Method for preparing IgM (Immunoglobulin M) calibrator

A calibrator and polyclonal antibody technology, which is used in the preparation, sampling, and instrumentation of test samples. It can solve problems such as non-specific adsorption and false serum results, and achieve the effect of good stability and high affinity.

Pending Publication Date: 2022-05-27
武汉原谷生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Non-specific adsorption may occur when stored for too long, and false results may occur when serum is used at low dilution, so a method for preparing IgM-type calibrator is needed

Method used

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  • Method for preparing IgM (Immunoglobulin M) calibrator
  • Method for preparing IgM (Immunoglobulin M) calibrator
  • Method for preparing IgM (Immunoglobulin M) calibrator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1. Acquisition of hepatitis A polyclonal antibody

[0036] Obtain 5 mg of hepatitis A antigen for immunizing goats. The immunization procedure was 2 mg for the first time, followed by 1 mg each time, and the immunization interval was 2 weeks. Seven days after the completion of the fourth dose of immunization, the goats were bled, and the polyclonal serum of the goats was collected by centrifugation.

[0037] The polyclonal antibody was purified from polyclonal serum by proteinG and eluted with 100mM Gly pH3.0. The eluate was collected and dialyzed into PBS pH7.6.

[0038] Centrifuge at 12000rpm for 10min, take the supernatant, which is the anti-hepatitis A goat polyclonal antibody, freeze-dry and store.

[0039] 2. Acquisition of Hepatitis A Polyclonal Antibody Fab

[0040] Take goat anti-hepatitis A polyclonal antibody 100mg, papain 5mg, then add 200ul 0.5M cysteine, 200ul 0.1M EDTA pH7.0 and 0.1M phosphate buffer system (pH7.6), then place at 37 ℃, 150rpm constan...

Embodiment 2

[0050] Compared with the natural hepatitis A IgM antiserum, the prepared hepatitis A Fab-human IgM polymer can react with the hepatitis A natural antigen. Use the enzyme-linked immunoassay kit for comparison as follows:

[0051] Natural Hepatitis A Serum 1 Natural Hepatitis A Serum 2 Natural Hepatitis A Serum 3 Hepatitis A Fab-Human IgM Polymer (Negative Serum Diluted 1:10)

[0052]

[0053] Through the above findings, the prepared hepatitis A Fab-human IgM can react well with the natural hepatitis A antigen and has a high titer, the negative value is similar to the serum negative value, and has no background, and can be used as a calibrator of hepatitis A IgM class.

Embodiment 3

[0055] In order to further compare the stabilizing effects of the hepatitis A Fab-human IgM prepared by the present invention, the negative blank serum was used to dilute this product, and multiple freeze-thaw experiments and long-term stability experiments were performed respectively, and the enzyme-linked immunoassay kit was used for comparison as follows The long-term stable effect of this product.

[0056]

[0057] Repeated freeze-thaw results

[0058] Long-term stability results (≤-20°C)

[0059]

[0060] Through the above two tables, it is found that the hepatitis A Fab-human IgM prepared by the present invention has more than 80% activity after repeated freezing and thawing three times compared with natural hepatitis A IgM serum, and the activity loss of natural hepatitis A IgM is all at 50% after repeated freezing and thawing three times. % or more; when stored at ≤-20°C, the activity of hepatitis A Fab-human IgM polymer was still ≥85% after 6 months of storage,...

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Abstract

The invention discloses a method for preparing an IgM calibrator, and relates to the field of IgM calibrators, the method comprises the following steps: 1, obtaining a sheep polyclonal antibody: immunizing a sheep according to a fixed immunization program, and extracting serum when the titer is qualified; 2, purifying the sheep polyclonal antibody: purifying the extracted sheep polyclonal antibody serum by using affinity chromatography; 3, cutting the sheep polyclonal antibody: cutting the sheep polyclonal antibody IgG by using an enzyme, purifying a Fab fragment in the sheep polyclonal antibody, and using the purified Fab fragment for subsequent coupling; 4, coupling of the human IgM and the Fab fragment: coupling the human IgM and the Fab fragment of the sheep polyclonal antibody by using a chemical method; 5, detection: carrying out affinity test and aging test on a product obtained by coupling the human IgM and the Fab fragment of the sheep polyclonal antibody; the prepared specific IgM is added into negative serum according to a certain concentration to obtain the IgM antibody serum, and the IgM antibody serum can be used for research and development of in-vitro diagnosis IgM kits and calibration products of the specific IgM serum.

Description

technical field [0001] The invention relates to the technical field of IgM calibrator, in particular to a method for preparing IgM calibrator. Background technique [0002] IgM mainly exists in the blood of organisms and plays a major role in preventing bacteremia. The determination of IgM antibodies has a high clinical diagnostic value for certain infectious diseases such as hepatitis A. [0003] IgM antibody is the first antibody secreted in the immune response. Once infected, it is produced rapidly. After a period of time, the amount of IgM antibody gradually decreases and disappears. They initiate the complement cascade upon binding to antigen. They also link invaders to each other, clustering them into clumps that can be swallowed by macrophages. IgM in the mother cannot pass through the placenta. If IgM is found in the blood of the fetus or newborn, it means that intrauterine infection has occurred. [0004] IgM accounts for 5% to 10% of the total serum immunoglobu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/34
CPCG01N1/286G01N1/34G01N2001/2893G01N2001/2873
Inventor 熊雨胜张薇周欢
Owner 武汉原谷生物科技有限责任公司
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