CN107328944B - A kind of blood clotting and hemagglutination-inhibition test screening technique - Google Patents
A kind of blood clotting and hemagglutination-inhibition test screening technique Download PDFInfo
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Abstract
The invention discloses a kind of blood clottings and hemagglutination-inhibition test screening technique, comprising the following steps: the dilution of antigen and serum；Hemagglutination test；Hemagglutination-inhibition test screening disposably detects 80 parts of blood serum samples, filters out positive sample and finally determine that immune antiboidy is horizontal by using 1 block of dilution plate and 3 deblocking reaction plates in hemagglutination-inhibition test screening, and comparing conventional method, can to save antigen half more.The present invention detects more sample with less antigen, achievees the purpose that detect antibody level height, detection method is simple, testing result is accurate and reliable, detection efficiency is high, greatly reduces workload, reduces the demand to experimenter, it is nervous that very good solution laboratories detect funds, detection reagent is limited, the big problem of amount detection, and screening technique of the present invention is particularly suitable for base-level countryside animal epidemic prevention quarantine mechanism, not only detection reagent has been saved, but also has accelerated detection speed.
Description
Technical field
The invention belongs to biological immunology technical field more particularly to a kind of blood clottings and hemagglutination-inhibition test screening technique.
Background technique
In veterinary immunology experiment, hemagglutination test (HA) is mainly used to measure the HA-HI test of antigen and with the presence or absence of blood
Solidifying property antigen, and hemagglutination-inhibition test (HI) is then used as a kind of antibody detection technology, is widely used in veterinary laboratories monitoring, beast
Cure outpatient service, large and medium-sized poultry-farm.Necessary technology means of the laboratory inspection as basic animal epidemic prevention and quarantine mechanism, meet dynamic
The demand of object epidemic prevention and quarantine work.In practical applications, HA and HI test can be used for virus purification, identification, parting, detect antigen
Antibody, immunological identification etc., such as: poultry-farm is usually by the antibody after the vaccine immunities such as bird flu, newcastle disease, egg-decreasing syndrome
Level, as the measurement whether reasonable foundation of immune programme.Currently, common HI screening technique is although easy to operate, quick, quasi-
Really, but detection multiple sample needs to carry out test of many times, using more antigen, many and diverse, heavy workload is tested, for base
For veterinary laboratories, the effect of animal epidemic prevention detection is given full play to, routine testing is combined with temporary detecting, cause of disease
It learns detection and antibody test combines, the dynamic analysis rule for promptly and accurately grasping animal epidemics grasps immune antiboidy and detects water
Flat, reason is searched in analysis, summing up experience rule accomplish to have the prevention and control of animal epidemic it is perspective, it is timely to the animal epidemic of generation
It was found that resolute disposition etc., this high request, tight standard job specification make base's veterinary laboratories inhibit examination carrying out blood clotting
Occurs the problem that lab assistant is insufficient, workload is excessive during testing, while experiment fees are limited, resource distribution is low, detection
Reagent is limited, it is difficult to be efficiently completed animal epidemic prevention quarantine in time.In order to solve this problem, pass through practice summary, hair
Bright people finds out a kind of blood clotting and blood clotting Inhibition test screening technique, and more sample can be detected with less antigen, is reached
The purpose of antibody level height is detected, detection method is simple, and testing result is accurate and reliable, and detection efficiency is high, greatly reduces work
It measures, reduces the demand to experimenter.
Summary of the invention
The purpose of the present invention is to provide a kind of blood clottings and hemagglutination-inhibition test screening technique, it is intended to solve base animal doctor reality
Room is tested when carrying out hemagglutination-inhibition test, required antigen dosage is big, and more experimenter is needed to carry out test operation, workload
Excessive, time-consuming and laborious problem.
The invention is realized in this way a kind of blood clotting and hemagglutination-inhibition test screening technique, comprising the following steps:
Step 1: dilution
1 × PBS solution is prepared, after antigen and serum are opened, the product specification 1ml or 2ml as shown on every bottle label
PBS solution sufficiently dissolves, spare；
1 × the PBS solution is NaCl 8g, NaH2PO4·2H2O 1.561g, Na2HPO4·12H2O 3.582g, adds water
To 1000ml, adjust ph value to 7.2；
Step 2: hemagglutination test is carried out
(1) in V-type micro-reaction plate, every hole adds 0.025ml PBS solution；
(2) 0.025ml antigen is added in the 1st hole, successively makees the two-fold dilution of 0.025ml to the 11st hole；
(3) 0.025ml PBS solution is added in every hole；
(4) 0.025ml 1% (V/V) chicken erythrocyte suspension is added in every hole；
(5) reaction plate is shaken on the oscillator 1-2 minutes or tapping reaction plate mixed reactant, 20-25 DEG C stands 40
Minute or 2-8 DEG C stand 60 minutes, when the red blood cell of control wells be in significant button-type when determine result；
(6) result judgement: reaction plate is tilted, and observation red blood cell trickles whether there is or not trickling in tear sample entirely without tear sample
The highest extension rate of (100% agglutination) is hemagglutinative titer；
Step 3: hemagglutination-inhibition test screening is carried out
(1) hemagglutinative titer measured according to hemagglutination test in step 2 (6) calculates and prepares 4 unit antigens；
(2) one block of dilution plate is selected, it may be assumed that 1:8U type dilutes plate, is denoted as A plate；Select three deblocking reaction plates, it may be assumed that 1:8V type is empty
Reaction plate is denoted as B plate；First piece of V-type reaction plate, is denoted as C plate；2nd piece of V-type reaction plate, is denoted as D plate；A plate, B plate, C plate and D
Plate is 96 holes, and the arrangement in hole is 8 rows × 12 column；
(3) 0.070ml PBS solution is added in every hole in A plate, and 0.025ml PBS solution is added in antigen control hole；
(4) 0.025ml PBS solution is added in the complete every hole of plate in C plate and D plate；
(5) every hole is added 0.010ml and is detected blood serum sample in A plate, blood serum sample uses S1, S11, S21 respectively ... S71；
S2,S12,S22……S72；S3,S13,S23……S73；……；S9,S19,S29……S79；S10,S20,S30……S80
It indicates, is detected blood serum sample addition sequence and presses S1, S2, S3 ... S10；S11,S12,S13……S20；……；S71,S72,
S73 ... S80 is sequentially added and is mixed；Rifle point is changed, full plate adds 80 parts of samples, and does the positive, feminine gender, red blood cell, antigen control；
A. 0.025ml sample is taken by S1, S11, S21, S31, S41, S51, S61, S71 sequence from A plate with 8 hole pipettors
Mixed liquor successively moves into not plus in the hole S1, S11, S21, S31, S41, S51, S61, S71 of the B plate of PBS solution, remaining each column hole
According to said method make identical processing, B plate concentration is in 1:8 at this time；
B. 0.025ml is taken by S1, S11, S21, S31, S41, S51, S61, S71 sequence from B plate with 8 hole pipettors respectively
Sample mixed liquor successively moves into the hole S1, S11, S21, S31, S41, S51, S61, S71 for having added the C plate of 0.025ml PBS solution
In, according to said method make identical processing, by the 2nd column, the 3rd column, the 4th column, the 5th column, the 6th column, the 7th column, the 8th column, the 9th in B plate
Column, the 10th column hole correspond respectively to remaining 3rd column of C plate, the 5th column, the 7th column, the 9th column and the column of D plate the 1st, the 3rd column, the 5th
Then 2nd column of C plate and D plate, the 4th column, the 6th column, the 8th column, the 10th column are respectively corresponded C plate and D by column, the 7th column, the 9th column hole
The 1st column, the 3rd column, the 5th column, the 7th column, the 9th column of plate do doubling dilution, blow and beat 18-20 mixing, discard 0.025ml；C at this time
Plate and D plate concentration become 1:16 and 1:32；
(6) every hole adds the 4 unit antigens of 0.025ml on B plate, C plate and D plate respectively, and 20-25 DEG C stands 40 minutes, or
Person is 2-8 DEG C and stands 60 minutes；
(7) every hole is added the chicken erythrocyte suspension of 0.025ml 1% (V/V) on B plate, C plate and D plate respectively, it is light shake it is mixed
After even, 20-25 DEG C stands 40 minutes or 2-8 DEG C standing 60 minutes, determines when control wells red blood cell is in significant button-type
As a result；
(8) positive tested blood serum sample is filtered out, and is detected blood serum sample with the positive filtered out and re-starts blood clotting suppression
Test operation processed determines that immune antiboidy is horizontal.
Further, in step 3, the blood clotting of the tested blood serum sample inhibits potency < 1:8 to be judged to negative serum；Blood
Solidifying that potency=1:8 is inhibited to be judged to Suspect serum, blood clotting inhibits potency >=1:16 to be judged to positive serum.
Further, the sample of the Suspect serum should be examined 1 time again, examine blood clotting again and potency >=1:8 is inhibited to be judged to positive blood
Clearly, blood clotting inhibits potency < 1:8 to be judged to negative serum.
The advantages and positive effects of the present invention are: a kind of blood clotting provided by the invention and hemagglutination-inhibition test screening side
Method, detecting 80 parts of serum at most needs 3 deblocking reaction plates, 1 block of dilution plate, wherein plus antigen only needs plus 3 deblocking reaction plates, compare tradition
Method can save that antigen half is more, and realization detects more sample with less antigen, reach the mesh of detection antibody level height
, detection method is simple, and testing result is accurate and reliable, and very good solution laboratories detect funds anxiety, and detection reagent has
Limit, the big problem of amount detection, especially in base-level countryside, immune antiboidy level is lower, and experimenter is insufficient, detects antibody work
Work amount is excessive, time-consuming and laborious, very useful using screening technique of the present invention, has not only saved detection reagent, but also accelerates detection speed
Degree, detection efficiency is high, greatly reduces workload, reduces the demand to experimenter.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
With reference to the accompanying drawing and specific embodiment is further described application principle of the invention.
A kind of blood clotting and hemagglutination-inhibition test screening technique, comprising the following steps:
Step 1: dilution
1 × PBS solution is prepared, after antigen and serum are opened, the product specification 1ml or 2ml as shown on every bottle label
PBS solution sufficiently dissolves, spare；
1 × the PBS solution is NaCl 8g, NaH2PO4·2H2O 1.561g, Na2HPO4·12H2O 3.582g, adds water
To 1000ml, adjust ph value to 7.2；
Step 2: hemagglutination test is carried out
(1) in V-type micro-reaction plate, every hole adds 0.025ml PBS solution；
(2) 0.025ml antigen is added in the 1st hole, successively makees the two-fold dilution of 0.025ml to the 11st hole；
(3) 0.025ml PBS solution is added in every hole；
(4) 0.025ml 1% (V/V) chicken erythrocyte suspension is added in every hole；
(5) reaction plate is shaken on the oscillator 1-2 minutes or tapping reaction plate mixed reactant, 20-25 DEG C stands 40
Minute or 2-8 DEG C stand 60 minutes, when the red blood cell of control wells be in significant button-type when determine result；
(6) result judgement: reaction plate is tilted, and observation red blood cell trickles whether there is or not trickling in tear sample entirely without tear sample
The highest extension rate of (100% agglutination) is hemagglutinative titer；
Step 3: hemagglutination-inhibition test screening is carried out
(1) hemagglutinative titer measured according to hemagglutination test in step 2 (6) calculates and prepares 4 unit antigens；
(2) one block of dilution plate is selected, it may be assumed that 1:8U type dilutes plate, is denoted as A plate；Select three deblocking reaction plates, it may be assumed that 1:8V type is empty
Reaction plate is denoted as B plate；First piece of V-type reaction plate, is denoted as C plate；2nd piece of V-type reaction plate, is denoted as D plate；A plate, B plate, C plate and D
Plate is 96 holes, and the arrangement in hole is 8 rows × 12 column；
(3) 0.070ml PBS solution is added in every hole in A plate, and 0.025ml PBS solution is added in antigen control hole；
(4) 0.025ml PBS solution is added in the complete every hole of plate in C plate and D plate；
(5) every hole is added 0.010ml and is detected blood serum sample in A plate, blood serum sample uses S1, S11, S21 respectively ... S71；
S2,S12,S22……S72；S3,S13,S23……S73；……；S9,S19,S29……S79；S10,S20,S30……S80
Indicate, table 1 is that 1:8U type dilutes 80 parts of sample layout figures of plate selective mechanisms (A plate), be detected blood serum sample addition sequence by S1,
S2,S3……S10；S11,S12,S13……S20；……；S71, S72, S73 ... S80 are sequentially added and are mixed；Rifle point is changed,
Full plate adds 80 parts of samples, and does the positive, feminine gender, red blood cell, antigen control；
A. 0.025ml sample is taken by S1, S11, S21, S31, S41, S51, S61, S71 sequence from A plate with 8 hole pipettors
Mixed liquor successively moves into not plus in the hole S1, S11, S21, S31, S41, S51, S61, S71 of the B plate of PBS solution, remaining each column hole
According to said method make identical processing, B plate concentration is in 1:8 at this time；
B. 0.025ml is taken by S1, S11, S21, S31, S41, S51, S61, S71 sequence from B plate with 8 hole pipettors respectively
Sample mixed liquor successively moves into the hole S1, S11, S21, S31, S41, S51, S61, S71 for having added the C plate of 0.025ml PBS solution
In, according to said method make identical processing, by the 2nd column, the 3rd column, the 4th column, the 5th column, the 6th column, the 7th column, the 8th column, the 9th in B plate
Column, the 10th column hole correspond respectively to remaining 3rd column of C plate, the 5th column, the 7th column, the 9th column and the column of D plate the 1st, the 3rd column, the 5th
Then 2nd column of C plate and D plate, the 4th column, the 6th column, the 8th column, the 10th column are respectively corresponded C plate and D by column, the 7th column, the 9th column hole
The 1st column, the 3rd column, the 5th column, the 7th column, the 9th column of plate do doubling dilution, blow and beat 18-20 mixing, discard 0.025ml；C at this time
Plate and D plate concentration become 1:16 and 1:32；Table 3 is 80 parts of sample layout figures of the 1st piece of V-type reaction plate selective mechanisms (C plate), table
4 be 80 parts of sample layout figures of the 2nd piece of V-type reaction plate selective mechanisms (D plate)；
(6) every hole adds the 4 unit antigens of 0.025ml on B plate, C plate and D plate respectively, and 20-25 DEG C stands 40 minutes, or
Person is 2-8 DEG C and stands 60 minutes；
(7) every hole is added the chicken erythrocyte suspension of 0.025ml 1% (V/V) on B plate, C plate and D plate respectively, it is light shake it is mixed
After even, 20-25 DEG C stands 40 minutes or 2-8 DEG C standing 60 minutes, determines when control wells red blood cell is in significant button-type
As a result；
(8) positive tested blood serum sample is filtered out, and is detected blood serum sample with the positive filtered out and re-starts blood clotting suppression
Test operation processed determines that immune antiboidy is horizontal.
Concrete outcome determines are as follows: the blood clotting of tested blood serum sample inhibits potency < 1:8 to be judged to negative serum；Blood clotting inhibits effect
Valence=1:8 is judged to Suspect serum, and blood clotting inhibits potency >=1:16 to be judged to positive serum；The sample of Suspect serum should be examined 1 time again, weight
Inspection blood clotting inhibits potency>=1:8 to be judged to positive serum, and blood clotting inhibits potency<1:8 to be judged to negative serum.
The test operation layout of A plate is as shown in table 1 below:
1 1:8U type of table dilutes 80 parts of sample layout figures of plate selective mechanisms (A plate)
The test operation layout of B plate is as shown in table 2 below:
2 1:8V type reaction plate 80 parts of sample layout figures of selective mechanisms (B plate) of table
The test operation layout of C plate is as shown in table 3 below:
80 parts of sample layout figures of the 1st piece of V-type reaction plate selective mechanisms of table 3 (C plate)
The test operation layout of D plate is as shown in table 4 below:
80 parts of sample layout figures of the 2nd piece of V-type reaction plate selective mechanisms of table 4 (D plate)
80 parts of serum are detected in conventionally employed blood clotting and hemagglutination-inhibition test screening technique at least needs 7 deblocking reaction plates,
Many and diverse, heavy workload is tested, and the present invention 80 parts of serum of detection at most need 3 deblocking reaction plates, 1 block of dilution plate, wherein plus antigen
It needs plus 3 deblocking reaction plates, comparing conventional method can save that antigen half is more, and it is tight that very good solution laboratories detect funds
, detection reagent is limited, and the big problem of amount detection, especially in base-level countryside, immune antiboidy level is lower, while testing people
Member is less, very useful using screening technique of the present invention, and detection accuracy is high, has not only saved detection reagent, but also accelerate detection
Speed.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (3)
1. a kind of blood clotting and hemagglutination-inhibition test screening technique, which is characterized in that method includes the following steps:
Step 1: dilution
1 × PBS solution is prepared, after antigen and serum are opened, the product specification 1ml or 2ml PBS as shown on every bottle label
Solution sufficiently dissolves, spare；
1 × the PBS solution is NaCl 8g, NaH2PO4·2H2O 1.561g, Na2HPO4·12H2O 3.582g, adds water to
1000ml adjusts ph value to 7.2；
Step 2: hemagglutination test is carried out
(1) in V-type micro-reaction plate, every hole adds 0.025ml PBS solution；
(2) 0.025ml antigen is added in the 1st hole, successively makees the two-fold dilution of 0.025ml to the 11st hole；
(3) 0.025ml PBS solution is added in every hole；
(4) 0.025ml 1% (V/V) chicken erythrocyte suspension is added in every hole；
(5) reaction plate being shaken on the oscillator 1-2 minutes or tapping reaction plate mixed reactant, 20-25 DEG C stands 40 minutes,
Either 2-8 DEG C stands 60 minutes, determines result when the red blood cell of control wells is in significant button-type；
(6) result judgement: reaction plate is tilted, and observation red blood cell is whether there is or not trickling in tear sample, entirely without the trickling of tear sample
The highest extension rate of 100% agglutination is hemagglutinative titer；
Step 3: hemagglutination-inhibition test screening is carried out
(1) hemagglutinative titer measured according to hemagglutination test in step 2 (6) calculates and prepares 4 unit antigens；
(2) one block of dilution plate is selected, it may be assumed that 1:8U type dilutes plate, is denoted as A plate；Select three deblocking reaction plates, it may be assumed that the reaction of 1:8V type sky
Plate is denoted as B plate；First piece of V-type reaction plate, is denoted as C plate；2nd piece of V-type reaction plate, is denoted as D plate；A plate, B plate, C plate and D plate are equal
For 96 holes, the arrangement in hole is 8 rows × 12 column；
(3) 0.070ml PBS solution is added in every hole in A plate, and 0.025ml PBS solution is added in antigen control hole；
(4) 0.025ml PBS solution is added in the complete every hole of plate in C plate and D plate；
(5) every hole is added 0.010ml and is detected blood serum sample in A plate, blood serum sample uses S1, S11, S21 respectively ... S71；S2,
S12,S22……S72；S3,S13,S23……S73；……；S9,S19,S29……S79；S10, S20, S30 ... S80 come
It indicates, is detected blood serum sample addition sequence and presses S1, S2, S3 ... S10；S11,S12,S13……S20；……；S71,S72,
S73 ... S80 is sequentially added and is mixed；Rifle point is changed, full plate adds 80 parts of samples, and does the positive, feminine gender, red blood cell, antigen control；
A. 0.025ml sample is taken to mix by S1, S11, S21, S31, S41, S51, S61, S71 sequence from A plate with 8 hole pipettors
Liquid successively moves into not plus in the hole S1, S11, S21, S31, S41, S51, S61, S71 of the B plate of PBS solution, this is pressed in remaining each column hole
Method makees identical processing, and B plate concentration is in 1:8 at this time；
B. 0.025ml sample is taken by S1, S11, S21, S31, S41, S51, S61, S71 sequence from B plate with 8 hole pipettors respectively
Mixed liquor successively moves into the hole S1, S11, S21, S31, S41, S51, S61, S71 for having added the C plate of 0.025ml PBS solution, presses
The method makees identical processing, by the 2nd column, the 3rd column, the 4th column, the 5th column, the 6th column, the 7th column, the 8th column, the 9th column, the in B plate
10 column holes correspond respectively to remaining 3rd column of C plate, the 5th column, the 7th column, the 9th column and the column of D plate the 1st, the 3rd column, the 5th column, the 7th
Then column, the 9th column hole respectively correspond the of C plate and D plate to the 2nd column of C plate and D plate, the 4th column, the 6th column, the 8th column, the 10th column
1 column, the 3rd column, the 5th column, the 7th column, the 9th column do doubling dilution, blow and beat 18-20 mixing, discard 0.025ml；C plate and D at this time
Plate concentration becomes 1:16 and 1:32；
(6) every hole adds the 4 unit antigens of 0.025ml on B plate, C plate and D plate respectively, and 20-25 DEG C stands 40 minutes, either
2-8 DEG C stands 60 minutes；
(7) every hole is added the chicken erythrocyte suspension of 0.025ml 1% (V/V) on B plate, C plate and D plate respectively, it is light shake mix after,
20-25 DEG C stands 40 minutes or 2-8 DEG C standing 60 minutes, determines result when control wells red blood cell is in significant button-type；
(8) positive tested blood serum sample is filtered out, and is detected blood serum sample with the positive filtered out and re-starts blood clotting inhibition examination
Operation is tested, determines that immune antiboidy is horizontal.
2. blood clotting as described in claim 1 and hemagglutination-inhibition test screening technique, which is characterized in that described tested in step 3
The blood clotting of blood serum sample inhibits potency < 1:8 to be judged to negative serum；Blood clotting inhibits potency=1:8 to be judged to Suspect serum, blood clotting suppression
Potency >=1:16 processed is judged to positive serum.
3. blood clotting as claimed in claim 2 and hemagglutination-inhibition test screening technique, which is characterized in that the sample of the Suspect serum
It should examine again 1 time, examine blood clotting again and potency>=1:8 is inhibited to be judged to positive serum, blood clotting inhibits potency<1:8 to be judged to negative serum.
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| 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 |
| CN110118873A (en) * | 2019-04-23 | 2019-08-13 | 天津市宝坻区人民医院 | Disposable fertilizer reaches outer striking experiment reaction plate |
| CN113533718A (en) * | 2021-06-02 | 2021-10-22 | 威海市环翠区动物疫病预防控制中心(威海市环翠区动物卫生检疫中心) | Hemagglutination test detection method for avian influenza and newcastle disease |
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| CN102012430B (en) * | 2010-11-05 | 2013-09-04 | 中国农业科学院哈尔滨兽医研究所 | Avian influenza H5N1 subtype Re-5 strain hemagglutination inhibition antigen standard substance and preparation method |
| CN102735853B (en) * | 2012-06-07 | 2014-06-04 | 广州市华南农大生物药品有限公司 | Method for determining titer of swine flu inactivated vaccine |
| CN103513032B (en) * | 2013-08-06 | 2015-08-12 | 云南省畜牧兽医科学院 | Chicken nose tracheae bird bacillus hemagglutination inhibition antibody detection kit, preparation method and application |
| CN104730259A (en) * | 2015-03-12 | 2015-06-24 | 青岛易邦生物工程有限公司 | Method for detecting hemagglutination inhibition antibody of chicken infectious bronchitis |
| CN106033038A (en) * | 2015-03-12 | 2016-10-19 | 青岛易邦生物工程有限公司 | A method of processing serum used for avian infectious bronchitis hemagglutination inhibition assays |
| CN105866427B (en) * | 2016-04-22 | 2018-10-16 | 北京市农林科学院 | A kind of composition and its application in infectious bronchitis of chicken antibody determination |
| CN106749639A (en) * | 2016-11-23 | 2017-05-31 | 临沂大学 | A kind of new H5 subtype avian influenzas Yolk antibody replaces the method for serum antibody monitoring |
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