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Kit for quantitatively detecting HCV-aAg concentration in human serum by virtue of polystyrene microspheres

A polystyrene microsphere, quantitative detection technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., to achieve the effect of shortening the reaction time, short time consumption, and wide linear range

Active Publication Date: 2012-05-09
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

In the search results, there is no use of polystyrene microspheres as a solid phase carrier and liquid phase hybridization as a reaction mode to prepare a kit for detecting the concentration of HCV-cAg in human serum to quantitatively detect the concentration of HCV-cAg in serum. to report

Method used

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  • Kit for quantitatively detecting HCV-aAg concentration in human serum by virtue of polystyrene microspheres
  • Kit for quantitatively detecting HCV-aAg concentration in human serum by virtue of polystyrene microspheres
  • Kit for quantitatively detecting HCV-aAg concentration in human serum by virtue of polystyrene microspheres

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Step 1 Preparation of Microsphere Suspension

[0026] (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 pH7.4 Microspheres were suspended in PBS.

[0027](2) Cross-linking of microspheres: Add 100 μg of anti-HCV-cAg monoclonal antibody to the activated microspheres, dilute to 500 μl with 0.01M pH7.4 PBS, wrap in tin foil, and shake at room temperature for 4 hours in the dark , centrifuged at 15000r / min for 5min, discarded the supernatant, was...

Embodiment 2

[0047] 1. Kit for quantitative detection of HCV-cAg concentration in human serum by polystyrene microspheres

[0048] The kit consists of 1 piece of 96-well filter plate, 2 bottles of standard products, 1 bottle of sample diluent, 1 bottle of microsphere suspension, 1 bottle of enzyme conjugate, 1 bottle of substrate A, 1 bottle of substrate B, 1 bottle of 20× It consists of washing liquid, 5 self-adhesive sealing sheets and 1 instruction manual.

[0049] 2. Application of the kit for the quantitative detection of HCV-cAg concentration in human serum by polystyrene microspheres

[0050] (1) Dilution of standard substance: before use, each bottle of standard substance is diluted to 1ml with sample diluent, and the concentration is 50ng / ml. , 12.5ng / ml, 3.12ng / ml, 0.78ng / ml, 0.05ng / ml, 0.013ng / ml, the sample diluent was 0ng / ml.

[0051] (2) Adding samples: Shake and mix the microspheres at a medium speed before use, add 10 μl of microspheres containing about 50,000 HCV-cAg mon...

Embodiment 3

[0059] Evaluation of the detection kit for the quantitative detection of HCV-cAg concentration in human serum by polystyrene microspheres:

[0060] (1) Linear range: Dilute the HCV-cAg antigen in a 2-fold gradient with the sample diluent to the following serial concentrations: 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, 0.007ng / ml, and 0ng / ml were tested, and each concentration sample was tested three times, with the sample concentration as the abscissa and the relative luminous intensity as the ordinate Coordinate plotting, after statistical analysis, the linear range is 0.013ng / ml~50ng / ml.

[0061] (2) Minimum detection limit: divide the HCV-cAg-removed zero value serum into 20 parts for detection, measure its concentration, calculate the mean value and standard deviation, calculate the sum of 2 times of the mean value and standard deviation, and put it on the standard curve Find out that the lowest detection line of t...

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Abstract

The invention discloses a kit for quantitatively detecting HCV-cAg concentration in human serum by virtue of polystyrene microspheres, comprising a 96-hole filter plate, a standard product, sample diluent, microsphere suspension, enzyme conjugate, substrate A, substrate B, 20* cleaning solution, adhesive sticker sealing strip and operating instruction. In the kit disclosed by the invention, a solid phase carrier is changed into polystyrene microspheres from the traditional micropore board, and reaction between antigen and antibody takes place in liquid phase, thus the antigen and antibody canbeneficially maintain bioactivity and correct space conformation, and influence of surface tension and space obstacle of the traditional reaction mode to antigen antibody reaction can be eliminated, the two advantages are all beneficial to reaction of the antigen and the antibody, and reaction time and washing frequency can be effectively reduced; meanwhile, the kit disclosed by the invention hasthe characteristics of simple operation, high sensitivity, good specificity and low cost and has important clinical significance on diagnosis of 'window period' of a hepatitis C virus infestor.

Description

technical field [0001] The invention relates to a kit for quantitatively detecting the concentration of hepatitis C virus core antigen in serum, in particular to a quantitative detection of human serum by using polystyrene microspheres specifically coated with hepatitis C virus core antigen (HCV-cAg) antibody Kit for Hepatitis C Virus Core Antigen (HCV-cAg) Concentration in Medium. Background technique [0002] Hepatitis C is caused by hepatitis C virus (Hepatitis C Virus, HCV) infection, and is mainly transmitted through blood transfusion. Chronic infection, about 70% of hepatitis C may develop into chronic hepatitis, 20% into liver cirrhosis, 12% into liver cancer, the harm is very serious. There are about 170 million people in the world carrying HCV, and it is estimated that there are 38 million HCV-infected people in my country, and the number of new cases is rising every year, and the infection rate is about 3.2%. At present, the treatment method recognized at home an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/576G01N33/544G01N21/76
Inventor 朱之炜王佳颖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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