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Preparation method of antibody for bovine serum albumin and other impurities in vaccine and detection method

A technology for bovine serum albumin and antibody preparation, which is applied in the field of antibody preparation for detection of bovine serum albumin and neomycin, and achieves the effect of convenient and easy inspection method, high specificity and simple structure

Active Publication Date: 2020-10-30
ZHEJIANG INST FOR FOOD & DRUG CONTROL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, antibiotics such as neomycin may also be present in the vaccine, which is an impurity that needs to be detected

Method used

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  • Preparation method of antibody for bovine serum albumin and other impurities in vaccine and detection method
  • Preparation method of antibody for bovine serum albumin and other impurities in vaccine and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The preparation of embodiment 1 kit components

[0024] 1. Preparation of neomycin hapten

[0025] Take 680mg of neomycin sulfate, add 25ml of water to dissolve, add 6ml of 0.2mol / L potassium carbonate, stir well, add 1ml of methanol solution containing 151mg of 6-maleimido-hexanal, stir at room temperature for 8h, add borohydrogenation Sodium 55.2 mg, continue to stir for 2 hours, adjust the pH value to 7 with 6mol / L HCl, rotary evaporate to dryness, remove water, add 50ml of pyridine, fully stir to dissolve, filter, remove salt, organic phase rotary evaporate to dryness, add absolute ethanol 5 ml was recrystallized to obtain 470 mg of the hapten product maleimide-neomycin with a yield of 79.05%.

[0026] 2. Antigen preparation

[0027] Immunogen preparation—the neomycin hapten was coupled with bovine serum albumin (BSA) to obtain the immunogen.

[0028] Take bovine blood albumin (BSA) 100mg, add 0.05mol / L PB8.0 6ml to dissolve, add 7.8mg dithiothreitol (DDT), fully...

Embodiment 2

[0040] The formation of embodiment 2 ELISA kit

[0041] Set up the ELISA kit for detecting neomycin / bovine serum albumin, so that it contains the following components:

[0042] (1) Enzyme plates coated with a coating source;

[0043] (2) 6 bottles of neomycin standard solution, the concentrations are 0μg / L, 0.5μg / L, 1.5μg / L, 4.5μg / L, 13.5μg / L, 40.5μg / L; bovine serum albumin standard 6 bottles of solution, the concentrations are 0μg / L, 0.1μg / L, 0.3μg / L, 0.9μg / L, 2.7μg / L, 8.1μg / L;

[0044] (3) antibodies labeled with horseradish peroxidase;

[0045] (4) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0046] (5) The stop solution is 2mol / L sulfuric acid;

[0047] (6) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide preservative, and 0.1 to 0.3mol / L phosphate buffer, and the percentage is weight volume percentage;

Embodiment 3

[0048] The detection of analyte in embodiment 3 biological products

[0049] 1. Detection with kit

[0050] Number the corresponding microwells of the samples and standards in sequence, make 2 parallel wells for each sample and standard, and record the positions of the standard wells and sample wells. Add standard / sample 20μl-80μl per well to the corresponding microwell, then add enzyme conjugate working solution 20μl-80μl per well, shake gently to mix, cover the plate with a cover film and place it in a light-proof environment at 25°C React for 30 minutes. Dry the liquid in the wells, add 250 μl / well of washing working solution, wash thoroughly 4-5 times with an interval of 10 seconds, pour off the washing liquid in the wells, and pat dry with absorbent paper. Add 50 μl / well of substrate solution A, then add 50 μl / well of substrate solution B, shake and mix gently, cover the plate with a cover film, and place it in a dark environment at 25°C for 15 minutes. Add 50 μl / well ...

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Abstract

The invention provides a preparation method of an antibody for bovine serum albumin and other impurities in a vaccine, and relates to the following steps: directly immunizing animals to obtain a bovine serum albumin antibody, preparing neomycin immunogen from an impurity neomycin antibody, immunizing animals with the neomycin immunogen, and applying the obtained antibody to an enzyme-linked immunosorbent assay kit.

Description

technical field [0001] The invention relates to antibody preparation technology, in particular to an antibody preparation method for detecting bovine serum albumin and neomycin. The prepared antibody can be applied to a rapid detection method for determining the residual amount of such impurities in vaccines. Background technique [0002] Bovine serum albumin is widely used in basic, biochemical, teaching, scientific research, pharmaceuticals, biological products and other related laboratories, especially in the production process of biological products such as vaccines (such as cell and tissue culture, etc.), generally a certain amount of bovine serum albumin is used Serum, although the final products of each variety are removed through a certain process, a small amount of bovine serum may still remain. Bovine serum, as a sensitizing substance, may have serious consequences after vaccination. Therefore, the detection of residual bovine serum is an important indicator in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/44C12N5/20G01N33/535G01N33/543G01N33/68
CPCC07K16/18C07K16/44G01N33/543G01N33/535G01N33/68
Inventor 沈泓陈碧莲万宇平李超刘柱张瑜王宇邓祖跃陈晓玉何凯伦
Owner ZHEJIANG INST FOR FOOD & DRUG CONTROL
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