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Preparation method of E hepatitis rabbit-human chimeric antibody quality control substance

A technology of chimeric antibody and hepatitis E, which is applied in the field of clinical laboratory science and biology, can solve the problems of prolonging the preparation cycle of quality control substances, low antibody affinity and complexity, etc., and achieve the effect of easy high titer mass acquisition and high affinity

Inactive Publication Date: 2010-03-24
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation of such quality control substances involves hybridoma technology, recombinant DNA technology, etc., which is relatively complicated, prolonging the preparation cycle of quality control substances, and the affinity of such antibodies is lower than that of real human samples

Method used

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  • Preparation method of E hepatitis rabbit-human chimeric antibody quality control substance
  • Preparation method of E hepatitis rabbit-human chimeric antibody quality control substance
  • Preparation method of E hepatitis rabbit-human chimeric antibody quality control substance

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0054] Preparation of purification reagents:

[0055] (1) Neutralizing solution: 1M Tris-HCl, pH 9.0: Dissolve 12.11g Tris in 80ml of distilled water, add concentrated hydrochloric acid to adjust the pH to 9.0 (about 7.5ml), add water to make up to 100ml.

[0056] (2) Binding buffer: 20mM sodium phosphate, pH7.0, dissolve 3.8012g sodium phosphate in 500ml water, adjust the pH to 7.0 (about 1.5ml) with concentrated hydrochloric acid.

[0057] (3) Elution buffer: 0.58% (v; v) glacial acetic acid NaCl (0.15M) solution, NaCl (0.15M) solution: 4.383g NaCl was dissolved in 500ml of water, and then 1.45ml of glacial acetic acid was added.

[0058] Protein SDS-polyacrylamide gel electrophoresis reagent preparation (see Molecular Cloning Volume 2 P1716)

[0059] (1) 1×Tris-glycine electrophoresis buffer: can be prepared with 5× storage solution, add 15.1g Tris base and 94g glycine in 900ml deionized water, add 50ml 10% SDS (m / V), make up to 1000ml with deionized water.

[0060] (2) C...

Embodiment 1

[0072] Embodiment 1: the preparation of rabbit anti-human HEV-IgG

[0073] 1. Materials

[0074] 1. New Zealand white rabbits, healthy males, weighing about 2.5kg, from Beijing Xingfu Animal Farm.

[0075] 2. Antigen: NE2, batch number: 20060829, content 1.817mg / ml, Beijing Wantai Biological Pharmaceutical Co., Ltd.

[0076] 3. Hepatitis E IgM virus antibody diagnostic kit, Beijing Wantai Biopharmaceutical Co., Ltd.

[0077] 2. Method

[0078] 1. Serum Preparation

[0079] Blood was collected from the ear vein, and 1-2ml of blood was collected in a 1.5ml eppendorf centrifuge tube for future reference. Let the rabbit blood stand at room temperature for several hours, transfer the serum to a new centrifuge tube, centrifuge at 5000rpm for 10 minutes to collect the serum, put the serum in a 1.5ml tube, and store it at -70°C.

[0080] 2. Antigen Preparation

[0081] Take 10 μl of the original antigen solution and dilute it to 1 ml with 0.9% saline for injection (the content i...

Embodiment 2

[0145] Example 2: Antibody Crosslinking

[0146] 1. Method

[0147] 1. Rabbit anti-HEV-IgG cross-linked with human IgM

[0148] (1) Take (about 180μl) and (about 427μl) (1mgIgG and 1mgIgM) dialyzed with MES cross-linking buffer

[0149] (2) Equilibrate EDC to room temperature

[0150] (3) Mix the above two proteins after dialysis. (1mgIgG and 1mgIgM)

[0151] (4) Take 2.5 mg of EDC, dissolve it in 250 μl of water, and add 100 μl to the reaction system.

[0152] (5) React at room temperature for 2 hours.

[0153] (6) Dialyze with PBS overnight (1000ml) Dialyze overnight, during which the liquid is changed three times, and the interval is placed at 4 degrees, and stirred.

[0154] 2. Determination of chimeric antibody titer by ELISA

[0155] (1) Add 100 μl sample diluent to each well.

[0156] (2) Add negative control, EDC cross-linked product and positive control 10 μl in sequence (both make two wells).

[0157] (3) Incubate at 37°C for 30 minutes, wash plate 5 times.

...

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Abstract

The invention discloses an E hepatitis rabbit-human chimeric antibody quality control substance and preparation method thereof, belonging to the clinical examination and biotechnology field. The E hepatitis rabbit-human chimeric antibody quality control substance is characterized in that:the E hepatitis rabbit-human chimeric antibody quality control substance is chimeric antibody formed by cross-linking rabbit-anti-human HEV-IgG and human IgM using 1-ethyl-3[3-dimethyl amino propyl] carbodiimide. The innovation point of the invention is that: the chimeric antibody quality control substance for iImmunological analysis of HEV is suitable for EQA and IQC. The anti HEV IgM quality control substance is constructed using a chemical crosslink method for the first time at home and abroad. The quality control substance is repetitively produced on the premise of relative uniform and relatively stable and economic and easy to obtain a large amount at high titer without latent biology infection danger and reference to medical ethics problem and with higher affinity with the antigen. The E hepatitis rabbit-human chimeric antibody quality control substance is a good positive quality control substance.

Description

technical field [0001] The invention relates to a rabbit-human chimeric antibody quality control substance for specific IgM (anti-HEV) detection and a preparation method thereof, belonging to the fields of clinical laboratory science and biotechnology. Background technique [0002] Hepatitis E virus (HEV) was once known as a non-A non-B hepatitis virus transmitted through the digestive tract. The first outbreak in India in 1955 was thought to be caused by the hepatitis A virus. In the early 1970s, the HAV detection method was established, and the serum of the hepatitis patients at that time was re-tested. The results did not find that the anti-HAV IgM or IgG titer in the patient's serum increased, so it was determined that it was caused by the non-A non-B hepatitis virus transmitted in the digestive tract. Sincerely. In 1986, an epidemic of hepatitis E occurred in the southern part of Xinjiang, my country. About 120,000 people became ill and more than 700 died. It was the ...

Claims

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Application Information

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IPC IPC(8): G01N33/576
Inventor 李金明张括王露楠张瑞
Owner BEIJING HOSPITAL
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