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Kit capable of determining titer of neutralizing antibodies of varicella-zoster viruses and production method thereof

A herpes zoster virus and kit technology, which is applied in the field of kits for determining varicella-zoster virus neutralizing antibody titers, can solve the problems of inability to achieve high-throughput determination, limited sample volume, high cost, and the like

Active Publication Date: 2015-02-18
SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method also has obvious disadvantages: cell culture and virus culture are required, and a laboratory that meets biosafety standards is required; the experiment period is long, usually more than 10 days; the sample size for determination is limited, and high-throughput determination cannot be achieved
However, the FAMA method also has its own shortcomings: it requires an expensive fluorescence microscope; the fluorescence intensity is judged by human eyes, which is highly subjective, which is not conducive to the promotion among different laboratories; it still needs cell culture and virus culture
However, since the neutralizing antigen (viral glycoprotein) is located on the viral envelope and the cell membrane of infected cells, purification is very difficult, costly, and easily inactivated during the purification process

Method used

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  • Kit capable of determining titer of neutralizing antibodies of varicella-zoster viruses and production method thereof
  • Kit capable of determining titer of neutralizing antibodies of varicella-zoster viruses and production method thereof
  • Kit capable of determining titer of neutralizing antibodies of varicella-zoster viruses and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Optimal well-coated cell number studies. If the number of coated cells per well is too small and the amount of antigen is insufficient, the detection limit of the kit will be too low; if the number of coated cells is too large, the cells will cause light scattering, which will affect the measurement results of the absorbance value during the measurement process.

[0050] Human embryonic lung fibroblasts grown as dense monolayers were inoculated with VZV seeds. The inoculation ratio was: 200 plaque-forming units (PFU) of VZV virus were inoculated per 1000 cells. Cultured at 37°C and 5% carbon dioxide for 60 hours, the cytopathic rate was 70%, the cells were digested into single cells with 0.25% trypsin, and bovine serum albumin (final concentration 5%) was added to adjust the cell density to 3×10 4 pcs / ml, 6×10 4 pcs / ml, 1.2×10 5 pcs / ml, 2×10 5 pcs / ml, 4×10 5 pcs / ml, 6×10 5 pcs / ml, 1×10 6 pieces / ml. The above-mentioned cells of different concentrations...

Embodiment 2

[0060] Example 2 Research on the distribution of coated cells between wells and the shedding of coated cells after washing and shaking.

[0061] Human embryonic lung fibroblasts grown as dense monolayers were inoculated with VZV seeds. The inoculation ratio was: 200 plaque-forming units (PFU) of VZV virus were inoculated per 1000 cells. Cultured at 37°C and 5% carbon dioxide for 60 hours, the cytopathic rate was 70%, the cells were digested into single cells with 0.25% trypsin, and bovine serum albumin (final concentration 5%) was added to adjust the cell density to 6×10 4 pcs / ml, 1.2×10 5 pcs / ml, 2×10 5 pieces / ml. The above-mentioned cells of different concentrations were thoroughly mixed and suspended, and added to different wells of a 96-well plate (12 wells for each concentration were repeated), and 5 microliters of cell suspension was added to each well. Shake the 96-well plate horizontally to distribute the cell suspension evenly at the bottom of the well. Add 5 m...

Embodiment 3

[0065] Example 3 Kit preparation and sample test result analysis

[0066] 1. 96-well plate preparation

[0067] A. Human embryonic lung fibroblasts (MRC5, 75cm) growing into a dense monolayer 2 , about 3 x 10 6 cells) inoculated with varicella-zoster virus (VZV, oka strain) 3×10 5 PFU, cultured at 37°C and 5% carbon dioxide for 3 days, the microscopic examination of the cell lesion reached 70%, the cells were digested with 0.25% trypsin, dispersed into individual cells by pipetting, centrifuged, and the cells were washed with PBS, and 20ml containing 5% The PBS solution of bovine serum albumin resuspended the cells, and the count was 1.6×10 with a cell counting plate. 5 pieces / ml.

[0068] B. Prepare 40 96-well plates, and add the above-mentioned mixed cells into 96-well plates at 5 μl / well using a 12-channel pipette. Shake the 96-well plate horizontally to distribute the cell suspension evenly at the bottom of the well. Add 5 microliters of 4% glutaraldehyde solution ...

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Abstract

The invention relates to the technical field of in-vitro diagnostic reagents and particularly relates to a kit capable of determining the titer of neutralizing antibodies of varicella-zoster viruses and a production method thereof. The kit adopts cells infected by VZV as antigens, is coated and fixed in holes of a 96-pore plate, the neutralizing antibodies in the sample to be determined are captured by using a large amount of virus glycoprotein (neutralizing glycoprotein) carried on the surfaces of the cells, simultaneously the integrity of the cells is maintained; and the other virus antigens inside are not contacted with the sample, so that the specificity of the neutralizing antibodies in determination can be guaranteed. The kit can be directly used for fast determination of the titer of the neutralizing antibodies in VZV on an automatic biochemical analyzer or an enzyme labeling instrument; the determination process is simple and fast, the flux of the determined sample is high and quantitative determination can be realized.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnostic reagents, in particular to a kit capable of measuring the titer of varicella-zoster virus neutralizing antibodies and a production method thereof. technical background [0002] Varicella-zoster virus (VZV) is the pathogen of human chickenpox and herpes zoster. It is highly contagious and widely spread. It is one of the common contact infectious diseases in childhood. %above. Susceptible individuals refer to individuals who do not have VZV neutralizing antibodies in their bodies, or whose neutralizing antibody levels do not reach the protective level. In general, people who have suffered from chickenpox and who have been vaccinated against varicella-zoster will produce VZV neutralizing antibodies above the protective level. However, the varicella-zoster vaccine is not yet included in the scope of immunization in my country, and the penetration rate is low; and the varicella disease us...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/531
CPCG01N33/531G01N33/54313
Inventor 朱孟沼马杰巩艳艳陈振菅长永马山
Owner SHANDONG TAIBANG BIOLOGICAL PROD CO LTD
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