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One-step homogeneous-phase H-FABP detection kit and preparation and use method thereof

A kit and chemiluminescent detection technology, applied in the field of one-step homogeneous H-FABP detection kits, can solve the problems of inability to balance sensitivity and linearity, reduce detection repeatability, etc., and achieve clinical detection convenience, large market value, The effect of high sensitivity

Inactive Publication Date: 2016-09-28
NANJING PERLONG MEDICAL EQUIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CMIA method is improved from the ELISA method. Compared with the ELISA method, it has the characteristics of simple operation and fast detection speed. However, the CMIA method is a heterogeneous reaction, and the operation process needs to be cleaned, which reduces the repeatability of the detection.
Latex turbidimetry uses the principle of antigen-antibody reaction to determine the content of H-FABP in serum. This method cannot balance sensitivity and linearity.

Method used

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  • One-step homogeneous-phase H-FABP detection kit and preparation and use method thereof
  • One-step homogeneous-phase H-FABP detection kit and preparation and use method thereof
  • One-step homogeneous-phase H-FABP detection kit and preparation and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of anti-H-FABP antibody-coupled luminescent microspheres:

[0045] (1) Add 0.1 mg of anti-H-FABP monoclonal antibody to an ultrafiltration tube, centrifuge for 10 minutes, wash with HEPES buffer (pH7.4) for 6 times, and dilute the antibody to 1 mg / ml for use. Wash 0.1 mg of luminescent microspheres twice with HEPES buffer (pH7.4), centrifuge, and remove the supernatant;

[0046] (2) Add the antibody solution to the luminescent microspheres and resuspend;

[0047] (3) Add 2.0μl 10% Tween 20, 8μl 400mM NaCNBH 3 , add HEPES buffer to make up the volume to 200 μl, shake and react at 37°C for 24 hours;

[0048] (4) Prepare 65mg / ml CMO with 800mM NaOH, add 8μl of the solution to the reaction system; shake and react at 37°C for 0.5 hours, centrifuge, and remove the supernatant;

[0049] (5) Add 150 μl Tris-HCl (pH8.0) buffer to resuspend, centrifuge, remove supernatant, repeat once;

[0050] (6) After the last centrifugation, resuspend the microspheres with a m...

Embodiment 2

[0062] Preparation of anti-H-FABP antibody-coupled luminescent microspheres:

[0063] (1) Add 0.1 mg of anti-H-FABP polyclonal antibody to an ultrafiltration tube, centrifuge for 10 minutes, wash with HEPES buffer (pH7.4) for 6 times, and dilute the antibody to 1 mg / ml for use. Wash 10 mg of luminescent microspheres twice with HEPES buffer (pH7.4), centrifuge, and remove the supernatant;

[0064] (2) Add the antibody solution to the luminescent microspheres and resuspend;

[0065] (3) Add 3μl 10% Tween 20, 12μl 400mM NaCNBH 3 , add HEPES buffer to make up the volume to 200 μl, shake and react at 37°C for 48 hours;

[0066] (4) Prepare 65 mg / ml CMO with 800 mM NaOH, add 12 μl of the solution to the reaction system; shake and react at 37 ° C for 1.5 hours, centrifuge, and remove the supernatant;

[0067] (5) Add 250 μl Tris-HCl (pH8.0) buffer to resuspend, centrifuge, remove supernatant, repeat once;

[0068] (6) After the last centrifugation, resuspend the microspheres with...

Embodiment 3

[0080] (1) Add 0.1 mg of anti-H-FABP monoclonal antibody to an ultrafiltration tube, centrifuge for 10 minutes, wash with HEPES buffer (pH7.4) for 6 times, and dilute the antibody to 1 mg / ml for use. Wash 1 mg of luminescent microspheres twice with HEPES buffer (pH7.4), centrifuge, and remove the supernatant;

[0081] (2) Add the antibody solution to the luminescent microspheres and resuspend;

[0082] (3) Add 2.5μl 10% Tween 20, 10μl 400mM NaCNBH 3 , add HEPES buffer to make up the volume to 200μl, shake and react at 37°C for 36 hours;

[0083] (4) Prepare 65 mg / ml CMO with 800 mM NaOH, add 10 μl of the solution to the reaction system; shake and react at 37 ° C for 1 hour, centrifuge, and remove the supernatant;

[0084] (5) Add 200 μl Tris-HCl (pH8.0) buffer to resuspend, centrifuge, remove supernatant, repeat once;

[0085] (6) After the last centrifugation, resuspend the microspheres with a mixture of PBS buffer (pH7-8) and bovine serum albumin, with a final concentrati...

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Abstract

The invention discloses a one-step homogeneous-phase H-FABP oxygen channel chemiluminescence detection kit. The kit mainly comprises luminous microspheres resistant to H-FABP antibody coupling, photosensitive microspheres resistant to H-FABP antibody coupling, analysis and buffer liquid and a reaction hole. The invention further discloses a preparation method of the one-step homogeneous-phase H-FABP oxygen channel chemiluminescence detection kit. The method includes the steps of preparation of luminous microspheres resistant to H-FABP antibody coupling, preparation of photosensitive microspheres resistant to H-FABP antibody coupling and preparation of analysis and buffer liquid. Finally, the invention discloses a use method of the kit. The kit has the advantages of being high in sensitivity, good in specificity, broad in detection range, good in repeatability, easy to operate, free of cleaning and the like, it is convenient for the kit to be used for clinical detection, and the kit is used for monitoring the heart disease, can raise the accurate rate of diagnosis of myocardial infarction and has great market value.

Description

technical field [0001] The invention relates to the field of H-FABP content detection, in particular to a one-step homogeneous H-FABP detection kit and a preparation and use method thereof. Background technique [0002] Acute myocardial infarction (AMI) is one of the diseases with high morbidity and mortality in the world. According to recent data from the American Heart Association, there were 7.2 million AMI patients in the United States in 2003. But at the same time, AMI is one of the diseases with a high misdiagnosis rate. Some patients who do suffer from AMI have not been properly treated, resulting in a high mortality rate; while other non-AMI patients have received unnecessary treatment, causing unnecessary cost. The World Health Organization recommends that AMI can be diagnosed if two of the three indicators of typical chest pain, ECG changes and abnormal myocardial enzymes are met. However, many AMI patients do not have typical clinical symptoms and abnormal ECG ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543
CPCG01N33/6893G01N33/54313G01N2333/47G01N2800/324
Inventor 李明明黄宝福淳林
Owner NANJING PERLONG MEDICAL EQUIP
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