Animal influenza antibody blocking test strip

A technology for detecting test strips and antibodies, applied to the field of poultry disease antibody detection devices, can solve the problems of census unsuitable for large-scale samples, high cost, time-consuming, etc. Easy and fast effects

Inactive Publication Date: 2019-07-26
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HI test is a classic method for influenza antibody detection, which can effectively evaluate the level of vaccine immune antibody and immune protection effect, but non-specific lectins and non-specific inhibins in serum, red blood cells and subjective judgments all affect the accuracy of HI test
Indirect ELISA has high sensitivity, is suitable for a large number of serological monitoring, can be standardized and the results are easy to analyze, but it cannot distinguish neutralizing and non-neutralizing antibodies, and cannot accurately evaluate the immune protection effect
The VN test is the most sensitive and specific method for detecting neutralizing antibodies. The level of influenza neutralizing antibodies is directly related to immune protection. However, this technical method is not only time-consuming, cumbersome and expensive, but virus culture must be performed in a Class III biosafety laboratory ( P3) The operation, technical and safety measures are extremely demanding, and it is impossible to achieve a large number of real-time detection of clinical samples
Therefore, although the above-mentioned detection methods can detect the level of influenza antibodies, they all have complex test operations, time-consuming, require specific professional skills and equipment, etc., are expensive, are often limited to laboratories, and are difficult to popularize and promote at the grassroots level. Not suitable for censuses with large batches of samples
In addition, there are large differences between the current widespread influenza virus epidemic strains and the vaccine strains commonly used in my country in terms of genetics, immune response and protective efficacy, and virulent influenza infections still occur in immunized animals from time to time. The large-scale application of serological markers and supporting differential diagnosis methods in my country makes it difficult to distinguish attenuated virus immunity from wild virus infection through antibody detection techniques such as hemagglutination inhibition test (HI) and enzyme-linked immunosorbent assay (ELISA). Conducive to the control and purification of influenza

Method used

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  • Animal influenza antibody blocking test strip
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  • Animal influenza antibody blocking test strip

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0053] (1) Preparation of influenza virus immune antigen

[0054] Inoculate 9-11-day-old SPF chicken embryos with different subtypes of influenza virus (H1N1, H3N2, H5N1, H7N9, H9N2) through the allantoic cavity, collect the allantoic fluid at 36-48 hours, and purify the virus antigen by differential centrifugation at 4°C 3000r / Centrifuge at low speed for 30 min to remove impurities, ultracentrifuge at 60,000 r / min at 4°C for 1 h, and resuspend the pellet with 7 mL of 0.01 mol / L PBS (pH 7.2). The HA titer of influenza virus was determined to be 2 by hemagglutination assay (HA). -12 above. The influenza virus was inactivated by adding 0.02% β-propiolactone (BPL) at a final concentration of 37° C. for 9 hours to the allantoic fluid of the influenza virus to prepare the inactivated virus.

[0055] (2) Preparation of anti-influenza virus monoclonal antibody

[0056] (2.1) Establishment of hybridoma cell lines

[0057] Use the inactivated influenza virus or vaccine as the immu...

Embodiment 1

[0127] Example 1: Monitoring the level of maternal antibodies to animal influenza, collecting animal serum, taking 100 μL of serum samples and adding 300 μL of PBS or saline for dilution to prepare a serum solution to be tested, and using animal influenza antibody blocking detection test paper according to the operation method (16) Detection and result judgment, detection of animal influenza maternal antibody level. Two red bands (test line and quality control line) "||" appear on the test paper, and the color intensity of the test line T is equivalent to that of the blank control, which is negative for influenza antibodies, indicating that the serum sample to be tested does not contain influenza maternal antibodies; Two red bands "||" appeared, but the detection line T was significantly weaker than that of the blank control, which was weakly positive for influenza antibodies, indicating that the serum samples to be tested contained low levels of influenza maternal antibodies, ...

Embodiment 2

[0128] Example 2: Evaluation of the immune effect of animal influenza vaccines. 2 to 3 weeks after immunization with animal influenza inactivated vaccines, collect serum from immunized animals, take 100 μL of serum samples and add 300 μL of PBS or normal saline for 1:2, 1:4, 1:8 …Double ratio dilution, prepare the serum solution to be tested, use the animal influenza antibody blocking test paper to perform detection and result judgment according to the operation method (16), and detect the animal influenza immune antibody level. Two red bands (test line and quality control line) "||" appear on the test paper, and the color intensity of the test line T is equivalent to that of the blank control, which is negative for influenza antibodies, indicating that the serum sample to be tested does not contain influenza neutralizing antibodies; Two red bands "||" appeared, but the detection line T was significantly weaker than that of the blank control, which was weakly positive for influ...

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Abstract

The invention discloses an animal influenza antibody blocking test strip, which comprises a supporting plate, an antigen pad, a conjugate pad, a detection membrane and a water absorption pad, whereinthe antigen pad is used for adsorbing an influenza virus detection antigen; the conjugate pad is used for adsorbing a colloidal gold labeled anti-influenza virus monoclonal antibody mAb1; the detection film contains detection line T"|'' and quality control line C"|'' prints; the detection line T is an anti-influenza virus monoclonal antibody mAb2 or anti-influenza virus polyclonal antibody pAb1 print; and the quality control line C is an anti-mouse IgG rabbit antibody pAb2 or staphylococcus aureus SPA print. According to the test strip, the rapid detection of the neutralizing antibody of influenza virus is realized, the real-time monitoring of an animal influenza maternal antibody and the immune antibody level and the immune evaluation of an immune effect of a vaccine can be realized, thetest strip is easy to operate and can be operated by everyone, the requirements of people at different levels can be better met, and the test strip is easy to popularize and apply in a wide range, andhas a wide market prospect and relatively great economic and social benefits.

Description

technical field [0001] The invention relates to a poultry epidemic antibody detection device, in particular to an animal influenza antibody blocking detection test paper. Background technique [0002] Influenza virus, referred to as influenza virus, is a zoonotic infectious disease pathogen that causes influenza in humans and animals. It can cause acute upper respiratory tract infection and spread rapidly through the air. Periodic pandemics occur everywhere. Influenza virus is a single-stranded negative-sense RNA virus belonging to the family Orthomyxoviridae (Orthomyxoviridae) and the genus Influenza virus. The virus genome consists of 8 segmented RNA segments, which encode polymerase (PB2, PB1, PA) (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M1, M2), and nonstructural protein (NS1, NS2) and other 10 viral proteins, according to their nucleoprotein (NP) and matrix The difference of protein (M) can divide influenza virus into three types: A, B, and C. Amo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68G01N33/577G01N33/558G01N33/531C07K16/10C07K14/11C07K1/22C12N15/70
CPCC07K14/005C07K16/1018C12N15/70C12N2760/16122C12N2760/16151C12N2800/22G01N33/531G01N33/558G01N33/56983G01N33/577G01N33/6854G01N2333/11
Inventor 李青梅张改平王彦红郭军庆刘肖石建州李鸽孙亚宁杨继飞柴书军赵东
Owner HENAN ACAD OF AGRI SCI
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