Anti-vancomycin monoclonal antibody and application thereof

A monoclonal antibody, vancomycin technology, applied in biochemical equipment and methods, instruments, microorganisms, etc., can solve the problems of high experimental equipment and technical requirements, cumbersome operation, not suitable for rapid detection of large quantities of samples, etc. Controllable and repeatable effects

Pending Publication Date: 2021-01-22
BIOLOGY INST OF HEBEI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The instrument method is sensitive and accurate, but the operation is cumbersome and requires high experimental equipment and technology, so it is not suitable for rapid detection of large batches of samples

Method used

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  • Anti-vancomycin monoclonal antibody and application thereof
  • Anti-vancomycin monoclonal antibody and application thereof
  • Anti-vancomycin monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 Preparation and Identification of Vancomycin Artificial Antigen of the Present Invention

[0046] 1. Preparation of Vancomycin Artificial Antigen

[0047] (1) Preparation of vancomycin hapten

[0048] Weigh 25 mg of vancomycin and 20 mg of succinic anhydride, add 0.2 mL of pyridine and 0.6 mL of dimethyl sulfoxide (DMSO), stir and react at 80°C for 4 hours, dry the solvent at 37°C, and the solid is a hapten.

[0049] (2) Preparation of vancomycin immunogen

[0050] Take 20mg hapten, dissolve it in 2mL N,N-dimethylformamide, take 30mg each of carbodiimide and N-hydroxysuccinimide, dissolve it in 0.2mL 0.01M PBS, and add it to the hapten solution. Stir at room temperature for 24 h to obtain reaction solution I. Weigh 50mg of bovine serum albumin, dissolve it in 3mL of 0.01M PBS, then add it dropwise to reaction solution I, stir at room temperature for 24h, dialyze with 0.01M PBS at 4°C for 3d, aliquot and store at -20°C.

[0051] (3) Preparation of Vancom...

Embodiment 2

[0058] Example 2 Preparation of vancomycin monoclonal antibody of the present invention

[0059] 1. Animal immunization: immunize 6-8 week-old female Blab / c mice with bovine serum albumin as the carrier protein, and immunize once every 2 weeks. The immunization process is shown in Table 1. After the third immunization, the tail was docked and blood was taken for determination Titer and inhibition rate, select the mouse with the best result to prepare for fusion;

[0060] Table 1 Immunization flow chart

[0061]

[0062] 2. Cell fusion: the fused mice were bleed from the eyeballs, and the serum was used as a positive control. After the neck was killed, the spleen was taken out under aseptic conditions to prepare splenocytes, which were fused with SP2 / 0 cells by PEG at a ratio of 5:1. The final cell suspension was added to a 96-well plate already covered with feeder cells, and placed in a 37°C, 5% CO 2 cultivated in an incubator;

[0063] 3. Screening of positive hybridoma...

Embodiment 3

[0065] Embodiment three Identification of the characteristics of the vancomycin monoclonal antibody of the present invention

[0066] (1) Potency determination

[0067] The indirect ELISA method is used, and the specific steps are as follows: Coating: Dilute the coating material with carbonate buffer solution to a concentration of 2 μg / mL, 100 μL / well in a 96-well microtiter plate, overnight at 4°C; washing: return the coated plate to room temperature , pour off the coating solution, add 300 μL of washing solution to each well, let stand for 1 min each time, wash 3 times, and pat dry for the last time; sealing: add 200 μL of washing solution containing 10% calf serum to each well, 37°C for 1 hour; Remove the blocking solution, wash 3 times, and pat dry; add primary antibody: use the washing solution to dilute the monoclonal antibody at 1:2000 times, add 100 μL to each well, and set a blank control well (PBS) and a negative control (negative serum ), placed at 37°C for 45 minu...

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Abstract

The invention relates to an anti-vancomycin monoclonal antibody and application thereof, and belongs to the technical fields of immunology and veterinary drug residue analysis. The anti-vancomycin monoclonal antibody is secreted by a hybridoma cell strain with a preservation number of CCTCC NO:C2020117, has the titer of 1:256000, the subtype of IgG1, the affinity constant Ka of 4.23x10<8> L / mol and the vancomycin inhibition rate IC50 of 1.14 [mu]g / L, and has no cross reaction with other structural analogues. The monoclonal antibody can be used for preparing an enzyme linked immunosorbent assaykit and a colloidal gold chromatographic test strip for detecting vancomycin so as to achieve the purpose of rapidly and sensitively detecting vancomycin drug residues in animal-derived foods.

Description

technical field [0001] The invention relates to an anti-vancomycin monoclonal antibody, a hybridoma cell line producing the monoclonal antibody, and the application of the antibody in detecting vancomycin. The invention belongs to the technical field of immunology and the technical field of veterinary drug residue analysis. Background technique [0002] Vancomycin (VCM) is a glycopeptide antibiotic with the molecular formula C 66 h 75 C l2 N 9 o 24 , with weak alkalinity, soluble in water, slightly soluble in methanol, insoluble in acetone, butanol and ether and other organic reagents. Vancomycin can inhibit the growth and reproduction of bacteria, has a strong antibacterial effect on various Gram-positive cocci and bacilli, and has a good effect on the treatment of superbugs such as superbugs. It is the best class of antibiotics currently known , the so-called last-line drug. The country has strict regulations on the use of this type of drug, but some criminals have ...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577G01N33/543
CPCC07K16/44G01N33/577G01N33/54313C07K2317/35C07K2317/92
Inventor 李春生李玉静刘鹏茹贾春旭刘静静张静吴萌
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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