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Myoglobin enzymatic chemiluminescence immunodetection method and kit

A human myoglobin and detection method technology, applied in the human myoglobin enzymatic chemiluminescence immunoassay method and kit, the field of enzymatic chemiluminescence detection and analysis of human myoglobin, can solve the problem that there is no chemiluminescence analysis method, measurement Human myoglobin research and application, to achieve the effect of small batch-to-batch difference, improved sensitivity and specificity, and high specificity

Inactive Publication Date: 2013-04-10
BEIJING YUANDE BIO MEDICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is no research and application on the measurement of human myoglobin by chemiluminescence analysis.

Method used

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  • Myoglobin enzymatic chemiluminescence immunodetection method and kit
  • Myoglobin enzymatic chemiluminescence immunodetection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 Preparation of horseradish peroxidase (HRP) labeled antibody

[0044] Concentrate the anti-human myoglobin monoclonal antibody to 2mg / mL. Use the modified sodium iodate method for labeling, the specific steps are:

[0045] (1) 5mg of HRP (purchased from Sigma, USA) was dissolved in 0.5ml double distilled water, and newly prepared 0.06Mol / L NaIO was added 4 0.5ml aqueous solution, mix well, set at 4°C for 30min;

[0046] (2) Add 0.5ml of 0.16Mol / L ethylene glycol aqueous solution, and let stand at room temperature for 30min;

[0047] (3) Add 2.5ml of an aqueous solution containing 5mg of purified anti-human myoglobin monoclonal antibody (clone number 7001), mix well, put it into a dialysis bag, and slowly stir and dialyze against 0.05Mol / L pH 9.5 carbonate buffer 6h (or overnight), make it combine;

[0048] (4) Take out the solution in the dialysis bag, add NaBH 4 Solution (5mg / ml) 0.2ml, mix well, set at 4°C, 2h;

[0049] (5) Slowly add an equal volum...

Embodiment 2

[0051] Example 2 Adsorption immobilization of coated antibody

[0052] Dilute anti-human myoglobin monoclonal antibody (clone number 7004) to 2.5 μg / ml with 0.02M, pH7.4 phosphate buffer solution (PBS solution), add polystyrene microwell plate, 100 μL / well, 4 °C Overnight, wash three times with washing solution, block with 300 μL / well of 1% bovine serum albumin at room temperature for 2 hours, and then pat dry for later use.

[0053] The preparation method of washing liquid is: sodium chloride (NaCl) 2g, sodium dihydrogen phosphate (NaH 2 PO 4 .12H 2 O) 2.9g, dipotassium hydrogen phosphate (K 2 HPO 4 ) 0.2g, Potassium Chloride (KCl) 0.2g, Triton X-100 0.5mL, Tween-20 0.5mL, Bronidox-L 5g, make 1000mL with purified water, use Filter through a 0.2 μm filter and store at room temperature.

Embodiment 3

[0054] Example 3 Affinity immobilization of coated antibodies

[0055] Dilute avidin with 0.02M, pH7.4 phosphate buffer solution to 1.0 μg / ml, add to a polystyrene microwell plate, 100 μL / well, overnight at 4°C, wash with washing solution (preparation method is the same as in Example 2) Three times, add biotin-labeled anti-human myoglobin antibody (clone number 7004) (see Thermofisher for biotin labeling method) Instructions for use of Sulfo-NHS-Biotin, product number is 21217), concentration 1.0 μg / ml, 100 μL / well, overnight at 4°C, washed three times with washing solution, blocked with 1% bovine serum albumin 300 μL / well at room temperature for 2 hours, then photographed Dry and set aside.

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Abstract

Belonging to the technical field of immunodetection and analysis, the invention specifically provides a myoglobin enzymatic chemiluminescence immunodetection method. The method includes the steps of: forming a solid phase-antibody-antigen-enzyme-labeled secondary antibody immune sandwich composite and detecting the immune sandwich composite by means of chemiluminescence. The invention also provides a myoglobin enzymatic chemiluminescence immunodetection kit. The detection method and the detection kit provided in the invention are suitable for detection and analysis of myoglobin, and have the advantages of large detection range, and high sensitivity and specificity.

Description

technical field [0001] The invention belongs to the technical field of immunological detection and analysis, and specifically provides a human myoglobin enzymatic chemiluminescence immunological detection method and a kit, which are suitable for the enzymatic chemiluminescent detection and analysis of human myoglobin. Background technique [0002] Human myoglobin (Myo) is a low-molecular-weight cytoplasmic protein widely present in skeletal muscle and muscle cells, and its content in the former is higher than that in the latter. When acute myocardial ischemia (acute myocardial ischemia, AMI) occurs, Myo is significantly increased, and can last up to 12 hours, and has a high sensitivity. However, since skeletal muscle diseases, renal dysfunction, inflammation, systemic lupus erythematosus, and dermatomyositis can all cause Myo to increase, its specificity as a diagnostic indicator for AMI is low. However, due to its high sensitivity, it is generally used as a screening index...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/76
Inventor 唐磊朱琳琳吴旭东孙旭东杨晓林吴晓东
Owner BEIJING YUANDE BIO MEDICAL ENG
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