Method capable of detecting four kinds of type I diabetes autoimmune antibodies simultaneously

An autoimmune antibody and diabetes technology, applied in the field of medical testing, can solve problems such as cross-reaction, false-positive reaction, tissue sample processing is not fast enough, inactivation of degradable enzymes and salts, and impact on results, etc., to achieve stable, reliable and accurate measurement values High efficiency, quick and easy method

Active Publication Date: 2014-08-06
SHANDONG DONGXING HUIZHI BIOTECH
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Problems solved by technology

However, this method sometimes has cross-reactions, false-positive reactions, and the tissue sample is not pr

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  • Method capable of detecting four kinds of type I diabetes autoimmune antibodies simultaneously

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[0048] Examples:

[0049] The method of the present invention that can simultaneously detect four type I diabetes autoimmune antibodies includes the following steps:

[0050] 1. Construction of fusion vector of luciferase gene and multi-antigen gene

[0051] (1) Select the target sequence of luciferase gene and multi-antigen gene (GAD65 antigen gene sequence, IA-2a antigen gene sequence, IA-2b antigen gene sequence and insulin antigen gene sequence), carry out PCR amplification, PCR amplification Reaction conditions program: 90℃~95℃ denaturation for 5-10 minutes; then 93℃~98℃denature for 45~60 seconds, 45℃~50℃ annealing for 30~45 seconds, 65℃~70℃ extension for 50~60 seconds, proceed 25-30 cycles; the last 70℃~75℃ extension for 10-15 minutes, 0~4℃ heat preservation.

[0052] Introduce restriction enzymes at the kpnl site (GGTACC) to obtain the target fragment and perform gel electrophoresis for identification;

[0053] Multiple antigens are antigens that can produce antibodies in the s...

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Abstract

The invention provides a method capable of detecting four kinds of type I diabetes autoimmune antibodies simultaneously, and belongs to the technical field of medical testing. The method comprises the following steps: fusing a luciferase gene with a multi-antigen gene to form a fusion vector, then guiding the fusion vector into a lactation cell system by using a cell transformation technology to perform the expression of luciferase-multi-antigen fusion protein; splitting a genetic recombinant cell, so as to obtain the luciferase-multi-antigen fusion protein; then by using a immune precipitation, mixing a dilution of the luciferase-multi-antigen fusion protein with patient serum to be subjected to specific combination to obtain a fusion protein-antibody compound, and further adding protein A agarose to capture the fusion protein-antibody compound; adding a luciferase substrate to detect the fluorescence intensity. The method is quick, simple, convenient, high in accuracy, high in sensitivity, high in signal-to-noise ration, and stable and reliable in measured value, and solves the problems such as radioactive ray radiation and pollution caused by the radioactive markers used in the prior art.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a method capable of simultaneously detecting four types of type I diabetes autoimmune antibodies. Background technique [0002] Generally, type I diabetes is an autoimmune disease. The body is induced by infection (especially viral infection), poison and other factors to produce abnormal self-humoral and cellular immune responses, resulting in damage to islet β cells and decreased insulin secretion. Rely on exogenous insulin to supplement to maintain life, so type 1 diabetes is also called insulin-dependent diabetes. [0003] This form of diabetes is common in children and young adults, but it can occur in people of any age and accounts for approximately 10% of all diabetes cases. It can be seen that early diagnosis of type 1 diabetes and prediction in high-risk groups are of great significance. [0004] The diagnosis of diabetes is mainly through the detection of aut...

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Application Information

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IPC IPC(8): G01N21/64G01N33/533
Inventor 唐东起李世武吴琦苏红霞
Owner SHANDONG DONGXING HUIZHI BIOTECH
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