Enzymatic chemiluminescence immunoassay method for human heart-type fatty acid binding protein and reagent kit

A technology for fatty acid binding and detection methods, applied in the fields of chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, which can solve the research and application of measuring human cardiac fatty acid binding proteins, without chemiluminescence analysis, etc. problem, to achieve the effect of small batch-to-batch difference, high affinity, and strong specificity

Inactive Publication Date: 2012-10-17
BEIJING YUANDE BIO MEDICAL ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is no research and application on the measuremen...

Method used

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  • Enzymatic chemiluminescence immunoassay method for human heart-type fatty acid binding protein and reagent kit
  • Enzymatic chemiluminescence immunoassay method for human heart-type fatty acid binding protein and reagent kit

Examples

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Embodiment 1

[0044] Example 1 Preparation of horseradish peroxidase (HRP)-labeled antibody

[0045] Concentrate the anti-human cardiac fatty acid binding protein monoclonal antibody to a concentration of 2 mg / mL. Use the modified sodium periodate method for labeling, the specific steps are:

[0046] (1) Dissolve 5mg of HRP (purchased from Sigma, USA) in 0.5ml of double-distilled water, and add newly prepared 0.06Mol / L NaIO 4 0.5ml aqueous solution, mix well, set at 4°C for 30min;

[0047] (2) Add 0.5ml of 0.16Mol / L ethylene glycol aqueous solution and let stand at room temperature for 30min;

[0048] (3) Add 2.5ml of an aqueous solution containing 5mg of purified anti-human cardiac fatty acid binding protein monoclonal antibody (clone number 22), mix well, put it into a dialysis bag, and slowly stir against 0.05Mol / L pH 9.5 carbonate buffer Dialyze for 6h (or overnight) to make it combine;

[0049] (4) Take out the solution in the dialysis bag and add NaBH 4 Solution (5mg / ml) 0.2ml, m...

Embodiment 2

[0052] Example 2 Adsorption immobilization of coated antibody

[0053] Dilute the anti-human cardiac fatty acid binding protein monoclonal antibody (clone number 28) to 2.5 μg / ml with 0.02M, PH7.4 phosphate buffer solution (PBS solution), add polystyrene microwell plate, 100 μ L / well, 4 overnight at ℃, washed three times with washing solution, blocked with 1% bovine serum albumin 300 μL / well at room temperature for 2 hours, and then patted dry for later use.

[0054] The washing liquid preparation method is: sodium chloride (NaCl) 2g, sodium dihydrogen phosphate (NaH 2 P0 4 .12H 2 0) 2.9g, dipotassium hydrogen phosphate (K 2 HP0 4 ) 0.2g, potassium chloride (KCl) 0.2g, Triton X-1000.5mL, Tween-200.5mL, Bronidox-L 5g, make 1000mL with purified water, and use 0.2μm Filter and store at room temperature.

Embodiment 3

[0055] Example 3 Affinity immobilization of coated antibodies

[0056] 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 (the preparation method is the same as in Example 2) Three times, add biotin-labeled anti-human cardiac fatty acid binding protein antibody (clone number 22) (for biotin labeling method, refer to Thermofisher’s 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 for 2 hours at room temperature Pat dry and set aside.

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Abstract

The invention belongs to the technical field of immunoassay analysis and particularly provides an enzymatic chemiluminescence immunoassay method for a human heart-type fatty acid binding protein. The method comprises the steps of forming solid phase-antibody-antigen-enzyme labelled secondary antibody immune sandwich complexes and detecting the immune sandwich complexes with the method of chemiluminescence. The invention also provides an enzymatic chemiluminescence immunoassay reagent kit for the human heart-type fatty acid binding protein. The detection method and the detection reagent kit are suitable for detecting and analyzing the human heart-type fatty acid binding protein and have the advantages of large detection range, high sensitivity and high specificity.

Description

technical field [0001] The invention belongs to the technical field of immunoassay and analysis, and specifically provides an enzymatic chemiluminescence immunoassay method and kit for human cardiac fatty acid binding protein, which are suitable for enzymatic chemiluminescence detection and analysis of human cardiac fatty acid binding protein. Background technique [0002] Human cardiac fatty acid binding protein (FABP) is an important intracellular fatty acid binding protein. Its cytoplasmic protein with a smaller molecular weight of 15kD exists in a large amount in myocardial tissue, and its content in myocardium is 10 times higher than that in skeletal muscle, and its content in kidney, liver and small intestine is very low. It intervenes in the intracellular transport of free fatty acids in cardiomyocytes and provides cardiomyocytes with energy. Myocardial ischemia leads to necrosis of cardiomyocytes, and h-FABP leaks from cardiomyocytes into the blood, thereby increasi...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/76
Inventor 唐磊朱琳琳孙旭东杨晓林吴晓东
Owner BEIJING YUANDE BIO MEDICAL ENG
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