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Enzyme-linked immunosorbent assay kit for detecting protein A and other impurities in biological product and application of enzyme-linked immunosorbent assay kit

An enzyme-linked immunosorbent reagent and biological product technology, applied in animal/human proteins, biological testing, albumin peptides, etc., can solve the problems of hindering antibody binding, inaccurate test results, difficult quality control, etc. Simple processing and low pre-processing requirements

Pending Publication Date: 2021-06-04
ZHEJIANG INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the detection of residual ProA, because ProA has the characteristics of being able to bind to IgG molecules, which hinders its combination with the corresponding antibody, often resulting in inaccurate detection results, so this item is not only an important indicator for the quality control of recombinant Fc fusion proteins and monoclonal antibodies , has always been a difficulty in quality control
In addition, antibiotics such as gentamicin may also exist in biological products, which are impurities that need to be detected

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting protein A and other impurities in biological product and application of enzyme-linked immunosorbent assay kit
  • Enzyme-linked immunosorbent assay kit for detecting protein A and other impurities in biological product and application of enzyme-linked immunosorbent assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Preparation of kit components

[0024] 1. Preparation of Gentamicin Hapten

[0025] Take 1.39g of gentamicin, add 80ml of pure water to dissolve, take 0.344g of 4,4'-difluoro-3,3'-dinitrodiphenylsulfone, add 30ml of methanol to dissolve, add it to the aqueous solution of gentamicin , add 1.38g of anhydrous potassium carbonate, stir at room temperature for 3h, stop the reaction, add 300ml of ethyl acetate to extract, stand still, separate the water phase, evaporate the organic phase to dryness, and recrystallize with 20ml of absolute ethanol to obtain fluoronitrobenzene- Gentamicin hapten product 0.34g, yield 43.9%.

[0026] 2. Antigen preparation

[0027] Preparation of immunogen—The hapten of gentamicin was coupled with bovine serum albumin (BSA) to obtain the immunogen.

[0028] Get 29 mg of fluoronitrobenzene-gentamycin hapten product, add DMF 2ml to dissolve, obtain hapten solution A liquid; get bovine serum albumin (BSA) 50 mg, add 0.05M PB buffer s...

Embodiment 2

[0040] Embodiment 2 The formation of ELISA kit

[0041] An enzyme-linked immunosorbent assay kit for detecting gentamicin / protein A was set up to include the following components:

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

[0043] (2) 6 bottles of gentamicin standard solution, the concentrations are 0μg / L, 0.1μg / L, 0.3μg / L, 0.9μg / L, 2.7μg / L, 8.1μg / L; protein A standard solution 6 bottles, the concentrations are 0mg / L, 0.2mg / L, 0.6mg / L, 1.8mg / L, 5.4mg / L, 16.2mg / 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] Example 3 Detection of analytes in 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 of sto...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit for detecting protein A and other impurities in a biological product, which comprises an elisa plate coated with a coating antigen, a protein A standard substance solution, a gentamicin standard substance solution, a protein A antibody, a gentamicin antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a washing solution; the coating antigen is a gentamicin coupling antigen, the enzyme conjugate is an enzyme-labeled protein A antibody and an enzyme-labeled gentamicin antibody, and the protein A antibody and the gentamicin antibody are both obtained by immunizing an animal with an immunogen. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the content of impurities such as protein A in biological products, is simple and convenient to operate, low in cost and high in sensitivity, can be used for on-site monitoring and is suitable for screening a large number of samples.

Description

technical field [0001] The invention relates to an ELISA kit for detecting impurities such as protein A in biological products and its application, in particular to a protein A immunization method, a gentamicin artificial antigen molecular structure and an ELISA kit for detecting protein A , used to determine the residual amount of such impurities in biological products. Background technique [0002] The relevant regulations of WHO and ICH and the three volumes of the Chinese Pharmacopoeia 2010 edition all describe and stipulate the removal of impurities and the residue limit. Protein A (Protein A, ProA) is one of the earliest discovered molecules that bind to immunoglobulins. As an important medium for affinity chromatography, it is widely used in the purification process of Fc fusion proteins and monoclonal antibodies. ProA affinity Chromatography is used for purification. The advantage is high performance and high purity. The disadvantage is that it can leach ProA, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/577G01N33/543G01N33/53C07K14/765C07K1/107
CPCG01N33/581G01N33/577G01N33/54393G01N33/9446C07K14/765G01N2333/31Y02A50/30
Inventor 沈泓周明昊孙晗陶巧凤万宇平何凯伦邓祖跃王宇陈晓玉
Owner ZHEJIANG INST FOR FOOD & DRUG CONTROL
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