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Kit for quantitatively detecting GFAP concentration in human serum by polystyrene microsphere

A polystyrene microsphere, quantitative detection technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., to achieve the effects of short time consumption, shortened reaction time, and reduced washing times

Active Publication Date: 2012-04-11
山东莱博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the search results, there is no report on using polystyrene microspheres as a solid phase carrier and using liquid phase hybridization as a reaction mode to prepare a kit for detecting GFAP concentration in human serum to quantitatively detect GFAP concentration in serum

Method used

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  • Kit for quantitatively detecting GFAP concentration in human serum by polystyrene microsphere
  • Kit for quantitatively detecting GFAP concentration in human serum by polystyrene microsphere
  • Kit for quantitatively detecting GFAP concentration in human serum by polystyrene microsphere

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Effect test

Embodiment 1

[0031] Step 1 Preparation of Microsphere Storage Solution

[0032](1) Activation of microspheres: after the polystyrene microspheres are uniformly dispersed, take 5.0×10 6 Add 100 μl washing buffer (PBS, 0.05% Tween) to the microspheres, shake at high speed for 15 seconds, suspend the microspheres with 80 μl activation buffer after washing, add 10 μl carbodiimide (EDC, 50 mg / ml), and quickly add 10 μL Sulfo- NHS (50mg / ml), shake at high speed for 30s, shake at room temperature for 20min in the dark, then wash twice with 500μl 0.01M pH7.4PBS, centrifuge at 12000r / min for 3min each time, discard the supernatant, and finally wash with 100μL 0.01M pH7.4PBS suspended microspheres.

[0033] (2) Cross-linking of microspheres: Add 100 μg of anti-GFAP monoclonal antibody to the activated microspheres, dilute to 500 μl with 0.01 MpH7.4 PBS, wrap in tin foil, shake at room temperature for 4 hours in the dark, 15000 r / min Centrifuge for 5min, discard the supernatant, wash once with 500μ...

Embodiment 2

[0056] 1. A kit for the quantitative detection of GFAP concentration in human serum by polystyrene microspheres, consisting of a 96-well filter plate, 2 bottles of standard products, 1 bottle of sample diluent, and polystyrene with anti-GFAP antibody coupled to its surface Microspheres, 1 bottle of microsphere storage solution, 1 bottle of enzyme conjugate, 1 bottle of substrate A, 1 bottle of substrate B, 20× washing solution, 20 self-adhesive coverslips and 1 instruction manual.

[0057] 2. Application of the kit for the quantitative detection of GFAP concentration in human serum by polystyrene microspheres

[0058] (1) Dilution of the standard product: Dilute each bottle to 1ml with the sample diluent before use, and after standing for 10 minutes, mix it upside down repeatedly. ml, 12.5ng / ml, 3.12ng / ml, 0.78ng / ml, 0.05ng / ml, the sample diluent is 0ng / ml.

[0059] (2) Adding samples: shake and mix the microspheres at a medium speed before use, add 10 μl of microspheres cont...

Embodiment 3

[0067] Evaluation of the detection kit for the quantitative detection of GFAP concentration in human serum by polystyrene microspheres:

[0068] (1) Linear range: Dilute the GFAP antigen in a 2-fold gradient with the sample diluent to the following serial concentrations: 100ng / ml, 50ng / ml, 12.5ng / ml, 6.25ng / ml, 3.13ng / ml, 1.56ng / ml, 0.39ng / ml, 0.10ng / ml, 0.025ng / ml, 0.013ng / ml, 0ng / ml were tested, each concentration sample was tested 3 times, the abscissa was the sample concentration, and the relative luminous intensity was plotted as the ordinate , after statistical analysis, the linear range is 0.025ng / ml~100ng / ml.

[0069] (2) Minimum detection limit: divide the GFAP-removed zero-value serum into 20 parts for detection, measure its concentration, calculate the average value and standard deviation, calculate the sum of 2 times the average value and standard deviation, and detect on the standard curve The lowest detection line of the present invention is 17pg / ml.

[0070] (...

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Abstract

The invention discloses a kit for quantitatively detecting GFAP concentration in human serum by polystyrene microsphere, comprising a 96-hole filter plate, a standard product, a sample diluent, a polystyrene microsphere coupled with an anti-GFAP antibody on the surface, a microsphere storage solution, an enzyme compound, a substrate A, a substrate B, a 20*cleaning solution, an adhesive sticker strip and an operating manual. The kit provided by the invention changes the solid phase carrier made of traditional microporous plate into the polystyrene microsphere, generates the antigen antibody reaction in the liquid phase to keep a good bioactivity and a correct spatial configuration, and eliminates the effect on the antigen antibody reaction by the surface tension and the steric hindrance ofthe traditional reaction mode, and effectively shortens the reaction time and the rinsing times, besides, the invention has the characteristics of simple operation, high sensitivity, good specificityand low cost and has important clinical significance for the diagnosis of the acute cerebral hemorrhage early disease degree.

Description

technical field [0001] The invention relates to a kit for quantitatively detecting the concentration of glial fibrillary acidic protein (GFAP) in serum, in particular to a kit for quantitatively detecting the concentration of glial fibrillary acidic protein (GFAP) in human serum by using anti-GFAP antibody-specifically coated polystyrene microspheres. GFAP concentration kit. Background technique [0002] Epidemiological surveys show that cerebrovascular disease, heart disease, and malignant tumors constitute the three major causes of human death. Compared with developed countries in the West, the morbidity and mortality of cerebrovascular diseases in my country are significantly higher than those of cardiovascular diseases. It is estimated that there are about 2 million new stroke patients in the country every year; about 1.5 million patients die of stroke every year; the number of surviving patients is 6 million to 7 million, and 70% to 80% of the survivors are left with p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 王佳颖朱之炜郑祖惠丁兴龙韩娟张增丽李夫东王恒江长林
Owner 山东莱博生物科技有限公司
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