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A homogeneous enzyme immunoconjugate and its preparation method and application

A homogeneous enzyme immunoassay and conjugate technology, applied in biological testing, instruments, measuring devices, etc., can solve problems such as low coupling efficiency and insufficient reliability of test results, achieve high coupling efficiency and reduce the probability of interference , the effect of mild reaction conditions

Active Publication Date: 2022-02-18
HANGZHOU BOPU MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the fact that the enzyme conjugates used in the process of measuring specific antigen antibodies in serum by homogeneous enzyme immunoassays are easily affected by individual health conditions, resulting in insufficient reliability of test results and low coupling efficiency It provides a homogeneous enzyme immunoconjugate that can avoid the possible interference of glucose-6-phosphate dehydrogenase in the human body on the results, improve the reliability of the results, and improve the coupling efficiency during the synthesis process. its preparation method

Method used

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  • A homogeneous enzyme immunoconjugate and its preparation method and application
  • A homogeneous enzyme immunoconjugate and its preparation method and application
  • A homogeneous enzyme immunoconjugate and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] CG-MDH: Dissolve sodium glycocholate (200mg) in 20mL pH 7.6, 100mM phosphate buffer, weigh 15mg hemocyanin (KLH) and malate dehydrogenase MDH (10mg), dissolve in pH 7.6 , in 100mM phosphate buffer, stirred at room temperature for 10 minutes, then added 160mg of water-soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) powder, kept at 2°C Stir under light and temperature for 120 minutes. After the reaction is over, put it into a dialysis bag (free permeable molecular size <14KD), dialyze at 4°C, change the solution every 6 hours, and continue for 6 times to obtain the required enzyme coupling. Substance CG-MDH.

Embodiment 2

[0038] CG-MDH: Dissolve sodium glycocholate (200mg) in 20mL pH 7.6, 100mM phosphate buffer, weigh 10mg bovine serum albumin (BSA) and MDH (30mg), dissolve in pH 7.6, 100mM phosphoric acid In salt buffer, stir at 8°C for 15 minutes, then add 200 mg of water-soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) powder, and stir gently at room temperature for 180 Minutes, after the reaction is over, put it into a dialysis bag (free permeable molecule size <14KD), dialyze at 4°C, change the liquid once every 6 hours, and continue for 6 times to obtain the required enzyme conjugate CG-MDH .

Embodiment 3

[0040] CG-FDH: Dissolve sodium glycocholate (200mg) in 20mL pH 7.6, 100mM phosphate buffer, weigh 5mg hemocyanin (KLH) and FDH (50mg), dissolve in pH 6, 100mM phosphate buffer , stirred at room temperature for 15 minutes, then added 250 mg of water-soluble dicyclohexylcarbodiimide powder (DCC), and stirred gently at 2°C for 8 hours in the dark. Dialyze at 4°C and change the medium every 6 hours for 8 consecutive times to obtain the desired enzyme conjugate CG-FDH.

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Abstract

The invention relates to the technical field of homogeneous enzyme immunoassay, in particular to a coupling product obtained by dehydrating a small molecule hapten and an enzyme under the action of a coupling agent. Firstly, the small molecule hapten is dissolved in phosphate buffer; then a coupling agent, enzyme and protein are added to the above small molecule hapten solution for reaction to obtain a homogeneous enzyme immunoconjugate. The invention overcomes the defect that the enzyme conjugate used in the process of determining the specific antigen antibody in the serum by the homogeneous enzyme immunoassay is easily affected by the health status of the individual, resulting in insufficient reliability of the test result and low coupling efficiency , the present invention has the ability to reduce the interference of individual health conditions on the test and improve the reliability of test results; enzymes and proteins are directly combined with small molecule haptens under the action of coupling agents without other bridging molecules, the coupling efficiency is high and the reaction conditions Mild; the prepared enzyme conjugate has the advantages of good water solubility and less loss of enzyme activity.

Description

technical field [0001] The invention relates to the technical field of homogeneous enzyme immunoassay, in particular to a homogeneous enzyme immunoconjugate and its preparation method and application. Background technique [0002] Homogeneous enzyme immunoassay technology is a competitive immunoassay technology based on a liquid-phase homogeneous reaction system. This technology is based on the principle of antigen-antibody reaction, that is, the combination of an enzyme-labeled hapten and its corresponding antibody produces steric hindrance to inhibit enzyme activity. , and when the sample contains free small molecule hapten, it will compete with the enzyme-labeled hapten to bind to the antibody, and the enzyme-labeled hapten bound to the antibody will be released, and its activity will also be enhanced. By establishing the absorbance value and The standard curve of the concentration of the calibrator of the analyte is used to calculate the content of the unknown sample. T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/531G01N33/535
Inventor 王轶雄杨晓东缪刘曹静
Owner HANGZHOU BOPU MEDICAL TECH
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