the instrument and the method are not suitable for rapid detection of large-batch samples. The immunoassay method comprises enzyme-linked immunosorbent assay (ELISA) and colloidal Gold Immunochromatography (GICA), has the characteristics of high sensitivity, good specificity, low cost, convenient and quick operation and the like, and is suitable for screening large-batch samples. Therefore, the preparation of the anti-vancomycin monoclonal antibody and the establishment of the rapid detection method of vancomycin immunity in animal derived food have important significance for promoting the health development of food industry and ensuring the human body health.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide an anti-vancomycin monoclonal antibody, wherein the monoclonal antibody has specificity to vancomycin drugs and is generated by a hybridoma cell strain VCM-3A10 with the preservation number of CCTCC NO: C2020117. Furthermore, the monoclonal antibody is applied to detecting vancomycin drug residues in animal derived food.

The technical problem of the invention is realized by the following technical scheme.

An anti-vancomycin monoclonal antibody is generated by a hybridoma cell strain with the preservation number of CCTCC NO: C2020117. The hybridoma cell strain is named as a hybridoma cell strain AOZ-3A10, and is preserved in China center for type culture Collection with the address: wuhan, Wuhan university, China, with the preservation number of CCTCC NO: C2020117, and the preservation date of 2020, 8 and 28 days.

The titer of the anti-vancomycin monoclonal antibody is 1:256000, and the subtype is IgG₁Affinity constant Ka 4.23X 10⁸L/mol, p-vancomycinInhibition ratio IC₅₀1.14 mu g/L, has 100.00 percent of cross reaction rate with vancomycin and has no cross reaction with other analogues (bacitracin A, virginiamycin, colistin and the like);

the anti-vancomycin monoclonal antibody is applied to preparing a non-diagnosis-purpose detection product for detecting vancomycin in a biological sample.

In the application, the non-diagnostic detection product is an enzyme linked immunosorbent assay kit or a colloidal gold chromatography test strip.

Further, an enzyme-linked immunoassay kit for detecting vancomycin, which comprises the vancomycin-resistant monoclonal antibody of

claim

1 or 2.

A colloidal gold chromatography test strip for detecting vancomycin, which comprises the vancomycin-resistant monoclonal antibody of

claim

1 or 2.

A method for preparing the anti-vancomycin monoclonal antibody comprises the following steps:

1. the preparation method of the vancomycin artificial antigen comprises the following steps

(1) Preparation of haptens

Weighing 25mg of vancomycin and 20mg of succinic anhydride, dissolving with 0.6mL of dimethyl sulfoxide, adding 0.2mL of pyridine, stirring at 80 ℃ for reaction for 4h, and drying at 37 ℃ to obtain a solid serving as a hapten.

(2) Preparation of immunogens

20mg of hapten is dissolved in 2mL of N, N-dimethylformamide, 30mg of carbodiimide and 30mg of N-hydroxysuccinimide are dissolved in 0.2mL of 0.01M PBS and then added into the hapten solution, and the mixture is stirred at room temperature for 24 hours to obtain reaction liquid I. Weighing 50mg of bovine serum albumin, dissolving in 3mL of 0.01M PBS, dripping into the reaction solution I, stirring at room temperature for 24h, dialyzing with 0.01M PBS at 4 ℃ for 3d, subpackaging, and storing at-20 ℃. Obtaining immunogen VCM-BSA.

(3) Preparation of coating antigen

Ovalbumin 40mg was used instead of bovine serum albumin and the other steps were followed for immunogen preparation. Obtaining the coating antigen VCM-OVA.

2. The preparation method of the vancomycin monoclonal antibody comprises the following steps:

(1) animal immunization: selecting carrier protein as immunogen of bovine serum albumin, immunizing female Blab/c mice of 6-8 weeks old, immunizing for 1 time at interval of 2 weeks, cutting tail after three times of immunization, taking blood to determine titer and inhibition rate, selecting mice with optimal results, enhancing immunity, and preparing for fusion;

(2) cell fusion: fusing splenocytes of the mouse selected in the step (1) with SP2/O cells stored in a laboratory, measuring supernatant by an indirect ELISA method, selecting positive high holes, and subcloning the positive holes by a limiting dilution method until a hybridoma cell strain generating a single vancomycin-resistant monoclonal antibody is established;

(3) large-scale preparation of monoclonal antibodies: selecting a female Blab/c mouse with a larger individual, preparing a large amount of ascites by an in vivo induced ascites method, purifying the ascites by caprylic acid-ammonium sulfate precipitation, dividing the ascites into small tubes, and storing at-20 ℃;

3. the characteristic identification of the vancomycin monoclonal antibody comprises the following steps:

(1) potency assay

Mixing the raw materials in a ratio of 1: 40000 dilution and coating of original coating detection plate, diluting the purified monoclonal antibody at 1:200, 1:400, 1:800, … … 1:800000, adding into the hole of the enzyme label plate, adding HRP labeled goat-anti-mouse secondary antibody after reaction, and finally developing with TMB, the result shows that the titer of the purified vancomycin monoclonal antibody reaches 1 when the concentration is 1 mg/ml: 256000.

(2) subtype determination

Subtype determination was carried out using a murine monoclonal antibody subtype identification kit purchased from Sigma, and the result showed that the subtype of the vancomycin monoclonal antibody was IgG 1.

(3) Affinity assay

The result of determining the affinity constant of the vancomycin monoclonal antibody by a non-competitive enzyme-linked immunosorbent assay shows that the affinity constant Ka is 4.23 multiplied by 10⁸L/mol。

(4) Determination of inhibition Rate

The inhibition was determined by indirect competition ELISA as a percentage of absorbance B/B0% (B for A450 in different concentrations of standard and B0 forA450 of zero concentration standard solution) is ordinate, logarithm of different concentration standard solutions [ lg (VCM) ]]Inhibition curves of the antibodies are plotted on the abscissa. The result shows that the antibody has inhibiting rate IC on vancomycin₅₀It was 1.14. mu.g/L.

(5) Specific assay

The cross-reactivity of the antibody with vancomycin and its analogs (bacitracin A, virginiamycin and colistin) was determined by competition ELISA, and the antibody with vancomycin CR (%) was 100% and with its analogs CR (%) was less than 0.1%.

The monoclonal antibody provided by the invention can be applied to detecting vancomycin in a sample, and is mainly applied to preparing an enzyme linked immunosorbent assay kit and colloidal gold test strip paper for vancomycin detection.

The quality of the anti-vancomycin monoclonal antibody provided by the invention has strong controllability and repeatability, and provides experimental basis for further establishment and application of a specific and sensitive immunoassay method through preliminary analysis and identification of the characteristics of the obtained antibody.

Drawings

FIG. 1 ultraviolet scanning identification chart of vancomycin artificial immune antigen

FIG. 2 gel electrophoresis identification chart of vancomycin artificial immune antigen

FIG. 3 is a diagram showing the subtype determination of the vancomycin monoclonal antibody of the present invention

FIG. 4 is a graph showing the determination of the affinity of the vancomycin monoclonal antibody of the present invention

FIG. 5 is a graph showing the measurement of the inhibitory rate of the vancomycin monoclonal antibody of the present invention

The hybridoma cell strain for producing the vancomycin-resistant monoclonal antibody is named as a hybridoma cell strain AOZ-3A10, and is preserved in China center for type culture Collection with the address: wuhan, Wuhan university, China, with the preservation number of CCTCC NO: C2020117, and the preservation date of 2020, 8 and 28 days.

Detailed Description

The present invention is further described in detail with reference to the following specific embodiments, which are not intended to limit the scope of the present invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example one preparation and identification of the vancomycin artificial antigen

1. Preparation of vancomycin artificial antigen

(1) Preparation of vancomycin hapten

Weighing 25mg vancomycin and 20mg succinic anhydride, adding 0.2mL pyridine and 0.6mL dimethyl sulfoxide (DMSO), stirring at 80 ℃ for reaction for 4h, drying the solvent at 37 ℃, and taking the solid as hapten.

(2) Preparation of vancomycin immunogens

20mg of hapten is dissolved in 2mL of N, N-dimethylformamide, 30mg of carbodiimide and 30mg of N-hydroxysuccinimide are dissolved in 0.2mL of 0.01M PBS and then added into the hapten solution, and the mixture is stirred at room temperature for 24 hours to obtain reaction liquid I. Weighing 50mg of bovine serum albumin, dissolving in 3mL of 0.01M PBS, dripping into the reaction solution I, stirring at room temperature for 24h, dialyzing with 0.01M PBS at 4 ℃ for 3d, subpackaging, and storing at-20 ℃.

(3) Preparation of vancomycin coatingen

20mg of hapten is dissolved in 2mL of N, N-dimethylformamide, 30mg of carbodiimide and 30mg of N-hydroxysuccinimide are dissolved in 0.2mL of 0.01M PBS and then added into the hapten solution, and the mixture is stirred at room temperature for 24 hours to obtain reaction liquid I. Weighing ovalbumin 40mg, dissolving in 3mL of 0.01M PBS, dripping into the reaction solution I, stirring at room temperature for 24h, dialyzing with 0.01M PBS at 4 ℃ for 3d, subpackaging, and storing at-20 ℃.

2. Identification of vancomycin artificial antigen

(1) Ultraviolet spectrophotometry method for identification

Vancomycin and BSA were diluted appropriately with PBS, the protein concentration of BSA was determined, VCM-BSA was diluted to the same concentration as BSA, and UV scanning was performed at a wavelength of 220-700 nm. The result is shown in figure 1, the characteristic absorption peak of BSA is 278nm, the characteristic absorption peak of VCM is 282nm, the absorption peaks of the synthesized products of the BSA and the VCM are 278-280 nm, and the peak shapes are closer to those of BSA, so that the successful synthesis of immunogen VCM-BSA is judged.

(2) SDS-PAGE identification

Preparing 12% separation gel and 5% concentration gel according to a conventional SDS-PAGE method, and after the voltage is 30V and a protein band enters the separation gel, adjusting the voltage to 90V to continue electrophoresis for about 2 hours. Dyeing and decoloring. The results are shown in FIG. 2, the coupling product has obvious difference with BSA, bandwidth and tail, and the success of VCM-BSA coupling is judged.

EXAMPLE two preparation of the vancomycin monoclonal antibody of the present invention

1. Animal immunization: immunizing a female Blab/c mouse with 6-8 weeks old by using immunogen with bovine serum albumin as carrier protein, immunizing for 1 time at intervals of 2 weeks, wherein the immunization process is shown in table 1, performing tail breaking and blood drawing after three-immunization to determine titer and inhibition rate, and selecting the mouse with the best result for fusion;

TABLE 1 immunization scheme

2. Cell fusion: fused mice are bled by eyes, serum is used as positive control, spleens are taken out under aseptic condition after cervical removal and sacrifice, splenocytes are prepared and fused with SP2/0 cells according to the proportion of 5:1 by PEG, fused cell suspension is added into a 96-well plate which is paved with feeder cells, the 96-well plate is placed at 37 ℃ and 5% CO₂Culturing in an incubator;

3. screening positive hybridoma cell strains: the fused cells were checked for contamination the next day, and HT medium was used at 10d after fusion. And (3) screening positive holes by using indirect ELISA and indirect competitive ELISA 2-3d after liquid change, selecting holes with strong positive, high inhibition rate, single clone and good cell state as much as possible, performing subcloning by a limiting dilution method, and simultaneously performing amplification culture and cryopreservation until a single monoclonal cell strain secreting vancomycin resistant is established.

4. Large-scale preparation of monoclonal antibodies: adopting a method of inducing ascites in mice, taking healthy Balb/c female mice, injecting 0.5mL of paraffin oil into each mouse, adjusting cloning positive hybridization after 7 daysTumor cell 10⁶and/mL, injecting 1mL into the abdominal cavity of each mouse, taking ascites after 7-9 days, centrifuging, removing fat, purifying by an octanoic acid-ammonium sulfate and Protein A affinity chromatography column, freeze-drying, and storing at-20 ℃.

EXAMPLE three characterization of the vancomycin monoclonal antibody of the invention

(1) Potency assay

The indirect ELISA method is adopted, and the specific steps are as follows: coating: diluting the coated antigen with carbonate buffer solution until the concentration is 2 mug/mL, and keeping the concentration of the coated antigen in a 96-well enzyme label plate at 100 mug/well for overnight at 4 ℃; washing: recovering the coated plate to room temperature, decanting off coating solution, adding 300 μ L of washing solution into each well, standing for 1min each time, washing for 3 times, and drying for the last time; and (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and keeping the temperature at 37 deg.C for 1 h; pouring off the confining liquid, washing for 3 times, and patting dry; adding a primary antibody: diluting the monoclonal antibody with washing solution at a ratio of 1:2000 times, adding 100 μ L per well, setting blank control well (PBS) and negative control (negative serum), and standing at 37 deg.C for 45 min; pouring out primary antibody, washing for 3 times, and patting dry; adding an enzyme-labeled secondary antibody: adding 100 μ L of HRP enzyme-labeled goat anti-mouse IgG diluted by 10000 times into each well, and standing at 37 deg.C for 30 min; pouring out the secondary antibody, washing for 3 times, and patting dry; color development: adding 100 mu L of substrate developing solution into each hole, and reacting for 15min at 37 ℃ in a dark place; and (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction; and (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm).

And (4) judging a result: and (A sample well-A blank)/(A negative control-A blank) is not less than 2.1, and when A450nm of the negative control is less than 0.2, the dilution factor of the antibody is the antibody titer. The titer of the anti-vancomycin monoclonal antibody 3A10 at the concentration of 1mg/ml reaches 1: 256000.

(2) subtype determination

Subtype determination was carried out using a murine monoclonal antibody subtype identification kit purchased from Sigma. The monoclonal antibody secreted by the hybridoma cell is obviously different from secondary antibodies of different subclasses in color development, wherein the A450nm value of the secondary antibody with IgG1 is the highest, the color development of the secondary antibody with IgM is weak, the secondary antibody with IgG2a, IgG2b, IgG3 and IgA hardly develops color, and the antibody type secreted by the cell is mainly IgG 1. The results are shown in FIG. 3, where the vancomycin monoclonal antibody subtype is IgG 1.

(3) Affinity assay

The affinity constant (Ka) was determined by a non-competitive ELISA method. The method comprises the following specific steps: coating: diluting the coated antigen with carbonate buffer solution until the concentration is 0.8, 0.1, 0.05 and 0.01 mu g/mL, respectively coating the 96-well enzyme label plate at 100 mu L/well, and standing overnight at 4 ℃; pouring out the coating solution, washing for 3 times, and patting dry; and (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and keeping the temperature at 37 deg.C for 1 h; pouring off the confining liquid, washing for 3 times, and patting dry; adding a monoclonal antibody: diluting the monoclonal antibody with washing solution at a ratio of 100 μ g/mL, adding 100 μ L per well, and keeping the temperature and humidity at 37 deg.C for 45 min; pouring out primary antibody, washing for 3 times, and patting dry; adding an enzyme-labeled secondary antibody: adding 100uL of goat anti-mouse IgG labeled with HRP enzyme and diluted by 10000 times into each hole, and standing at 37 ℃ for 30 min; pouring out the secondary antibody, washing for 3 times, and patting dry; color development: adding 100 mu L of substrate developing solution into each hole, and reacting for 15min at 37 ℃ in a dark place; and (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction; and (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm). Calculating Ka according to the following formula

Ka＝(n-1)/2(n Ab’-Ab)

In the formula: ab is the antibody concentration which generates a half-absorbance value when the antigen concentration is Ag; ab 'is the concentration of the antibody which generates a half-absorbance value when the antigen concentration is Ag'; n is the dilution factor between Ag and Ag', the result of determining the affinity constant of the vancomycin monoclonal antibody by the non-competitive ELISA method is shown in FIG. 5, and the affinity constant Ka is 4.23 × 10⁸L/mol。

(4) Determination of inhibition Rate

The indirect competitive ELISA method is adopted, and the specific steps are as follows: coating: diluting the coated antigen with carbonate buffer solution until the concentration is 2 mug/mL, and keeping the concentration of the coated antigen in a 96-well enzyme label plate at 100 mug/well for overnight at 4 ℃; pouring out the coating solution, washing for 3 times, and patting dry; and (3) sealing: adding 200 μ L of lotion containing 10% calf serum into each well, and keeping the temperature at 37 deg.C for 1 h; pouring off the confining liquid, washing for 3 times, and patting dry; adding monoclonal antibody and standard substance: diluting the standard mother solution with PBS to 8.1, 2.7, 0.9, 0.3, 0.1 and 0 μ g/L, adding 50 μ L of monoclonal antibody into each well, adding 50 μ L of monoclonal antibody with certain dilution times into each well, setting blank control well (PBS) and negative control (50 μ L of monoclonal antibody +50 μ L of lotion), and keeping the temperature and moisture at 37 ℃ for 45 min; pouring out primary antibody, washing for 3 times, and patting dry; adding an enzyme-labeled secondary antibody: adding 100uL of HRP enzyme-labeled goat anti-mouse IgG diluted by 10000 into each hole, and standing at 37 ℃ for 30 min; pouring out the secondary antibody, washing for 3 times, and patting dry; color development: adding 100 mu L of substrate developing solution into each hole, and reacting for 15min at 37 ℃ in a dark place; and (4) terminating: adding 50 mu L of stop solution into each hole to stop the reaction; and (3) detection: the absorbance value at a wavelength of 450nm was measured (A450 nm).

The absorbance percentage B/B0% (B represents A450 of standard solution with different concentration, B0 represents A450 of standard solution with zero concentration) is taken as the ordinate, and the logarithm of the standard solution with different concentration is taken [ lg (VCM)]Inhibition curves of the antibodies are plotted on the abscissa. The results are shown in FIG. 3, and the inhibitory rate IC of the antibody to vancomycin₅₀It was 1.14. mu.g/L.

(5) Specific assay

The Cross-reactivity of the anti-VCM monoclonal antibody with bacitracin A, virginiamycin, colistin and other substances is detected by a competitive ELISA method, and the specificity of the anti-VCM monoclonal antibody is judged according to Cross reaction rate (CR). CR ═ IC of VCM₅₀IC of each structural analog₅₀) X 100%, see table 2, the antibody with vancomycin CR (%) is 100%, and with its analogue CR (%) is less than 0.01%.

TABLE 2 VCM antibody Cross-reaction results

EXAMPLE four applications of the vancomycin monoclonal antibody of the invention

The embodiment is an application example of the monoclonal antibody 3A10 in establishing an enzyme-linked immunoassay kit for detecting vancomycin residues and a colloidal gold chromatography test strip method, and can be used for detecting vancomycin in animal tissues and milk.

1. Sample pretreatment

(1) Pretreatment of tissue (meat, liver, aquatic product) sample tissue

After tissue homogenization, accurately weighing 5.0g (accurate to 0.01g) of a sample into a 50mL centrifuge tube, adding 10mL of methanol-water solution (5:5, V/V) and 400 mu L of formic acid solution, homogenizing and extracting for 2min, vortexing, ultrasonically extracting for 30min, freezing and centrifuging for 2min at 8000r/min, transferring the supernatant into another centrifuge tube, repeatedly extracting the residues once again according to the steps, centrifuging, combining the extracting solutions, and detecting.

(2) Pretreatment of urine samples

Accurately weighing 5.0g (accurate to 0.01g) of sample into a 50mL centrifuge tube, adding 10mL of acetonitrile and 0.1mL of trichloroacetic acid (mass fraction is 8%), freezing and centrifuging at 8000r/min for 2min, transferring the supernatant into another centrifuge tube, repeatedly extracting the residue once again according to the steps, centrifuging, combining the extracting solutions, and detecting.

2. The antibody is applied to the detection of an enzyme-linked immunoassay kit method

The detection principle of the kit is an indirect competitive ELISA method.

And (3) detection: coating the vancomycin coating antigen on a micropore plate, taking out a lath as required, and placing the lath on an ELISA plate; mixing vancomycin monoclonal antibody working solution and enzyme-labeled anti-antibody working solution according to a required amount in a ratio of 5: 1; sequentially adding 50ul of a standard substance or a sample to be detected, 50ul of mixed solution of vancomycin monoclonal antibody working solution and enzyme-labeled anti-antibody working solution into the holes, lightly oscillating and uniformly mixing, and incubating for 30min in a dark place at 25 ℃; spin-drying the liquid in the holes, washing with 250 ul/hole of washing working solution (20 times diluted by deionized water) for 4 times, and drying; mixing the color development liquid A and the color development liquid B according to the proportion of 1:1, adding 100ul per hole for color development, and incubating for 15min in a dark place at 25 ℃; adding 50ul of stop solution to stop the reaction, measuring by using dual-wavelength 450nm/630nm under an enzyme-labeling instrument at 450nm, and carrying out quantification or qualitative determination according to a standard curve.

And (4) judging a result: (a) quantitative analysis: respectively calculating the average absorbance values of the standard substance and the sample to be detected, dividing the absorbance value (B) of the standard substance or the sample by the absorbance value of the standard substance of 0, and multiplying by 100% to obtain the percent absorbance value, wherein the percent absorbance value is equal to (B/B)₀) X 100%. And drawing a standard curve by taking the percent absorbance value as a vertical coordinate and the logarithm of the standard concentration as a horizontal coordinate. Substituting the percent absorbance value of the sample to be measured into the standard curve to obtain the corresponding concentration, and multiplying the corresponding concentration by the dilutionThe multiple is the actual content of the sample. (b) And (3) qualitative analysis: and comparing the average absorbance value of the sample to be tested with the absorbance value of the standard substance to obtain the concentration range of the sample to be tested, wherein the test range is 0.1ug/ml-8.1 ug/ml.

(3) Method for detecting colloidal gold chromatography test paper strip by using antibody

The reaction principle adopts a competition method to carry out semi-quantitative detection on vancomycin, vancomycin existing in a sample is firstly combined with an antibody 3A10 marked by gold particles in the process of moving upwards along a test strip, a coating antigen fixed on an NC membrane and the vancomycin compete to combine with a gold-labeled antibody at the same time, the color development strength of a T position (a detection line) is inversely proportional to the content of residual vancomycin in the sample, if no vancomycin remains in the sample, all the gold-labeled antibodies react with the coating antigen, and the T line of the test strip develops color.

And (3) detection: and taking out the VCM colloidal gold chromatography test strip, inserting the sample end into the sample solution to be detected, wherein the insertion depth is not more than the mark line, taking out the test strip in about 10-20 seconds, horizontally placing, observing for 3-5 minutes to judge the detection result, and invalidating the result after 10 minutes.

And (4) judging a result: a brownish red line is displayed at the position (quality control line) of a corresponding quality control area C on the coating film, and a brownish red line is not displayed at the position T of the detection area, which indicates that the detection result is positive, and indicates that VCM is contained in the sample to be detected; a two-day brown-red line is displayed at the position T, C on the coating film, the result is negative, and the VCM is not contained in the sample to be tested; and when the quality control area C does not show a brownish red strip, the test paper is judged to be invalid whether the detection area T shows a brownish red strip or not.

The above-described embodiments are intended to illustrate the technical idea and advantages of the invention, and the invention may also be subject to other variants, as known to the skilled person, which serve merely as illustrations of the scope of protection of the invention described above, and to the skilled person in the art who is within the scope of protection of the invention defined by the present invention there are many conventional variants and other embodiments, which are all within the scope of protection of the invention covered by the present invention.

Claims

1. The anti-vancomycin monoclonal antibody is characterized in that the monoclonal antibody is generated by a hybridoma cell strain VCM-3A10 with the preservation number of CCTCC NO: C2020117.

2. The anti-vancomycin monoclonal antibody of claim 1, wherein the antibody titer is 1:256000, subtype is IgG₁Affinity constant Ka 4.23X 10⁸L/mol, inhibition ratio IC to vancomycin₅₀1.14 mu g/L, 100.00 percent of cross reaction rate with vancomycin and no cross reaction with other similar bacitracin A, virginiamycin, colistin and the like.

3. A hybridoma cell line VCM-3a10 which produces the anti-vancomycin monoclonal antibody according to claim 1 or 2, which is deposited with the chinese type culture collection at the address: wuhan, Wuhan university, China, with the preservation number of CCTCC NO: C2020117, and the preservation date of 2020, 8 and 28 days.

4. Use of the anti-vancomycin monoclonal antibody of claim 1 or 2 for the preparation of a test product for the non-diagnostic detection of vancomycin in a biological sample.

5. The use of claim 4, wherein the non-diagnostic test product is an enzyme linked immunosorbent assay kit or a colloidal gold chromatography strip.

6. An enzyme-linked immunoassay kit for detecting vancomycin, which comprises the vancomycin-resistant monoclonal antibody of claim 1 or 2.

7. A colloidal gold chromatography test strip for detecting vancomycin, which comprises the vancomycin-resistant monoclonal antibody of claim 1 or 2.