qx type ibv hemagglutination inhibition test antigen and its preparation method and application

A technology of hemagglutination inhibition test and antigen, which is applied in the biological field, can solve the problems of inability to carry out QX type IBV, decrease in accuracy rate, and distant evolutionary relationship, and achieve improved reliability and operability, high specificity, and high sensitivity Effect

Active Publication Date: 2020-06-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the hemagglutination inhibition test antigen of chicken infectious bronchitis virus (M41 strain) has been successfully prepared at home and abroad, because the strain belongs to the Mass type, it is far from the evolutionary relationship with the current domestic main epidemic strains (QX type), resulting in False negatives are often shown in production practice testing, so the accuracy rate of M41 strain hemagglutination inhibition test antigens in clinical testing is greatly reduced
Therefore, the detection of QX-type IBV cannot be carried out in the farm. Once an epidemic occurs, it will cause huge losses and easily cause the rapid spread of the virus, thereby causing greater harm.

Method used

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  • qx type ibv hemagglutination inhibition test antigen and its preparation method and application
  • qx type ibv hemagglutination inhibition test antigen and its preparation method and application
  • qx type ibv hemagglutination inhibition test antigen and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Preparation and Concentration of Example 1 IBV SZ Strain Virus Liquid

[0050] (1) Propagation of the IBV SZ strain: the allantois of the IBV SZ strain preserved at -80°C was passed through the allantoic cavity, according to 10 5 EID 50 Inoculate 9-11 day-old SPF chicken embryos, 0.2mL / embryo. Incubate in a constant temperature incubator at 37°C, observe once every 6 hours, discard the dead embryos within 24 hours, take out the chicken embryos after 36 hours of incubation, cool at 4°C for 10 hours, and aseptically collect the allantoic fluid of the chicken embryos. Viral allantoic fluid was used or stored in a -80°C refrigerator for later use.

[0051] (2) Concentration of IBV SZ strain: the IBV virus allantoic fluid is centrifuged at 8000rpm for 45min, and the supernatant is obtained; centrifuged at 40000rpm for 2 hours with a high-speed centrifuge, the supernatant is discarded, and the IBV virus fluid (supernatant) is used for precipitation after the first centrifug...

Embodiment 2

[0052] Example 2 Exploration of the action time of type I phosphatase C (PLC1)

[0053] Preparation of hemagglutination inhibitory antigen of IBV SZ strain: Add 200-fold concentrated IBV virus liquid to type I phosphatase C (PLC1) to make the final concentration of PLC1 2.5U / mL, mix well, and then place the virus liquid in 37°C constant temperature oscillation 200r / min for 2 hours, take it out at 4°C and place it for several days for treatment, every 3 days, measure the hemagglutination titer of the antigen, the test results show that the concentrated antigen HA titer of the IBV QX type SZ strain after PLC1 treatment Increased with the different storage time at 4°C ( figure 1 ), after 21 to 35 days, the titer of HA was the highest and remained stable, and the IBV-HI antigen was obtained. The results of hemagglutination titer determination are shown in Table 1 (the titer reaches 1:256 from 21 days to 35 days):

[0054] Table 1 Hemagglutination titer of IBV SZ strain hemagglut...

Embodiment 3

[0056] Example 3 Optimum reaction temperature and time optimization of IBV SZ strain HI antigen HA test

[0057] Utilize the antigen that embodiment 1 and 2 prepare, the condition determined with reference to embodiment is to the optimal reaction temperature of IBV SZ strain HI antigen HA test and the optimal operation method of time as follows:

[0058] IBV-HI antigen HA test operation:

[0059] ① Carry out on a 96-well microplate, from the 1st well to the 12th well, add 25 μL of normal saline to each well.

[0060] ② Add 25 μL of the HI antigen prepared in Example 1 to the first well on the left side, mix well, then pipette 25 μL into the second well, then serially dilute to the 12th well, and discard 25 μL.

[0061] ③ Add 25 μL of 0.5% fresh chicken erythrocyte suspension to each well successively from right to left, and shake on the shaker for 30 seconds.

[0062] Then place the hemagglutination plate under different conditions respectively, at 2-8°C for 30 minutes; at 2...

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Abstract

The invention relates to QX-type infectious bronchitis virus (IBV) hemagglutination inhibition test antigens, a preparation method thereof and application. A preparation method for an HI antigen comprises the following steps of inoculating 10<4>-10<7> EID<50> of QX-type IBV strains to chicken embryos, carrying out incubation for virus multiplication at a temperature of 37 degrees centigrade, after36-48h, taking out the chicken embryos, carrying out cooling for 6-16h at the temperature of 2-8 degrees centigrade, collecting chicken embryo allantoic liquid, uniformly mixing concentrated chickenembryo allantoic liquid with I-type phosphatase C, carrying out shaker action for 2-2.5h at the temperature of 37 degrees centigrade, and then carrying out placement for 21-35d at the temperature of 2-8 degrees centigrade to obtain antigens. The HI antigens and the detection method provided by the invention are characterized by high specificity, high stability, high sensitivity and high efficiency. Rapid detection can be carried out on clinical serum samples within 2h. The HI antigens and the detection method can be widely applied to quarantine and diagnosis of IB and detection of immunized antibodies. The HI method for detecting QX-type infectious bronchitis becomes feasible. A practical and scientific method for prevention of the IB is provided. The prevention and control over the disease in poultry production is guided well.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a QX type IBV hemagglutination inhibition test antigen. Background technique [0002] Infectious Bronchitis (IB) is an acute, highly contagious respiratory and genitourinary disease caused by Infectious Bronchitis Virus (IBV). The typical clinical features are: Sick chickens have dyspnea, tracheal rales and other respiratory symptoms. The QX-type virus strain will cause kidney swelling and paleness, showing "mottled kidneys", which will cause the death of chickens. Infection of the disease at the chick stage will also cause permanent damage to the reproductive system of hens. sexual harm. [0003] At present, a large number of public reports indicate that QX type (nephropathy type) has replaced Mass type as the main domestic IBV strain type, which has brought great harm to the poultry industry. Therefore, rapid and accurate monitor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/531
CPCG01N33/531G01N33/56983
Inventor 张国中赵静谢德琼颜世红
Owner CHINA AGRI UNIV
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