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A kind of colloidal gold test strip, kit and detection method for rapid detection of canine parvovirus antibody hemagglutination inhibition titer

A technology of colloidal gold test paper and canine parvovirus, which is applied to measuring devices, instruments, scientific instruments, etc., to achieve the effects of shortening operation time, expanding the scope of use, and reducing requirements

Active Publication Date: 2017-07-28
北京世纪元亨动物防疫技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no simple and rapid colloidal gold test strip that can be used for the determination of HI titer of CPV antibody in canine serum

Method used

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  • A kind of colloidal gold test strip, kit and detection method for rapid detection of canine parvovirus antibody hemagglutination inhibition titer
  • A kind of colloidal gold test strip, kit and detection method for rapid detection of canine parvovirus antibody hemagglutination inhibition titer
  • A kind of colloidal gold test strip, kit and detection method for rapid detection of canine parvovirus antibody hemagglutination inhibition titer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1, preparation of canine parvovirus therapeutic monoclonal antibody

[0040] 1. Hybridoma culture:

[0041]The hybridoma monoclonal antibody cell line (preservation number CGMCC No. 4304) was expanded and cultured, from 96 wells to 24 wells, and then expanded to T25 cell bottle expansion culture. Then the cells in the cell bottle were collected and injected into the peritoneal cavity of the mouse. After 8 to 9 days, the ascites was collected. The obtained ascites was centrifuged at 12,000 rpm for 30 minutes to remove grease and sediment. The supernatant was filtered with a 0.22 μm microporous membrane and frozen for later use.

[0042] 2. Purification of monoclonal antibodies:

[0043] Add an equal volume of PBS buffer to the above-mentioned filtered monoclonal antibody ascites, separate and purify with Protein G Sepharose 4B chromatographic column, collect the protein elution peak, dialyze with PBS with pH 7.2 overnight, and use a filter membrane with a molecu...

Embodiment 2

[0044] Embodiment 2, the colloidal gold test strip prepared by canine parvovirus therapeutic monoclonal antibody

[0045] 1. Preparation and identification of colloidal gold

[0046] Take 100ml of 0.01% chloroauric acid aqueous solution and heat it to boiling, accurately add 2ml of 1% trisodium citrate aqueous solution under stirring, the golden yellow chloroauric acid aqueous solution will turn purple within 2 minutes, continue to boil for 15 minutes, cool and distilled water Return to the original volume, the gold sol prepared in this way has the maximum absorption peak at 530±5nm. After cooling, add ultrapure water to restore to the original volume, and store at 4°C. The fired colloidal gold eye is purple-red, transparent and clear; its maximum absorption peak is 530nm as measured by a spectrophotometer, and its absorbance value is 0.954. The size of the fired colloidal gold was observed through an electron microscope to be uniform and scattered; the size of 100 colloidal...

Embodiment 3

[0065] Embodiment 3, the method for rapidly detecting CPV antibody HI titer in canine serum

[0066] 1. Determination of antigenic units

[0067] Dilute the standard antigen from 1:2 to 1:2048 with PBS, draw 80 μl drop by drop and slowly drop it into the sample hole of the test strip prepared above, and judge the result within 30 minutes. When red bands appear on the C line and T line at the same time, the antigen with the highest dilution factor is defined as 1 antigen unit. The highest dilution factor is 1:512, that is, when the standard antigen is diluted 512 times, it is 1 antigen unit.

[0068] 2. Determination of the amount of reactive antigen

[0069] Prepare standard antigens of 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 antigen unit (that is, the standard antigens are not diluted, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512 dilution); standard serum 1 and standard serum 2 were first diluted 10 times with PBS, and then diluted to 1 : 10240 for standby; use a...

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Abstract

The invention discloses a colloidal gold test strip for rapidly detecting the hemagglutination inhibition titer of a canine parvovirus antibody, and a kit and a detection method thereof. When the test strip prepared by using the CPV (canine parvovirus) therapeutic monoclonal antibody is used in the quantitative detection process, canine serum is double-diluted from 1:10, the diluted serum is neutralized with a CPV standard antigen with 16 antigen units, the neutralized serum is dripped to the test strip, the serum highest dilution ratio when the detection line (T line) of the test strip disappears is a test strip detection tilter (CGIA tilter), and the antibody HI titer in the canine serum is obtained through multiplying the CGIA tilter by 4; when a standard antigen with 32 antigen units is used, the HI tilter is obtained through multiplying the CGIA tilter by 8; when a standard antigen with 8 antigen units is used, the HI tilter is obtained through multiplying the CGIA tilter by 2; and when a standard antigen with 4 antigen units is used, the HI tilter equals to the CGIA tilter. The coincidence rate of the result of the method to the result of a hemagglutination inhibition detection method reaches 90.7%, and the coincidence rate of an antibody qualitative detection result reaches 98.8%, and the method can be used to evaluate the immunization effect of CPV vaccines.

Description

technical field [0001] The invention belongs to the field of veterinary biotechnology, and relates to a colloidal gold test strip, a kit and a detection method for rapidly detecting the hemagglutination inhibition titer of canine parvovirus antibody. Background technique [0002] Canine parvovirus (Canine Parvovirus, CPV) belongs to the Parvoviridae family and the genus Parvovirus. It is an autonomously replicating single-stranded DNA virus with a diameter of about 22nm, an icosahedral stereosymmetric structure, no capsule, and 32 capsomers. CPV can cause acute, contact, and highly fatal infectious diseases in dogs. The main clinical features are severe vomiting, hemorrhagic enteritis or non-suppurative myocarditis and a significant decrease in white blood cells. There are two clinical manifestations, hemorrhagic enteritis and myocarditis type. Puppies are the most susceptible, with a morbidity rate of 50-100% and a case-fatality rate of 10-50%. It is one of the most import...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558
CPCG01N33/558G01N33/56983G01N33/577G01N2333/015
Inventor 孙明陈西钊申屠芬琴马永缨杨欣艳
Owner 北京世纪元亨动物防疫技术有限公司
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