CN104730259A - Method for detecting hemagglutination inhibition antibody of chicken infectious bronchitis - Google Patents
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Abstract
The invention relates to the technical field of livestock vaccine, and in particular relates to a method for detecting a hemagglutination inhibition antibody of chicken infectious bronchitis. The method comprises the following steps: treating serum to be detected, namely, by taking kaoline suspension as a serum treatment liquid for treating serum to be detected, adding the supernate into chicken erythrocyte blood corpuscle mud so as to obtain serum which is diluted in a ratio of 1:4 and is to be detected; performing hemagglutination inhibition test, namely, adding 25mu l of the serum which is diluted in a ratio of 1:4 and is to be detected into 25mu l of antigen with 4HA units, and performing hemagglutination judgment. By adopting the negative serum treated by using a method for removing non-specific reaction of the serum, the infectious bronchitis hemagglutination inhibition evaluation titer is less than 1:4, and by adopting the infectious bronchitis positive serum treated by using the method, the infectious bronchitis hemagglutination inhibition evaluation titer is reduced by 0.5-1 titer, the non-specificity reaction is removed, and the accuracy of the infectious bronchitis hemagglutination inhibition experiment result is improved.
Description
Technical field
The invention belongs to poultry vaccine technical field, be specifically related to a kind of infectious bronchitis of chicken hemagglutination inhibition antibody detection method.
Background technology
Infectious bronchitis of chicken (IB) is the acute high degree in contact breathing problem of one caused by IBV (IBV), with tracheae rale, coughs, sneezes; Children chicken is had a running nose, and laying hen egg production reduces and the quality of egg drops to feature; Ill chicken often causes death because of respiratory tract, kidney or infection of digestive canal.Pass an antigenic characteristic, virus itself is coagulation, just must can discharge the erythrocytic acceptor of aggegation, occur coagulation through special processing.The efficacy test of current infectious bronchitis seedling mainly adopts attacks malicious guard method.Attack poison protection and there is loose malicious risk, therefore find serology alternative method very necessary, in serological method, blood clotting and blood clotting suppress simple possible the most.
The method of the at present both at home and abroad removal serum nonspecific reaction of research is a lot, as add chicken red blood cell process, 56 DEG C heat and add polypropylene glycol process etc., but these method results are all not good.The nonspecific reaction of infective bronchitis hemagglutination-inhibition test serum is just higher in addition, and the nonspecific reaction of removing it is relatively more difficult.
Summary of the invention
The object of the present invention is to provide a kind of method of infectious bronchitis of chicken hemagglutination-inhibition test serum treating fluid and process serum, is the method for removing serum nonspecific reaction in infective bronchitis hemagglutination-inhibition test.Adopt the present invention to remove the negative serum of the method process of nonspecific reaction in serum, infective bronchitis blood clotting suppresses to tire equal <1:4.
The technical solution used in the present invention is:
A kind of infectious bronchitis of chicken hemagglutination inhibition antibody detection method, comprises process and the hemagglutination-inhibition test of serum to be checked, it is characterized in that: concrete steps are as follows:
Step 1: the process of serum to be checked
(1) get porcelain earth 25g, add 0.85% physiological saline to 100ml, be the porcelain earth suspension of 25%, 2 ~ 8 DEG C save backup;
(2) get 40 μ l serum to be checked, add serum treating fluid prepared by 120 μ l (1), vibration mixing, room temperature places 30 minutes, then with 3000r/min centrifugal 10 minutes, get supernatant;
(3) add 2 μ l chicken red blood cell blood cell mud in gained supernatant, slight oscillatory mixes, and room temperature places 30 minutes, then with 3000r/min centrifugal 10 minutes, get the serum to be checked that supernatant is 1:4 dilution;
Step 2: hemagglutination-inhibition test
(1) get 96 hole V-type micro-reaction plates, every hole adds 25 μ l PBS;
(2) draw the serum to be checked of the 1:4 dilution that 25 μ l steps 1 obtain respectively, add in each respective aperture of first row of every block, and establish standard positive serum and negative serum control on every block plate, then 2 times of serial dilutions;
(3) respectively to the antigen 25 μ l added in each hole containing 4HA unit, 2 ~ 8 DEG C leave standstill 30 minutes;
(4) add 0.5% chicken erythrocyte suspension 25 μ l in every hole, mix gently, 2 ~ 8 DEG C leave standstill 40 minutes;
(5) result judges:
Reaction plate is tilted, in all seroreaction holes and red blood cell control wells, red blood cell is judged to blood clotting from the hole underflow person of dropping down at the same rate and suppresses, when negative serum HI tire≤2.0log2, positive serum HI tire to tire phase ratio error≤2.0log2 with regulation time, test can be set up, and tires to suppress the most high dilution of the serum of 4HA unit antigen completely as HI.
Preferably, the preparation method of the chicken red blood cell blood cell mud described in step 1 is: add anti-coagulants and adopt SPF chicken blood, wash, mix gently, 3500r/min, suck supernatant, so repeatedly wash 3 times with PBS or physiological saline, namely obtain chicken red blood cell blood cell mud.
Described anti-coagulants is Ah's test solution or liquaemin, is commercially available prod.SPF refers to the rank of chicken.
Beneficial effect of the present invention:
With the negative serum of method process removing nonspecific reaction in serum in the present invention, infective bronchitis blood clotting suppresses to tire equal <1:4; With the infective bronchitis positive serum of the method process, infective bronchitis blood clotting suppresses to tire reduction by 0.5 ~ 1 titre, eliminates nonspecific reaction, improves the accuracy of infective bronchitis hemagglutination-inhibition test result.
Embodiment
Embodiment 1
One, antigen preparation
1, breathing pattern IBV-M41 strain, Glandular Stomach Type IBV-YBX strain and kidney type IBV-T strain seed culture of viruses are used stroke-physiological saline solution 100 times dilution respectively, inoculation 10 age in days SPF chicken embryos in allantoic cavity, every embryo 0.1ml, puts 37 DEG C of hatching case hatchings, 24 hours photograph embryos 1 time, discard dead germ.Until 48 hours, no matter death whether, is all taken out, put 4 DEG C of refrigerator coolings.According to said method often kind of antigen prepares 3 batches.
Wherein, breathing pattern IBV-M41 strain and kidney type IBV-T strain seed culture of viruses, be purchased from China Veterinery Drug Inspection Office.Glandular Stomach Type IBV-YBX strain gathers voluntarily for the applicant.
2, collect allantoic fluid, load in 500ml centrifugal bottle, centrifugal 30 minutes of 3000rpm, gets supernatant.
3, loaded by supernatant in 40ml centrifuge tube, through 30000g, 4 DEG C centrifugal 3 hours.
4, supernatant discarded, often manages the Tris-HCL damping fluid adding 1M pH6.5, blows and beats, and mixing, by viral suspension collection in a pipe.
5, get viral suspension and add 2U/ml LecithinaseC mixed in equal amounts.
6, by virus mixture 37 DEG C of water-baths 2 hours, every vibration mixing in 15 minutes.Last 4 DEG C are spent the night, and are IBV-HA antigen.
7, respectively IBV-HA antigen is added 1% thimerosal, final concentration is 0.01%, and limit edged mixes, and mixes rear 4 DEG C of preservations.Obtain that M41-1 criticizes, M41-2 criticizes, M41-3 criticizes; YBX-1 criticizes, YBX-2 criticizes, YBX-3 criticizes; T-1 criticizes, T-2 criticizes, T-3 criticizes.
By freeze-dried mixed with stabilizing agent respectively for above 9 kinds of antigens.
Two, the process of serum to be checked
1, get porcelain earth 25g, add 0.85% physiological saline to 100ml, be the porcelain earth suspension of 25%, 2 ~ 8 DEG C save backup.
2, with 25% porcelain earth suspension as serum treating fluid, get 40 μ l serum to be checked during operation, add 120 μ l serum treating fluids, vibration mixing, room temperature places 30 minutes, then with 3000r/min centrifugal 10 minutes, gets supernatant.
3, with the process of blood cell mud, add about 2 μ l chicken red blood cell blood cell mud in 1.3.2 gained supernatant, slight oscillatory mixes, and room temperature places 30 minutes, then with 3000r/min centrifugal 10 minutes, get the serum to be checked that supernatant is 1:4 dilution.
Three, hemagglutination test
Get 96 hole V-type micro-reaction plates and hemagglutinative titer detection is carried out to the antigen of above-mentioned preparation, every hole adds 25 μ lPBS (0.01mol/L, pH value 7.0 ~ 7.4), first row adds 25 μ l antigens, and do 2 ~ 4 repeating holes, then antigen is carried out 2 times of serial dilutions, after dilution, every hole adds 25 μ l PBS again, finally add 0.5% chicken erythrocyte suspension 25 μ l again, mix with micro oscillator, 2 ~ 8 DEG C leave standstill 40 minutes result of determination, to make the most highly diluted multiple of the antigen of 100% red cell agglutination as judgement terminal.The HA-HI test of antigen prepared by result all at more than 9log2, the results detailed in Table 1.
Table 1 antigen blood clotting bioactivity result
Four, hemagglutination-inhibition test
(get 96 hole V-type micro-reaction plates, every hole adds 25 μ l PBS to detect IBV-M41 strain inactivated vaccine, IBV-YBX strain inactivated vaccine and IBV-T strain inactivated vaccine immune serum respectively with corresponding antigens by the following method; Draw 25 μ l respectively through the serum to be checked of above-mentioned process, add in each respective aperture of first row of every block, and establish standard positive serum and negative serum control on every block plate, then 2 times of serial dilutions; Respectively to the antigen 25 μ l added in each hole containing 4HA unit, 2 ~ 8 DEG C leave standstill 30 minutes; Add 0.5% chicken erythrocyte suspension 25 μ l in every hole, mix gently, 2 ~ 8 DEG C leave standstill 40 minutes; Result judges reaction plate to tilt, and in all seroreaction holes and red blood cell control wells, red blood cell is judged to blood clotting from the hole underflow person of dropping down at the same rate and suppresses.When negative serum HI tire≤2.0log2, positive serum HI tire to tire phase ratio error≤2.0log2 with regulation time, test can be set up.Tire to suppress the most high dilution of the serum of 4HA unit antigen completely as HI.) result immune group serum blood clotting blood clotting suppresses the >=4log2 that tires all., the results detailed in Table 2.
Table 2 hemagglutination-inhibition test result
From above result, the blood clotting result indifference that the blood clotting result detected with the homemade antigen in laboratory and import antigen detect; The blood clotting of untreated positive serum, negative serum suppresses to tire all higher than processed group, and suppress the to tire blood clotting suppression of more processed positive serum of the blood clotting of untreated positive serum is tired high 0.5 ~ 1 titre; The blood clotting of untreated negative serum suppresses to tire generally at 3Log2 ~ 4Log2, and the blood clotting of processed negative serum suppresses to tire all to be less than 1:4.These results suggest that, the method removing serum nonspecific reaction in the present invention obviously can remove nonspecific reaction.Improve the accuracy of test findings.
Above-described embodiment is illustrative instead of determinate, can list several embodiments, therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention according to institute's limited range.
Claims (2)
1. an infectious bronchitis of chicken hemagglutination inhibition antibody detection method, comprises process and the hemagglutination-inhibition test of serum to be checked, it is characterized in that: concrete steps are as follows:
Step 1: the process of serum to be checked
(1) get porcelain earth 25g, add 0.85% physiological saline to 100ml, be the porcelain earth suspension of 25%, 2 ~ 8 DEG C save backup;
(2) get 40 μ l serum to be checked, add serum treating fluid prepared by 120 μ l (1), vibration mixing, room temperature places 30 minutes, then with 3000r/min centrifugal 10 minutes, get supernatant;
(3) add 2 μ l chicken red blood cell blood cell mud in gained supernatant, slight oscillatory mixes, and room temperature places 30 minutes, then with 3000r/min centrifugal 10 minutes, get the serum to be checked that supernatant is 1:4 dilution;
Step 2: hemagglutination-inhibition test
Get 96 hole V-type micro-reaction plates, every hole adds 25 μ l PBS;
Draw the serum to be checked of the 1:4 dilution that 25 μ l steps 1 obtain respectively, add in each respective aperture of first row of every block, and establish standard positive serum and negative serum control on every block plate, then 2 times of serial dilutions;
Respectively to the antigen 25 μ l added in each hole containing 4HA unit, 2 ~ 8 DEG C leave standstill 30 minutes;
Add 0.5% chicken erythrocyte suspension 25 μ l in every hole, mix gently, 2 ~ 8 DEG C leave standstill 40 minutes;
Result judges:
Reaction plate is tilted, in all seroreaction holes and red blood cell control wells, red blood cell is judged to blood clotting from the hole underflow person of dropping down at the same rate and suppresses, when negative serum HI tire≤2.0log2, positive serum HI tire to tire phase ratio error≤2.0log2 with regulation time, test can be set up, and tires to suppress the most high dilution of the serum of 4HA unit antigen completely as HI.
2. infectious bronchitis of chicken hemagglutination inhibition antibody detection method according to claim 1, it is characterized in that, the preparation method of the chicken red blood cell blood cell mud described in step 1 is: add anti-coagulants and adopt SPF chicken blood, wash with PBS or physiological saline, mix gently, 3500r/min, suck supernatant, so repeatedly wash 3 times, namely obtain chicken red blood cell blood cell mud.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866427A (en) * | 2016-04-22 | 2016-08-17 | 北京市农林科学院 | Composition, and application thereof in infectious bronchitis antibody determination |
| CN106442986A (en) * | 2016-09-21 | 2017-02-22 | 华南农业大学 | Method for detecting regional SIV (swine influenza virus) subtype distribution |
| CN107328944A (en) * | 2017-06-14 | 2017-11-07 | 王琴 | A kind of blood clotting and hemagglutination-inhibition test screening technique |
| CN110007070A (en) * | 2019-04-13 | 2019-07-12 | 北京家禽育种有限公司 | A kind of HI test serum pre-dilution method |
| CN111198265A (en) * | 2018-11-20 | 2020-05-26 | 内蒙古正大食品有限公司 | Combined rapid detection method for various poultry antibodies |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA943066A (en) * | 1968-10-31 | 1974-03-05 | Betty L. Lanius | Hemagglutination inhibition test for viruses |
| CN101957362A (en) * | 2010-09-02 | 2011-01-26 | 洛阳普莱柯生物工程有限公司 | Efficacy test method of infectious bronchitis vaccines and application thereof |
| CN102093999A (en) * | 2010-12-14 | 2011-06-15 | 山东省农业科学院家禽研究所 | Indirect ELISA kit for detecting avian infectious bronchitis virus antibody |
-
2015
- 2015-03-12 CN CN201510108455.XA patent/CN104730259A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA943066A (en) * | 1968-10-31 | 1974-03-05 | Betty L. Lanius | Hemagglutination inhibition test for viruses |
| CN101957362A (en) * | 2010-09-02 | 2011-01-26 | 洛阳普莱柯生物工程有限公司 | Efficacy test method of infectious bronchitis vaccines and application thereof |
| CN102093999A (en) * | 2010-12-14 | 2011-06-15 | 山东省农业科学院家禽研究所 | Indirect ELISA kit for detecting avian infectious bronchitis virus antibody |
Non-Patent Citations (6)
| Title |
|---|
| 付朝阳，等: "鸡传染性支气管炎血凝抑制试验方法标准化的研究", 《中国畜禽传染病》, no. 5, 30 September 1994 (1994-09-30) * |
| 张兵: "鸡传染性支气管炎病毒（M41株）血凝抑制试验抗原的研制及应用", 《中国优秀硕士学位论文全文数据库 农业科技辑》, no. 9, 15 September 2008 (2008-09-15) * |
| 欧阳文军，等: "呼吸型IBV-M41血凝抗原的研制与应用", 《家禽科学》, no. 12, 31 December 2007 (2007-12-31) * |
| 王川庆，等: "猪细小病毒病的研究 Ⅱ猪群细小病毒HI抗体的血清学调查", 《中国畜禽传染病》, no. 2, 31 March 1995 (1995-03-31) * |
| 金岳，等: "猪细小病毒病微量血凝抑制试验方法的探讨", 《家畜传染病》, no. 4, 31 August 1985 (1985-08-31) * |
| 韩孝成，等: "猪细小病毒病血凝抑制试验的研究", 《中国畜禽传染病》, no. 3, 31 May 1996 (1996-05-31) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105866427A (en) * | 2016-04-22 | 2016-08-17 | 北京市农林科学院 | Composition, and application thereof in infectious bronchitis antibody determination |
| CN106442986A (en) * | 2016-09-21 | 2017-02-22 | 华南农业大学 | Method for detecting regional SIV (swine influenza virus) subtype distribution |
| CN106442986B (en) * | 2016-09-21 | 2018-05-18 | 华南农业大学 | A kind of method of the detection zone swine influenza virus subtype distribution of non-disease diagnose and treat purpose |
| CN107328944A (en) * | 2017-06-14 | 2017-11-07 | 王琴 | A kind of blood clotting and hemagglutination-inhibition test screening technique |
| CN111198265A (en) * | 2018-11-20 | 2020-05-26 | 内蒙古正大食品有限公司 | Combined rapid detection method for various poultry antibodies |
| CN110007070A (en) * | 2019-04-13 | 2019-07-12 | 北京家禽育种有限公司 | A kind of HI test serum pre-dilution method |
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Application publication date: 20150624 |