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Digoxin non-competitive chemiluminescence immunoassay kit and application thereof

A non-competitive chemiluminescence immunoassay technology, which is applied in the field of non-competitive chemiluminescence immunoassay kits for digoxin, can solve the problems of large zero-dose point error, long reaction incubation time, and difficult to distinguish reaction signals. Achieve the effect of improving detection sensitivity, high accuracy and high sensitivity

Pending Publication Date: 2020-08-07
AILEX TECH GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the sensitivity of competitive immunoassays is largely limited by antibody affinity constants, which generally do not exceed 10 for monoclonal antibodies to small molecule compounds. 12 mol / L, which determines that the competitive analysis mode is difficult to achieve the measurement level of sub-femtomolar concentration
In addition, the reaction signal at low concentration is difficult to distinguish from zero, which makes the relative error of the zero dose point larger, and the analysis process requires repeated detection, and the reaction incubation time is too long. Inferior to noncompetitive immunoassays in terms of precision, kinetics, and linear range of detection

Method used

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  • Digoxin non-competitive chemiluminescence immunoassay kit and application thereof
  • Digoxin non-competitive chemiluminescence immunoassay kit and application thereof
  • Digoxin non-competitive chemiluminescence immunoassay kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Preparation of digoxin anti-immune complex antibody

[0051] In this example, referring to the method of patent JP2793587, the immune complex formed by digoxin and anti-digoxigenin monoclonal antibody is used as the immunogen to prepare the anti-immune complex antibody, and the steps are as follows:

[0052] Digoxin and mouse anti-digoxigenin monoclonal antibody were preliminarily immunized in vitro, and the immune complex formed by combination was used as an antigen to immunize mice; 15 days, 35 days and 97 days after the initial immunization, booster immunization was carried out. After 3 days of final immunization, hybridomas with the ability to produce anti-monoclonal antibodies were established according to conventional methods;

[0053] Screen the binding capacity of the monoclonal antibodies produced by hybridomas, and select antibodies that are reactive to immune complexes but not to digoxin;

[0054] Purify the screened anti-immune complex antibody wi...

Embodiment 2

[0055] Example 2 Preparation of biotinylated anti-digoxigenin monoclonal antibody

[0056] Dissolve biotin-NHS in DMSO, add anti-digoxigenin monoclonal antibody at a molar ratio of 1:20, mix thoroughly and react at room temperature for 1.8 hours, and obtain biochemical anti-digoxigenin monoclonal antibody after dialysis;

[0057] The biochemical anti-digoxigenin monoclonal antibody was diluted to a working solution of 10 ng / mL with a pH=6.8 buffer containing 25 mM PBS, 0.1% BSA, and 0.1% Proclin300.

Embodiment 3

[0058] Example 3 kit assembly

[0059] Take 1 mL of streptavidin magnetic particles (particle size: 3 μm) with a concentration of 100 mg / mL, add them to 30 mL of buffer solution containing 25 mMPBS and 0.1% Proclin300 at pH=6.8, mix thoroughly and place on a magnetic separator for Magnetic separation, repeat 3 times until the supernatant is free of turbidity, discard the supernatant, and keep the magnetic particles;

[0060] Add magnetic particles to 25mM PBS, 0.1% BSA, 0.1% Proclin300 pH = 6.8 magnetic particle buffer to prepare a magnetic particle working solution with a concentration of 0.1%, which is designated as reagent R1;

[0061] The biotinylated anti-digoxigenin monoclonal antibody working solution prepared in Example 2 is designated as reagent R2;

[0062] The alkaline phosphatase anti-immune complex antibody working solution prepared in Example 1 is designated as reagent R3;

[0063] Reagents R1, R2 and R3 are combined to form a non-competitive chemiluminescence ...

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Abstract

The invention provides a digoxin non-competitive chemiluminescence immunoassay kit and application thereof. A constructed detection system comprises a coating antibody and a detection antibody, wherein the coating antibody is a monoclonal antibody of an anti-small molecule compound; the detection antibody is the antibody of an anti-immune complex; and the immune complex is formed by the small molecule compound and the monoclonal antibody of the small molecule compound. According to the invention, the digoxin monoclonal antibody is used as a coating antibody; the digoxin-anti-digoxin monoclonalantibody immune complex is formed by combining digoxin in a sample, then the anti-immune complex antibody is used as a detection antibody to construct a chemiluminiscence detection system, non-competitive detection of small molecule compounds is achieved, the detection sensitivity and accuracy are remarkably improved, and the linear range is remarkably widened.

Description

technical field [0001] The invention belongs to the technical field of immune detection, and relates to a non-competitive detection kit for small molecule compounds and its application, in particular to a digoxin non-competitive chemiluminescence immunological detection kit and its application. Background technique [0002] Digoxin is a biologically active substance extracted from digitalis, and it is a steroid glycoside with cardiotonic effect. The origin of digitalis preparations as cardiotonic drugs can be traced back to the 18th century. Today, for the treatment of heart failure with reduced ejection fraction (HFrEF) accompanied by left ventricular ejection fraction, its status remains strong and maintains Higher recommendation level. Especially for the case of upper ventricular arrhythmia accompanied by tachycardia, digitalis is one of the most effective therapeutic drugs. However, due to the narrow therapeutic concentration range of digoxin, it is easy to cause digit...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/577G01N21/76
CPCG01N33/543G01N33/577G01N21/76
Inventor 李子樵沈融
Owner AILEX TECH GRP CO LTD
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