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Kit for quickly detecting human AMH (Anti-Mullerian Hormone) by magnetic particle chemiluminescent immunoassay

A technology of chemiluminescence and magnetic particles, which is applied in the fields of chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, which can solve the problems of low sensitivity, linearity and stability, and relatively high environmental and operator influences. Large size, high maintenance and detection costs, to achieve the effect of increasing specific affinity, solving low sensitivity and non-specific reactions, reducing detection steps and reaction time

Inactive Publication Date: 2019-01-15
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to radioactive pollution, RIA has a great impact on the environment and operators, and at the same time, the intra-batch and inter-batch variation is large, which is difficult to automate; ELISA uses a macromolecular enzyme label, and the enzyme is easily inactivated. This detection method relies on colorimetry or polarized light. The technology suffers from too many interference factors, and its sensitivity, linearity and stability are not high; the ECLI method is a non-open system, and the reagents rely on imports, which are expensive, and the maintenance and detection costs are high, which limits its popularization and application

Method used

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  • Kit for quickly detecting human AMH (Anti-Mullerian Hormone) by magnetic particle chemiluminescent immunoassay
  • Kit for quickly detecting human AMH (Anti-Mullerian Hormone) by magnetic particle chemiluminescent immunoassay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of detection kit for AMH

[0024] The kit of the present invention includes: magnetic particles coated with goat anti-human AMH polyclonal antibody; horseradish peroxidase-labeled modified mouse anti-human AMH monoclonal antibody, AMH calibrator, concentrated washing solution and substrate;

[0025] 1. Preparation of magnetic particles coated with goat anti-human AMH polyclonal antibody:

[0026] Wash the magnetic particles with coating buffer (50mmol / L, pH7.6 phosphate buffer) 5 times, add 10% glutaraldehyde to activate; after washing, coat with the coating antibody diluted to 100-500μg / mL Shake and react for 2 hours; wash the solid phase carrier, and then block with pH4.4, 0.02mol / L buffer solution containing 50mmol / L acetic acid for 1 hour; add 0.5% Tween-20, 1 % BSA, 1% EDTA, 0.1% Procline300, 4% PEG6000 pH7.4, 0.02 mol / L phosphate buffer for storage, and store at 2-8°C for later use;

[0027] 2. Preparation of enzyme-labeled modified mouse a...

Embodiment 2

[0059] Embodiment 2 The usage method of kit of the present invention

[0060] The specific operation steps are:

[0061] 1. Reagent preparation

[0062] 1.1 Calibrators and magnetic particles: Take out the reagents and equilibrate to room temperature (20-25°C);

[0063] 1.2 Washing solution: Mix 10mL of concentrated washing solution and 990mL of purified water in a clean container, and use it as a working washing solution for later use. Purified water should be prepared by the user;

[0064] 1.3 Substrate: Take out and equilibrate to room temperature.

[0065] 2. Test operation

[0066] 2.1 Add 20 μL of AMH calibrator and test sample to 50 μL of magnetic particle antibody suspension in turn, and add 100 μL of enzyme-labeled antibody at the same time;

[0067] 2.2 Incubate the mixed solution with shaking for 15 minutes at room temperature;

[0068] 2.3 After incubation, wash 6 times with washing working solution;

[0069] 2.4 Add 100 μL of substrate to each reaction site...

Embodiment 3

[0071] Embodiment 3 Performance testing of the kit of the present invention

[0072] 1. Minimum detectable amount: Refer to the sensitivity of the test kit recommended by the CLSI EP-17A document, the minimum detectable amount of AMH is not higher than 0.005ng / mL, and the functional sensitivity is 0.01ng / mL.

[0073] 2. Linearity: AMH is between 0.01 and 25ng / mL, and the linear correlation coefficient r between the measured value and the theoretical value is greater than 0.99.

[0074] 3. Accuracy: AMH is between 0.01 and 25ng / mL; the relative deviation between the actual measured value and the theoretical value is within ±15.0% when testing the reference products of the enterprise.

[0075] 4. HOOK effect: When detecting AMH within 2000 ng / mL, there will be no hook effect in the luminescence value.

[0076] 5. Stability: The kit is placed at 37°C for 10 days from the date of production of the finished product (the validity period is 18 months); within the remaining validity ...

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Abstract

The invention discloses a kit for quickly detecting human AMH (Anti-Mullerian Hormone) by magnetic particle chemiluminescent immunoassay. The kit comprises a magnetic particle coated with a goat anti-human AMH polyclonal antibody, a modified mouse anti-human AMH monoclonal antibody labeled by enzyme, an AMH calibration product, concentration lotion and a substrate. The kit has the advantage that exiting chemiluminescent immunoassay which is quickly promoted and applied and is advanced is adopted. Compared with the prior art, the chemiluminescent immunoassay disclosed by the invention overcomesthe deficiency of RIA (radioimmunoassay) radioactive contamination and the defects of the instability and the narrow quantification range of an enzyme marker in an enzyme linked immunosorbent assay,meanwhile, the difficulty in an ELISA (Enzyme-Linked Immuno Sorbent Assay) method that a fluorescent marker is likely to be interfered by environment and the defects of the indirect marking and the like of an ECLI (Electro-Chemi-Luminescence Immunoassay) are overcome, the detection sensitivity and accuracy of the kit can be higher than an enzyme immunoassay method, a fluorescence method and the like by several orders of magnitude, detection steps and reaction time are reduced, an application range is wide, and the kit can be applied to an automatic and semi-automatic instruments, and has a good development prospect.

Description

technical field [0001] The invention relates to biological detection technology, in particular to a reagent kit for rapidly detecting human AMH by using magnetic particle chemiluminescence method. Background technique [0002] According to figures released by the World Health Organization (WHO), fertility problems afflict about 10% of couples worldwide, affecting as many as 80 million people, and the number is rising. Therefore, early assessment of female fertility is very necessary. [0003] Female fertility is currently assessed by ovarian reserve. Ovarian reserve function refers to the quality and quantity of follicle pool, and the ovarian reserve function of women of childbearing age declines with age. [0004] Currently, clinical indicators used to evaluate ovarian reserve include age, basic follicle-stimulating hormone (FSH), luteotropic hormone (LH), estradiol (E2), antral follicle Count (antral follicle count, AFC), anti-Mullerian hormone (Anti-MUllerian Hormone, ...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/543G01N33/76G01N21/76
Inventor 张跃峰刘功成渠海汤久停朱宇皇孙理智张创郑业焕付光宇
Owner AUTOBIO DIAGNOSTICS CO LTD
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