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Application of QX type IBV cellular adapted strain MJ to preparation of totivirus antigen ELISA inspection kit and kit

A technology for detecting kits and adapting strains, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of low specificity of ELISA detection kits, and achieve the effects of excellent sensitivity, low cost and strong specificity

Active Publication Date: 2019-04-05
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, in order to solve the problem of low specificity of the ELISA detection kit of the existing IBV, the present invention provides the application of a cell-adapted strain MJ of QX type IBV in the preparation of the whole virus antigen ELISA detection kit, using the The cell-adapted strain MJ has the characteristics of high specificity, no other virus antigens, and simple purification, and can prepare a highly sensitive and specific whole virus antigen indirect ELISA detection kit

Method used

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  • Application of QX type IBV cellular adapted strain MJ to preparation of totivirus antigen ELISA inspection kit and kit
  • Application of QX type IBV cellular adapted strain MJ to preparation of totivirus antigen ELISA inspection kit and kit
  • Application of QX type IBV cellular adapted strain MJ to preparation of totivirus antigen ELISA inspection kit and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] The whole virus antigen indirect ELISA detection kit of QX type IBV includes the following components:

[0098] A detection plate coated with an inactivated cell-adapted strain MJ (biological deposit number CGMCC No.14681);

[0099] The coating buffer: carbonate buffer at pH 9.6;

[0100] The antibody diluent: PBS buffer solution with a mass volume concentration of 2% skimmed milk;

[0101] The washing solution: PBS buffer (PBST) with a volume concentration of 0.05% Tween-20;

[0102] Described standard positive serum: anti-IBYZ isolate (preservation number is CGMCC No.14682) and the SPF chicken serum of M41 strain;

[0103] The standard negative serum: 7 days old negative SPF chicken serum;

[0104] The enzyme-labeled secondary antibody: HRP-labeled goat anti-chicken IgG antibody;

[0105] The chromogenic solution: TMB substrate chromogenic solution;

[0106] The stop solution is 2mol / L sulfuric acid solution.

[0107] (1) Preparation of inactivated QX type IBV c...

Embodiment 2

[0129] The method of using the whole virus antigen indirect ELISA detection kit of QX type IBV

[0130] (1) Dilute the serum to be tested with antibody diluent 1:200, add 100 μL / well primary antibody, and add standard positive serum and negative serum control at the same time, act at 37°C for 1 hour, wash the plate 5 times with 300 μL / well PBST;

[0131] (2) Dilute the HRP-labeled goat anti-chicken IgG secondary antibody with antibody diluent 1:10000, add 100 μL / well, react at 37°C for 1 hour, wash the plate 5 times with 300 μL / well PBST;

[0132] (3) Color development: add 100 μL / well of TMB color development solution, and protect from light at 37°C for 15 minutes;

[0133] (4) Termination: add 50 μL / well stop solution;

[0134] (5) Result determination:

[0135] OD on microplate reader 450nm Calculate the S / P value of the reading value:

[0136] S / P value = (OD of serum to be tested 450nm Mean - negative control OD 450nm mean) / (positive control OD 450nm Mean - negativ...

Embodiment 3

[0138] Take the inactivated virus supernatants prepared in Example 1 and Comparative Examples 1-4 respectively, dilute them with pH 9.6 carbonate buffer at a volume ratio of 1:40, and add them to 96-well microtiter plates respectively , after coating at 4°C for 24 hours, it reacted with standard positive serum and standard negative serum, and tested according to the method shown in Example 2, and the results are shown in Table 1.

[0139] Table 1 Comparison of ELISA readings of different antigen inactivation methods

[0140]

[0141] From the test results (Table 1), after inactivation at 56°C, the ELISA positive reading value was the lowest, and the positive serum reading value of the BEI inactivation method was the highest. down, but with the highest P / N value. It can be seen that the present invention chooses the β-propiolactone 1:1000 inactivation method to prepare the coating antigen detected by ELISA, which is easy to operate and has the best safety.

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Abstract

The invention provides application of a QX type IBV cellular adapted strain MJ to preparation of a totivirus antigen ELISA inspection kit and the kit, and belongs to the technical field of virus antibody inspection. According to the application of the QX type IBV cellular adapted strain MJ to the preparation of the totivirus antigen ELISA inspection kit, the preservation number of the QX type IBVcellular adapted strain MJ is CGMCC No. 14681, the cellular adapted strain MJ does not contain other viral antigens and is only in specific binding with an IBV positive serum, and cross reaction between the cellular adapted strain MJ and other virus positive serums is avoided, effective proliferation is conducted on a Vero cell, and the cellular adapted strain MJ is applied to effectively solve the problems that purification of IBV totivirus is difficult and non-specificity is high. The invention also provides the totivirus antigen indirect ELISA inspection reagent box constructed based on thecellular adaptive strain MJ. According to the totivirus antigen indirect ELISA inspection kit, detection sensitivity is high, specificity is strong, QX type IBV early infection can be inspected, andearly antibody level of chicken flock immune from attenuated vaccine can also be inspected.

Description

technical field [0001] The invention relates to the technical field of virus antibody detection, in particular to the application and kit of the cell-adapted strain MJ of QX type IBV in the preparation of whole virus antigen ELISA detection kit. Background technique [0002] Chicken infectious bronchitis (IB) is an acute and highly contagious respiratory infectious disease caused by chicken infectious bronchitis virus (IBV). The disease is widely prevalent in the world and is one of the main viral infectious diseases that seriously endanger the poultry industry. Because IBV is very easy to mutate, new mutant strains appear constantly, and the cross protection between different strains is weak, so that the disease often breaks out in immune and non-immune poultry flocks, causing huge economic losses and seriously restricting the The healthy development of my country's poultry industry. [0003] At present, the laboratory diagnosis methods of IBV mainly include: virus isolati...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/54306G01N33/56983
Inventor 姜逸周生唐梦君程旭俞燕赵秀美高明燕戴有理
Owner JIANGSU INST OF POULTRY SCI
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