CN102288771B - National standard positive serum for cow brucellosis and preparation method of same - Google Patents
National standard positive serum for cow brucellosis and preparation method of same Download PDFInfo
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Abstract
The invention relates to national standard positive serum for cow brucellosis and a preparation method of the same. The national standard positive serum for the cow brucellosis adopts positive serum naturally infected with the cow brucellosis in China as a candidate material and is prepared through potency screening, packaging and freeze-drying. Moreover, the national standard positive serum for the cow brucellosis obtained through VLA (verbundlenkerachse) is used for reference to carry out the alexin combination test, the test tube condensation test and the rose bengal precipitation test to determine the potency. The uniformity testing, stability testing, collaborative calibration and statistics are adopted to guarantee that the fixed potency is accurate and the national standard positive serum for cow brucellosis can be prepared.
Description
Technical field the present invention relates to the sick positive serum national standard of Brucella abortus, belongs to standard substance technology and veterinary biologics technical field.
Background technology Brucella abortus disease causes by ox type brucella (front title Bacillus abortus) usually, and its infection is worldwide trend, brought serious economic loss for a lot of countries and regions in the world.OIE (OIE) classifies them as three class zoonosis.At present, Serological testing is the common method of the sick diagnosis of Brucella abortus, for the world that promotes the sick diagnostic test of Brucella abortus is integrated and the antigen standardization, OIE has developed standard serum (can be that Britain defends bridge VLA acquisition from the brucella reference laboratory of OIE), also be referred to as the base standard product, various countries' use for laboratory international standard substance is traced to the source and is prepared national standard separately, be used to demarcating the sick complement fixation test (CFT) of Brucella abortus, tube agglutination test and rose bengal precipitation test.At present; can be (The Second International Standard for Anti-Brucella abortus Serum.IanDavidson, the C.Nancy Hebert & that prepared afterwards in 6 years of chamber immunity a head of cattle by experiment from the sick positive serum international standard substance of the Brucella abortus that VLA obtains; W.J.Brinley Morgan.Bull.Org.mond.Sante, Bull.Wld HIthiOrg.1969,40,129-140).Prepared by the positive serum that gathers field natural infection Brucella abortus disease by the present invention, make manufacturing cycle shorten, and has saved cost.And the quality requirements of standard items is high, preparation method and check or scaling method are strict, relevant preparation procedure and scaling method so far there are no the report.
Summary of the invention:
The objective of the invention is to set up the sick positive serum standard items of national Brucella abortus, be used to the sick complement fixation test (CFT) of the Brucella abortus of demarcating China, tube agglutination test and rose bengal precipitation test.
The present invention realizes by following technology path, namely that positive serum by gathering field natural infection Brucella abortus disease is as material standed for, by technological processs such as the screening of tiring, packing, freeze-drying, prepare the sick positive serum of Brucella abortus, with complement fixation test (CFT), tube agglutination test and rose bengal precipitation test, measure it and tire, demarcate definite value to prepare the sick positive serum national standard of Brucella abortus through Homogeneity Test, stability test and cooperation.
Detailed description of the present invention:
One, the preparation of the sick positive serum national standard of Brucella abortus material standed for
The ox of field natural infection Brucella abortus disease is counted head, asepticly according to a conventional method respectively takes blood and collects through in sterilizing graduated cylinder, puts 20 ℃ of left and right room temperatures 4 hours, and each contains blood volume cylinder sterile working pressurization copper after blood clotting.Standing, after serum is separated out, get supernatant, the centrifugal 20min separation of serum of 4000r/min.
Two, the evaluation of the sick positive serum national standard of Brucella abortus material standed for
1, steriling test
By " People's Republic of China's regulations " (The Ministry of Agriculture of the People's Republic of China, MOA. in 2000 version of People's Republic of China's regulations. Chemical Industry Press, 2001, the present invention is hereinafter to be referred as " rules ") prescriptive procedure carries out.
2, titration:
By the method for following " appendix A ", " appendix B ", " appendix C ", carry out respectively complement fixation test (CFT), tube agglutination test and rose bengal precipitation test and carry out titration.On replication more than 2 times is tired all identical basis, determine that according to the titration test findings complement fixation test (CFT) of this positive serum is tired, tube agglutination test is tired and rose bengal precipitation test is tired.
Sick complement fixation test (CFT) (Complement Fixation Test, the CFT) serum titer of appendix A Brucella abortus is measured
1 working fluid preparation
1.12 the hemolysin dilution of individual unit is diluted by the hemolysin operation instruction.
1.22 individual unit complement dilution.
1.2.1 complement titer determination (test the same day at every turn and all will measure the complement titre).
1) complement dilution in 1: 10.
2) prepare sensitized erythrocyte: in beaker, add 20mL 2.5% sheep red blood cell (SRBC) suspension, under magnetic agitation, 2 unit hemolysins of 20mL are slowly added, for abundant sensitization, after mixing, at room temperature use magnetic stirrer 15 minutes.
3) by table 1, carry out complement titre program.
Table 1 complement titre program
4) result of determination: produce complete hemolysis and the minimum pipe number of complement amount and be decided to be a proper order position, last pipe (pipe that contains more complement) is a complete unit, calculates complement extension rate X according to formula 1.
1 unit complement amount (Y)/10=40 (end reaction complement volume)/complement extension rate (X)
In formula, Y is a complete unit complement amount
1.2.22 individual unit complement dilution: carry out 2 unit complement dilutions by a complement extension rate X of unit.
1.3 antigen diluent: dilute by the antigen operation instruction.
2 complement fixation test (CFT)s:
2.1 tested positive serum and the dilution of international standard positive serum and the energy that goes out:
The energy 2.1.1 tested positive serum goes out, 30min namely heats in 58～59 ℃ of water-baths.During test, will after 10 times of dilutions of tested positive serum, carry out again the two-fold dilution, namely 1: 20,1: 40,1: 80,1: 160,1: 320,1: 640 (remarks: if dilution positive serum concentration is improper, need to make the appropriate adjustments according to test findings).
2.1.2VLA the international standard positive serum carries out 10 times of dilutions with physiological saline, i.e. positive serum 20 μ L+ physiological saline 180 μ L, go out can, namely in 58～59 ℃ of water-baths, heated 30 minutes.During test, 10 times of dilute serums carry out 5 times of dilutions again, namely 1: 10 positive serum 200 μ L+ physiological saline 800 μ L, obtain the international standard positive serum of 1: 50 times of dilution, the international standard positive serum of 50 times of dilutions carries out respectively 1: 2,1: 3,1: 4,1: 6,1: 8 and 1: 12 times dilution again and obtains the international standard positive serum dilution that final extension rate is 1: 100,1: 150,1: 200,1: 300,1: 400 and 1: 600 times.
2.2 carry out complement fixation test (CFT) by table 2
Table 2 complement fixation test (CFT) program
2.3 complement control: 2 complement two-fold dilutions of unit are 1 unit and 1/2 unit
Table 3 complement control preparation table
2.4 Evaluation of Complement Fixation Test is judged
Criterion: 100% haemolysis is the reacting hole complete hemolysis, and bottom deposits without red blood cell, and note is #; 75% haemolysis is the most of haemolysis of reacting hole, and a little red blood cell deposition is arranged at bottom, and during 75 degree inclination reaction plate, the deposition red blood cell does not trickle, and note is done +++; 50% haemolysis is that reacting hole has part haemolysis, and red blood cell deposition is arranged at bottom, trickling at once of deposition red blood cell during 75 degree inclination reaction plate, and note is done ++; 25% haemolysis is that reacting hole has part haemolysis, and red blood cell deposition is arranged at bottom, and during 75 degree inclination reaction plate, the deposition red blood cell trickles at once, note does+; Haemolysis is not haemolysis of reacting hole, and red blood cell deposition is arranged at bottom, and during 75 degree inclination reaction plate, the deposition red blood cell trickles at once, note does-.
When after reaction, each control wells result of observation is following, get final product log, otherwise complement is tired and should be redeterminated, and re-starts complement fixation test (CFT).Namely 2 unit complement control holes are 100% haemolysis, and 1 unit complement control hole is 50% haemolysis, and 1/2 unit complement control hole is not haemolysis.
While judging test findings, the extension rate in the tested seroreaction hole consistent with 1/200 times of dilution holes reaction result of international standard positive serum is tiring of tested serum.
The sick serum agglutination test (Serum agglutination Test, SAT) (test tube method) of appendix B Brucella abortus
1 working fluid preparation
1.1 antigen diluent is used in work: dilute by the antigen operation instruction.
1.2 tested positive serum national standard material standed for and the dilution of VLA international standard positive serum:
1.2.1 tested positive serum dilution: during test, tested positive serum is carried out to continuous two-fold dilution after with 10 times of dilutions of 0.5% carbolic acid physiological saline again, namely 1: 20,1: 40,1: 80,1: 160,1: 320,1: 640 (remarks: if dilution positive serum concentration is improper, need to make the appropriate adjustments according to test findings).
1.2.2VLA international standard positive serum dilution: during test, VLA international standard positive serum is carried out to 10 times of dilutions with 0.5% carbolic acid physiological saline, i.e. positive serum 20 μ L+0.5% carbolic acid physiological saline 180 μ L.During test, 10 times of dilute serums carry out 20 times of dilutions again, namely 1: 10 positive serum 200 μ L+0.5% carbolic acid physiological saline 3800 μ L, obtain the international standard positive serum of 1: 200 times of dilution, then carry out respectively the international standard positive serum dilution that 1: 2,1: 3,1: 4 and 1: 6 times dilution obtains 1: 400,1: 600,1: 800 and 1: 1200 times.
2 tube agglutination test methods
Each dilution of tested serum and work respectively add 0.5mL in test tube in antigen with the 1mL transfer pipet by tube agglutination test, shake up, and put 37 ℃ of incubator reactions and after 24 hours, take out the viewing test result.
Simultaneously, 20 times of dilution antigens are with after 0.5% carbolic acid physiological saline two-fold dilution, pressing table 1 production standard opacity tube in test.
Table 4 standard opacity tube preparation table
| Pipe number | 1 | 2 | 3 | 4 | 5 |
| 1/40 times of antigenic dilution (mL) | 0 | 0.25 | 0.5 | 0.75 | 1 |
| 0.5% carbolic acid physiological saline (mL) | 1 | 0.75 | 0.5 | 0.25 | 0 |
| Record symbol | # | +++ | ++ | + |
3 test findings criterion
By test tube and the contrast of standard opacity tube, observe the transparency of liquid column in test tube.Consistent with the transparency with the standard opacity tube, with "+,-" symbol record developmental tube result of corresponding opacity tube.If inconsistent with the transparency of standard opacity tube, with "+,-" symbol record developmental tube result of the close opacity tube of transparency, and according to the judgement to the transparency difference degree, in the upper left corner of above-mentioned symbol, make footmark as supplementary table will with "+,-" number, wherein "+" number means that transparency is strong, and "-" number means poor transparency.
While judging test findings, the extension rate of the tested positive serum reacting hole consistent with 1: 600 times of dilution holes reaction result of international standard positive serum is tiring of tested positive serum.
The red plate agglutination test of appendix C Brucella abortus sick tiger (Rose-Bengal Plate Agglutination Test, RBT)
1 working fluid preparation
1.1 during the dilution test of tested positive serum, with 0.5% carbolic acid physiological saline, tested positive serum is carried out to 10 times of dilutions, i.e. positive serum 50 μ L+0.5% carbolic acid physiological saline 450 μ L.Again by 10 times of dilute serum two-fold dilutions, namely 1: 20,1: 40,1: 80,1: 160,1: 320,1: 640 (if dilution positive serum concentration is improper, need to make the appropriate adjustments according to test findings).
1.2VLA the international standard positive serum carries out 10 times of dilutions with 0.5% carbolic acid physiological saline, i.e. positive serum 20 μ L+0.5% carbolic acid physiological saline 180 μ L.During test, 10 times of dilute serums carry out two-fold dilution to 80 times again.
2 rose bengal precipitation test methods (doing 2 repetitions when each blood serum sample is tested at every turn)
Glass plate or the white plaque of a standby rectangle cleaning, be divided into the grid that every lattice are about 4cm2, add respectively each dilution serum of 30 μ L and the red Avian tubercula plain agglutination test antigen of 30 μ L tiger in each lattice, with toothpick, the serum of each lattice and antigen are mixed, while mixing, each little lattice changes 1 toothpick.After antigen and serum mix, in 4min, record reaction result.
3 reaction result criterion
"-" means without aggegation, is the homogeneous powder redness; "+" means slightly can find aggegation, slightly has crimping to form, and between agglutinator, liquid takes on a red color; " ++ " means to form more obvious crimping, and aggegation interblock liquid is slightly limpid; " +++" mean that agglutinating reaction is stronger; " # " means that the aggegation piece is the flora shape, and between grumeleuse, liquid is limpid obviously.
The extension rate in tested seroreaction hole for "+" consistent with 1/40 times of dilution holes reaction result of standard positive serum is tiring of tested serum.
3, the selection of material standed for
If replication is more than 3 times continuously, test findings is in full accord, and the complement fixation test (CFT) of positive serum tire reach more than 1: 200 times, tube agglutination test tires and reaches more than 1: 600 times and rose bengal precipitation test is tired and reached more than 1: 40 times the material standed for that this positive serum namely can be used as the sick positive serum national standard of Brucella abortus.
4, packing, freeze-drying, sealing by fusing
Satisfactory material standed for is first through the aseptic filter membrane pressure filtration of 0.4 μ m, then through the aseptic filter membrane pressure filtration of 0.22 μ m; Use subsequently the bottleneck knockout (the packing precision for ± 0.01mL) aseptic subpackaged to the sterilizing Chang'an of 1mL small jar, after freeze-drying, vacuumize according to a conventional method, sealing by fusing.
Three, the check of the sick positive serum national standard of Brucella abortus
1. physical behavior freeze-drying positive serum national standard is faint yellow loose agglomerate, easily with the bottle wall, breaks away from, and dissolves rapidly after adding dilution.
2. steriling test carries out steriling test or pure check according to the method for " rules " regulation, answers asepsis growth.
3. vacuum tightness is measured according to the method for " rules " regulation and is carried out the vacuum tightness check, should be up to specification.
Residual moisture measure according to " Chinese veterinary pharmacopoeia " (the Chinese veterinary pharmacopoeia council. three ones of in 2005 versions of People's Republic of China's veterinary drug allusion quotation. Chinese agriculture publishing house, 2006, the present invention is called for short " Chinese veterinary pharmacopoeia ") with the vacuum drying method, carry out residual moisture mensuration, should be no more than 4% of standard code.
5. titration is randomly drawed the sample of specified quantity with Britain Wei Qiao (Veterinary Laboratories Agency, VLA) the sick positive serum international standard substance of Brucella abortus is reference, carries out respectively RBT test, SAT test and the red dull and stereotyped agglutination titer of its tiger of CFT test determination, tube agglutination is tired and complement fixation test (CFT) is tired.
6. Homogeneity Test is randomly drawed the sample of specified quantity, with the sick positive serum international standard substance of Britain Wei Qiao (VLA) Brucella abortus, make reference, carry out respectively RBT, SAT the test and the CFT test determination its tiger red dull and stereotyped agglutination titer, tube agglutination is tired and complement fixation test (CFT) is tired, test findings should be consistent.
7. stability test is measured with hot accelerated stability test, check under hot conditions, and the time that sample is placed, and using these data as foundation, for basis is done in the next group stability test.
8. definite value (cooperation is demarcated) relevant departments of China Veterinery Drug Inspection Office are responsible for as organization unit the staking-out work that cooperates, after developing programs, select to have more than 3 brucellosis patients is learned to the exper ienced laboratory of test, 3 samples of each use for laboratory, each sample repeats 2 times, with international standard substance, do reference simultaneously, each cooperation is demarcated unit and is demarcated according to the requirement of cooperation scaling scheme, and gives organization unit by experimental result.
(1), after organization unit arranges experimental result, determine cooperation demarcation numerical value.
(2) international unit is determined by the cooperation calibration result, calculates the international unit content of the sick positive serum national standard of 1mL Brucella abortus, usings this determined value as national standard.
Positive effect of the present invention:
The present invention relates to sick positive serum national standard of a kind of Brucella abortus and preparation method thereof.The sick positive serum national standard of Brucella abortus involved in the present invention is by utilizing positive serum by China's field natural infection Brucella abortus disease as material standed for, by technological processs such as the screening of tiring, packing, freeze-drying, prepare the sick positive serum of Brucella abortus, and measure it and tire and prepare the sick positive serum national standard of Brucella abortus as reference carries out complement fixation test (CFT), tube agglutination test and rose bengal precipitation test by take the sick positive serum international standard substance of Brucella abortus that VLA obtains.Method of the present invention makes standard items prepare science, rigorous, perfect more, makes each step scientific and precise according to the standard items preparation procedure, through cooperation, demarcate, and data statistics, the definite value that guarantees to tire is accurate.Prepared by the positive serum that gathers field natural infection Brucella abortus disease by the present invention, make manufacturing cycle greatly shorten (preparation time that has saved the immune serum of several years), has saved cost.
Embodiment 1
The preparation of the sick positive serum national standard of Brucella abortus
The preparation of 1 material standed for
4 of the milk cows of farm, Heilongjiang Province field natural infection Brucella abortus disease, take a blood sample in aseptic large graduated cylinder with the arteria carotis blood collection method, puts 20 ℃ of left and right room temperature 4h, and each contains blood volume cylinder sterile working pressurization copper after blood clotting.Standing, after serum is separated out, get supernatant, the centrifugal 20min separation of serum of 4000r/min.
2 material standed for screenings
2.1 steriling test: carry out steriling test or pure check according to the method for the regulation of " rules ", be asepsis growth.
2.2 titration
The standard serum of take carries out the red dull and stereotyped agglutination titer of tiger of 4 parts of serum of RBT test determination as reference, and test findings is in Table 5.Result shows: 4 parts of serum are the sick positive of Brucella abortus, and the red dull and stereotyped agglutination titer of the tiger of 3# serum is the highest.
The red dull and stereotyped agglutination titer measurement result of table 5 material standed for screening tiger
#, aggegation fully; +++, 75% aggegation; ++, 50% aggegation; +, 25% aggegation;-, not aggegation fully
The standard serum of take is tired as the tube agglutination that reference carries out 4 parts of serum of SAT test determination, and test findings is in Table 2.Result shows: 4 parts of serum are the sick positive of Brucella abortus, and the tube agglutination of 3# serum is tired the highest.
Table 6 material standed for screening tube agglutination titration result
#, aggegation fully; +++, 75% aggegation; ++, 50% aggegation; +, 25% aggegation;-, not aggegation fully
The standard serum of take is tired as the tube agglutination that reference carries out 4 parts of serum of CFT test determination, and test findings is in Table 7.Result shows: 4 parts of serum are the sick positive of Brucella abortus, and the CFT of 3# serum is the highest.
Table 7 material standed for screening complement fixation test (CFT) titration result
#, haemolysis not fully; +++, 25% haemolysis; ++, 50% haemolysis; +, 75% haemolysis;-, complete hemolysis
Comprehensive above-mentioned RBT, SAT and CFT found that, 4 parts of cow's serums are the sick positive serum of Brucella abortus, the red dull and stereotyped agglutination titer of the tiger of 3# serum wherein, tube agglutination is tired and CFT is all the highest, and the sick positive serum of Brucella abortus that is numbered 3# so select prepares the sick positive serum national standard of Brucella abortus
3 packing, freeze-drying, sealing by fusing
Satisfactory material standed for is first through the aseptic filter membrane pressure filtration of 0.4 μ m, then through the aseptic filter membrane pressure filtration of 0.22 μ m; Use subsequently the bottleneck knockout (the packing precision for ± 0.01mL) aseptic subpackaged to the sterilizing Chang'an of 1mL small jar, after freeze-drying, vacuumize according to a conventional method, sealing by fusing.
Product inspection and the cooperation of the sick positive serum national standard of embodiment 2 Brucella abortus material are demarcated
1. product inspection
(1) all ampoule freeze-drying of physical behavior positive serum is faint yellow loose agglomerate, easily with the bottle wall, breaks away from, dissolve rapidly after adding dilution, and after dissolving, be faint yellow clear liquid.
(2) steriling test carries out steriling test or pure check, asepsis growth according to the method for " rules " regulation.
(3) vacuum tightness is measured according to the method for " rules " regulation and is carried out the vacuum tightness check, up to specification.
(4) residual moisture is measured according to the regulation of " Chinese veterinary pharmacopoeia " and is carried out residual moisture mensuration with the vacuum drying method, and moisture is respectively 2.8%, 2.9%, 2.4% and 2.4% as a result, all lower than 4%, meets the requirements.
(5) titration is chosen at random 5 freeze-dry blood serums and be take standard serum it is tired as reference carries out respectively RBT, SAT and CFT test determination, the results are shown in Table 8.
The measurement result of tiring after the sick positive serum freeze-drying of table 8 Brucella abortus
(6) Homogeneity Test is chosen at random 15 freeze-dry blood serums and be take standard serum it is tired as reference carries out respectively RBT, SAT and CFT test determination, the results are shown in Table 9.Test findings shows, the red dull and stereotyped agglutination titer of the tiger of 15 freeze-dry blood serums, tube agglutination is tired and CFT is respectively 1: 160 "+", 1: 2400 " ++ ", 1: 800 " ++ ", and having good uniformity of this freeze-dry blood serum is described.
Homogeneity Test result after the sick positive serum freeze-drying of table 9 Brucella abortus
(7) the stability test sample of choosing at random specified quantity put respectively 56 ℃ of incubators, 37 ℃ of incubators and 25 ℃ of incubators at the appointed time sample thief carry out respectively CFT and SAT test determination it tired.Test findings is in Table 10.The stability test result shows, when 56 ℃ of incubators of freeze-dry blood serum preserved for 3 week, its CFT and tube agglutination were tired and are started to descend gradually, when 37 ℃ of incubators preserved for 8 week, its CFT and tube agglutination were tired and are started to descend gradually, and when 25 ℃ of incubators preserved for 9 week, its CFT and tube agglutination were tired and started to descend gradually.
The sick positive serum freeze-drying of table 10 Brucella abortus rear stability test findings
-, do not detect
2. cooperation is demarcated
The test findings that gathers four cooperation demarcation units shows (in Table 11), the CFT that the international standard substance of take records the sick positive serum national standard of this Brucella abortus as reference is tired as 1: 2400 " ++ ", the red dull and stereotyped agglutination titer of tiger and is 1: 160 "+" as 1: 800 " ++ ", tube agglutination, and the result that this calibration result is demarcated when developing this national standard is consistent.
Table 11 product inspection cooperation calibration result
The international standard substance of take is tired and the red dull and stereotyped agglutination titer of tiger is respectively 1: 800 " ++ ", 1: 2400 " ++ ", 1: 160 "+" as CFT, the tube agglutination of the sick positive serum national standard of the markization of tracing to the source Brucella abortus to be checked, international standard substance contains the 1000IU/ ampoule, according to international standard, by its tube agglutination test, calculating its international unit content is the 4800IU/ ampoule.
Claims (2)
1. the sick positive serum national standard of a Brucella abortus, it is characterized in that the sick complement fixation test (CFT) of sick its Brucella abortus of positive serum national standard of Brucella abortus involved in the present invention tires as 1:800, its result is judged to be " ++ ", tube agglutination test is tired as 1:2400, its result is judged to be " ++ " and rose bengal precipitation test is tired as 1:160, its result is judged to be "+", according to international standard, by its tube agglutination test, calculating its international unit content is the 4800IU/ ampoule.
2. the sick positive serum national standard of a kind of Brucella abortus as claimed in claim 1, is characterized in that it is the positive serum of field natural infection Brucella abortus disease that positive serum is taken from.
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