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Preparation of troponin I specific locus antibody and detection kit thereof

A troponin, specific site technology, applied in the field of troponin I specific site antibody preparation and detection kits, can solve the problem that specificity is difficult to meet clinical detection requirements and the like

Active Publication Date: 2014-07-23
南陵县建设投资有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem that there is sensitivity and specificity in the reagents for measuring troponin I (cTnI) in the above prior art, and it is difficult to meet the clinical detection requirements, so that the content of troponin I (cTnI) can be determined clinically. Determination standardization; the present invention is an antibody that acts on a specific site of human cardiac troponin I (cTnI) single peptide chain, and uses the antibody to prepare an immune latex reagent for measuring the content of cardiac troponin I (cTnI) in serum. The immunolatex reagent can be used for semi-automatic or fully automatic biochemical analysis instruments to accurately and specifically determine the content of cardiac troponin I (cTnI) in serum

Method used

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  • Preparation of troponin I specific locus antibody and detection kit thereof
  • Preparation of troponin I specific locus antibody and detection kit thereof
  • Preparation of troponin I specific locus antibody and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0068] image 3 The correlation of embodiment 3 measurement result and ELISA method

[0069] Figure 4 The correlation of embodiment 4 measurement result and ELISA method

[0070] Figure 5 The correlation of embodiment 5 measurement result and ELISA method

[0071] Positive significance of the present invention

[0072] The invention relates to the preparation of an antibody at a specific site of troponin I and a detection kit thereof. The present invention uses the fixed-point hydrolysis method to obtain three peptides of human cardiac troponin I (cTnI), and then performs animal immunization to obtain high-titer immune serum through immunogenic modification. The three peptides extracted by hydrolysis are used as Ligand, polyacrylamide is used as the ligand to prepare an affinity chromatography column, and the obtained high-titer serum is subjected to immunoaffinity chromatography to obtain a polyclonal antibody that specifically recognizes the peptide. The antibody has...

Embodiment 1

[0077] ——Preparation of latex microsphere reagent sensitized by anti-human cardiac troponin I antibody

[0078] 1. Take 20ml of 0.1M PBS buffer solution with pH=7.4, add purified human cardiac troponin I (cTnI, prepared by the applicant according to the conventional method), the preparation concentration is 100mg / L, first add proteinase K, the final activity The concentration is 50KU / L. After reacting for 1 hour, add proteinase K specific inhibitor (EGTA disodium salt), the final concentration is 100mmol / L, mix and react for 10min, then add carboxypeptidase Y, the final concentration is 25KU / L Mix well and react for 30min. Add carboxypeptidase inhibitor (diisopropylfluorophosphate) to a final concentration of 30mmol / L, mix and react for 20min.

[0079] 2. Add the above mixed solution to the TSKgel4000SW chromatographic column, elute with 0.1M PBS with pH=7.4, collect and detect the separated samples at a wavelength of 280nm (see figure 1 middle 1 lane). The relative molecul...

Embodiment 2

[0087] Goats were immunized with the modified polypeptide fragment obtained in Example 1, mixed with Freund's adjuvant at a concentration of 7 μg / ml for goat immunization, and after the initial immunization, a booster immunization was performed every 14 days for a total of four immunizations. Other steps are identical with embodiment 1.

[0088] Example 3

[0089] ——The ratio of each component in the kit

[0090] Reactant:

[0091]

[0092] Latex reagent: (1:2:1)

[0093]

[0094] Mix the three groups of latex microspheres according to the above ratio, add pure water to 1000ml

[0095] Calibrator:

[0096] PBS buffer at pH=5.8 20mM

[0097] Purified human cardiac troponin I 5ng / mL

[0098] According to the test kit of latex proportioning concentration in the present embodiment, see Table 2 for the test result of sample.

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Abstract

The invention relates to preparation of troponin I specific locus antibody and a detection kit thereof. A fixed point hydrolysis method is adopted to obtain the required three peptide sections of human cardiac troponin I (cTnI); the three peptide section are orderly subjected to immunogenicity modification and animal immunization so as to obtain high titer immune serum; the three peptide sections extracted in the hydrolysis are taken as the ligand and the polyacrylamide is taken as genin to prepare an affinity column; and finally the obtained high titer immune serum is subjected to immunoaffinity chromatography so as to obtain polyclonal antibodies having specific recognition on the three peptide sections. The antibodies have the advantages that: the affinity of the antibodies is higher than that of a corresponding monoclonal antibody, the specificity is equal to that of a corresponding monoclonal antibody, moreover, the preparation cost is lower, and the preparation process is simpler, compared to that of a monoclonal antibody. The obtained three polyclonal antibodies can be made into an immunity latex kit for a semi-automatic or automatic generation analysis instrument for detecting the content of cardiac troponin I (cTnI) in serum.

Description

technical field [0001] The invention relates to the preparation of an antibody at a specific site of troponin I and a detection kit thereof. The invention relates to the application of an antibody acting on a specific site of a single peptide chain of human cardiac troponin I (cTnI), and a method for preparing an immune latex reagent for measuring serum cardiac troponin I (cTnI) content by using the antibody. The kit prepared by the invention can be used in semi-automatic or full-automatic hair growth analysis instruments. Background technique [0002] Cardiac troponin I (cTnI) is one of the most sensitive and specific markers of myocardial injury. It is used as the gold standard for judging myocardial cell injury in acute myocardial injury and myocarditis, and is also used for risk stratification and prognosis of coronary artery syndrome. key biochemical markers. Troponin is a regulatory protein, the molecule is spherical, and consists of three peptides, troponin T (TnT),...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/68
CPCG01N33/531G01N33/54313G01N33/6887
Inventor 李传响
Owner 南陵县建设投资有限责任公司
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