CN1469126A - Method for detecting SARS coronavirus antibody and its reagent kit - Google Patents
Method for detecting SARS coronavirus antibody and its reagent kit Download PDFInfo
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- CN1469126A CN1469126A CNA03116661XA CN03116661A CN1469126A CN 1469126 A CN1469126 A CN 1469126A CN A03116661X A CNA03116661X A CN A03116661XA CN 03116661 A CN03116661 A CN 03116661A CN 1469126 A CN1469126 A CN 1469126A
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Abstract
The present invention discloses a method for detecting SARS coronavirus antibody and its kit. The specific detection steps are as follows: (1) coating a solid support with purified whole virus lysate; (2) adding a biological sample to be tested to the coated solid support and incubating under conditions suitable for the formation of an antigen-antibody immunological binding complex; (3) moderate washing to remove any unbound SARS coronavirus antibodies; (4) adding a labeled secondary antibody against the human antibody and incubating under conditions suitable for the formation of an antigen-antibody immune complex; (5) mild washing to remove any unbound secondary antibody; (6) quantitatively detecting the presence of the antigen-antibody immunological binding complex in the incubation mixture. The invention solves the problem of rapid diagnosis of SARS coronavirus infectors, has high accuracy, and provides a rapid and accurate diagnosis method for clinical detection of the SARS coronavirus infectors.
Description
Technical field
The present invention relates to the physianthropy health field, relate in particular to a kind of method and kit thereof that detects sars coronavirus antibody.
Background technology
Break out the SARS (Severe Acute Respiratory Syndrome) that causes the whole world death of people more than 150 end of the year 2002 all over the world, current popular SARS (Severe Acute Respiratory Syndrome) is a kind of acute respiratory infectious disease, be called severe acute respiratory syndrome (Severe Acute Respiratory Syndrome abroad, SARS), main by the closely air spittle and contact transmission closely, onset is anxious, propagation is fast, and case fatality rate is up to 4.9%.Relate to nearly 30 countries and regions, the world at present, global patient has reached 3389 many cases, and number of the infected still has ever-increasing trend.
The expert of U.S. CDC utilizes SARS patient's tissue samples, may be novel coronavirus by electron microscope separation and judgement SARS pathogen.Almost simultaneously, the existence of this novel coronavirus has also all been confirmed in the several laboratories that comprise China, Singapore, Canada, Germany.
On April 16th, 2003, the World Health Organization (WHO) announces, through 13 breadboard the working in concert in 10 countries and regions, the whole world, the pathogen of the SARS (Severe Acute Respiratory Syndrome) (English name is SARS, the hereinafter referred SARS) that a plurality of countries are infected fast in the whole world finally is identified.This is a kind of coronavirus, never finds in human body in the past.The SARS (Severe Acute Respiratory Syndrome) cause of disease, i.e. the separation of SARS virus and conclusive evidence, for this sick clinical diagnosis, treatment and prevent most importantly, this also is a problem to be solved by this invention.
Summary of the invention
The purpose of this invention is to provide a kind of method and kit thereof that detects sars coronavirus antibody.
Its concrete steps are as follows:
1) utilize the totivirus lysate bag of purifying by a kind of solid support;
2) biological sample to be detected is added on the solid support of bag quilt, hatch suitable formation under the condition of antigen-antibody immunity in conjunction with compound;
3) clean any unconjugated sars coronavirus antibody of removal;
4) adding is hatched under the condition that is fit to formation antigen-antibody immune complex through the second antibody at people's antibody of mark;
5) clean any unconjugated second antibody of removal;
6) detect in the mixtures incubated antigen-antibody immunity in conjunction with the existence of compound.
Its kit consists of:
1) envelope antigen: sars coronavirus totivirus lysate;
2) antigen coated microplate: adopt homemade 96 hole portable plates, and bag is by sars coronavirus totivirus lysate;
3) enzyme labelled antibody: the second antibody of horseradish peroxidase-labeled (IgG or IgM);
4) negative control and positive control serum: contain 0.05% thimerosal, 0.01% Sodium azide;
5) enzyme labelled antibody working fluid: with containing 20% sheep blood serum, the dilution of 0.05% thimerosal is diluted to suitable tiring with enzyme labelled antibody;
6) the 0.01M pH4.5 citrate buffer solution of substrate solution A:0.06% urea peroxide;
Substrate solution B:0.06%TMB0.01M pH4.5 citrate buffer solution;
7) stop buffer: 1M sulfuric acid;
8) cleansing solution: PBS contains 0.1% Tween-20;
9) indirect method detects sars coronavirus antibody kit instructions portion.
The invention solves clinical quick diagnosis problem, have higher accuracy, for this disease of clinical detection provides diagnostic method fast and accurately sars coronavirus the infected.
Description of drawings
Fig. 1 is the SDS-PAGE result schematic diagram of sars coronavirus totivirus lysate antigen;
Fig. 2 is enzyme mark second antibody cut-off attitude distribution schematic diagram when being IgG.
Embodiment
Biological sample of the present invention is meant from experimenter's blood of (comprising patient and negative control), serum, blood plasma, urine sample, body fluid, saliva and other secretion or excreta and tissue or cell extract.
At the second antibody of people's antibody can be (being RIA) with labelled with radioisotope, also can be (being ELISA) of carrying out mark with horseradish peroxidase, alkaline phosphatase, beta galactosidase or acetylcholinesterase.
Described second antibody can be anti-people-IgG or/and-IgM or/and-IgA antibody.These second antibody can be buied from the market, as sheep anti people-IgG or/and-IgM, the anti-people-IgG of goat or/and-IgM, mouse anti human-IgG or/and-IgM, rat anti people-IgG or/and-IgM, the anti-people-IgG of rabbit or/and-IgM etc.
Utilize different second antibody can detect antibody specific in the sample and whether exist, and the relative amount of different antibodies.Because the different times IgG that infects at sars coronavirus is different with the relative quantity of IgM in human body, utilizes different second antibody to carry out the course of disease that repeated detection helps to judge the patient.
Described " solid support " comprises the organic and inorganic polymer that this area is known, such as including, but not limited to: glucosan, natural or modified cellulose, tygon, polystyrene, polyacrylamide, agarose, latex, container inner wall such as test tube, titer plate, glass cylinder etc.
Can use the known several different methods bag in this area by solid support.For example: utilize the bifunctional reagent activation, referring to United States Patent (USP) 5399501.
Described " being fit to form the condition of antigen-antibody immune complex " is that this area is known, be meant the potpourri of under suitable temperature and time condition, hatching antigen and containing the sample of antibody, for example at about 0-50 degree centigrade, under preferred about 4-30 degree centigrade, mark about 20 minutes to about 48 hours, preferred mark about 20 minutes to about 4 hours.
After hatching formation antigen-antibody immune complex, with this area suitable wash solution washing check-out console several commonly used, to remove unconjugated antigen through mark.Used cleaning fluid is generally the about 5-8 of pH, is preferably the phosphate buffered solution of pH about 7.4.
Can adopt the formation of the known method detection by quantitative antigen antibody complex in multiple this area.According to the employed method of mark second antibody, select suitable detection by quantitative scheme.In embodiment preferred of the present invention, use horseradish peroxidase during labelled antigen, be A liquid and the diphenyl amine material substrate and the antigen-antibody immune complex effect that has the horseradish peroxidase that contains mark as B liquid so use with peroxide solutions, the colour developing back is that 450nm (reference wavelength 630nm) microplate reader reads light absorption value at wavelength.The reading of gained reading and negative control is compared, judged institute's test sample result originally.
Utilize sars coronavirus totivirus lysate of the present invention as antigen, the method that detects sars coronavirus antibody in the qautobiology sample by immunization comprises that also the conventional specific antigen that utilizes that uses detects other method of antibody in this area.
The present invention also relates to be used for these methods and detect sars coronavirus detection of antibodies kit.The kit that detects sars coronavirus antibody according to the present invention comprises: with the modified reorganization atypical pneumonia virus protein of the present invention of purifying as antigen coated solid support; With the explanation of using this kit.The second antibody that also comprises mark as indicated above.This second antibody can be anti-IgG antibody and/or anti-IgM and/or anti-IgA antibody.In above-mentioned detection kit, can also contain the corresponding scope of judging testing result that provides.Sars coronavirus totivirus lysate is to cultivate after adopting Vero-E6 cell inoculation virus, obtains through the ultrasonic degradation purifying.
The following example is explained the present invention in detail, but this does not limit the scope of the invention.The preparation and the evaluation of embodiment 1, sars coronavirus totivirus lysate
1, virus is directly inoculated Vero E6 after, observe pathology every day, when showing as gathering, round cell, refractive power, and when disseminated cell occurring and coming off, with cell-20C freeze repeatedly molten 2 this, the centrifuging and taking supernatant.
2, sars coronavirus purge process obtains the full lysate of sars coronavirus.
1) above-mentioned supernatant carries out ultrasonication (power 150W, ultrasonic time 2 seconds, 4 seconds quiescent intervals, 60 circulations);
2) 13000RPM is centrifugal 20 minutes;
3) insert bag filter dialyse (cut-off10000);
4) adopt the about 6-7 of PEG20000 concentrating sample doubly;
5) concentrating sample is transferred to clean tube;
6) ultrasonication (power 150W, ultrasonic time 2 seconds, 4 seconds quiescent intervals, 60 circulations);
7) 13000RPM is centrifugal 10 minutes; Remove insolubles;
8) adding solid urea to concentration is 6M;
9) ultrasonication (power 150W, ultrasonic time 2 seconds, 4 seconds quiescent intervals, 60 circulations);
10) 13000RPM is centrifugal 10 minutes, removes insolubles.Obtain sars coronavirus totivirus lysate.
3, purity identifies that cell liquid and totivirus lysate with VERO-E6 CELL Ultrasonic Pulverization carry out the SDS-PAGE electrophoretic determination simultaneously, concentrates gum concentration 5%, and resolving gel concentration is 10%, and the sample applied sample amount is 10 μ g.Control cells and middle poison cell band should have notable difference; Than living: with the antigen coated micropore lath of 1 μ g/ml, examine and determine with Quality Control P1 serum, it is a unit (IU) than work that definition A value equals 0.2 o'clock antigen, should be greater than 10IU than work.The SDS-PAGE of sars coronavirus totivirus lysate antigen the results are shown in accompanying drawing 1.Embodiment 2, sars coronavirus totivirus lysate bag are by solid support
96 orifice plate bars select for use the good polystyrene board of adsorbability (between the hole, between plate and difference between batch CV＜10%), selecting 0.05M pH9.6 carbonate buffer solution (CB) for use is coating buffer, and every hole adds coating buffer 100 microlitres that prepare antigen according to experimental design concentration, and the room temperature bag is spent the night.Discard antigen liquid then, bag is washed twice by plate with the 0.02M PBS cleansing solution (PBS-T) that contains 0.5%TW-20, pats dry, and 0.02M pH7.4 phosphate buffer (PBS) room temperature that adds 1% bovine serum albumin(BSA) (BSA) and 1% skimmed milk power was sealed 2 hours.Discard confining liquid, air-dry bag is by plate, the vacuum-packed 4 ℃ of preservations of aluminium foil bag.With the titration of tiring of internal control sars coronavirus serum, measure the sars coronavirus antigen valence that is used to wrap quilt with indirect method, the results are shown in Table 1, the tiring of sars coronavirus antigen that the result is identified for wrapping quilt is 1: 60.
Table 1. is used to wrap the sars coronavirus antigen valence titration results of quilt.
Embodiment 3, enzyme are marked horseradish peroxidase-labeled and evaluation with anti-human IgG, IgM
| Dilutability | ????1∶2 | ????1∶4 | ????1∶8 | ????1∶16 | ????1∶32 | ????1∶40 | ????1∶50 | ????1∶60 | ????1∶80 | ????1∶100 |
| Weak positive serum | ????1.842 | ????1.653 | ????1.324 | ????1.002 | ????0.825 | ????0.722 | ????0.683 | ????0.416 | ????0.225 | ????0.178 |
| Negative serum | ????0.242 | ????0.180 | ????0.134 | ????0.102 | ????0.083 | ????0.065 | ????0.062 | ????0.043 | ????0.012 | ????0.002 |
The enzyme mark is used for enzyme labeling with anti-human IgG, IgM behind assay approval.Horseradish peroxidase is available from Sigma company, RZ＞3.0.Labeling method adopts improvement sodium periodate method.
Activate HRP: take by weighing 5mg HRP, be dissolved in the 1ml 0.2M pH5.6 acetate buffer, add freshly prepared 0.1M NaIO4 0.25ml, abundant mixing, place fully reaction on the magnetic stirring apparatus, lucifuge reaction (150rpm) 20 minutes is fully dialysed 24 hours to remove micromolecular impurity for 4 ℃ with the acetate buffer of 1mM pH4.4.
Crosslinked with anti-human IgG/IgM antibody: add 1/4 volume 1.0M pH9.6 carbonate buffer solution among the HRP after the above-mentioned activation, regulate pH value to 9.6 (HRP concentration becomes 4mg/ml).Get the 1.0M pH9.6 carbonate buffer solution that the anti-human IgG/IgM of 1.25mg adds 1/4 volume and regulate pH value to 9.6, will resist human IgG/IgM solution slowly to join among the HRP of activation, room temperature, lucifuge was reacted 2 hours.
Reduction Shiff alkali: add the NaBH4 of 0.01ml 5mg/ml in the product after will be above-mentioned crosslinked, 4 ℃ of lucifuges left standstill 2 hours behind the abundant mixing.
Remove small molecular weight impurity: above-mentioned cross-linking products was fully dialysed 16 hours to the phosphate buffer (PBS) of 0.01MpH7.4.
Remove free unlabelled HRP: take out liquid from bag filter, slowly add isopyknic saturated (NH4) 2SO4, fully 4 ℃ of standing over night behind the mixing, centrifugal 4000rpm 20 minutes, collecting precipitation with the PBS dissolution precipitation of 0.01MpH7.4, was fully dialysed 16 hours to 0.01M pH7.4 PBS.
Labelled antibody is collected first peak through Sephadex G-50 column chromatography, merges sample hose, concentrates.
Preserve: add the equal-volume sterile glycerol ,-20 ℃ of preservations are standby.
The clarification of enzymic-labelled antibody outward appearance, muddy, the nothing precipitation of nothing.Active calibrating the results are shown in Table 2 with susceptibility and specificity that the HCV system detects enzymic-labelled antibody.The result shows that this enzyme labelled antibody susceptibility and specificity are good, can be used for the SARS system.
The antibody activity verification result of table 2. enzyme labeling.
Determining of antigen-antibody reaction condition that embodiment 4 is suitable and time
| Dilutability | 1∶1000 | ?1∶2000 | ?1∶4000 | ?1∶8000 |
| Weak positive serum | 1.448 | ?0.835 | ?0.355 | ?0.212 |
| Negative serum | 0.203 | ?0.141 | ?0.054 | ?0.042 |
Under the envelope antigen and enzyme mark second antibody concentration that the foregoing description is determined, adopt ELISA Plate and reaction buffer as the aforementioned, research is suitable for antigen-antibody reaction and forms the condition of immunity in conjunction with compound.Select for use weak, in, strong 3 positive serums and negative control, carry out the indirect method experiment according to the method for operating of describing in detail among the embodiment 6.By reacting respectively 20,30,60,90,120 minutes under 37 ℃ of conditions, labelled antibody reaction 20,30,60 minutes developed the color 10 minutes.
1) when enzyme mark second antibody was IgM, the OD value of being surveyed reached mxm. in the time of 60 minutes.So the choice reaction time is 60 minutes.
2) when enzyme mark second antibody was IgG, the OD value of being surveyed reached mxm. in the time of 30 minutes.So the choice reaction time is 30 minutes.The positive threshold value cut-off of embodiment 5 indirect method immune detection samples determines
1) enzyme mark second antibody is IgG:
According to the method for embodiment 6 described immune detection, detect 300 parts and be defined as negative normal blood donation person's serum through indirect elisa method, get the normal distribution value and carry out statistical study.Getting average is 0.021, and SD is 0.004.Setting positive threshold value is 0.18+ negative control average.
Detect 1000 parts of donors with normal serum specimen at random, S/Co ratio is carried out the normal state statistical study, detect patients serum's sample of 10 parts of clinical definite SARS simultaneously, analyze the S/Co value, set the Cut-off value.Detect 1000 parts of donors with normal serum specimen at random, negative control OD value is lower than 0.05 by 0.05 calculating, is higher than 0.05 by calculated with actual values, calculates the A value, and the A value is carried out the normal state statistical study, and average is 0.05, and standard deviation (SD) is 0.044.See Fig. 2 with average+3 times standard deviation=0.05+3 * 0.044=0.182Cut-off value=its normal distribution of 0.13+ negative control A value.
Set the Cut-off value in view of the above for judging dividing value.As positive greater than Cutoff, on the contrary negative.Patients serum's sample that to detect 10 parts of clinical definites simultaneously be SARS is all greater than the Cut-off value.
2) in like manner, when enzyme mark second antibody was IgM, calculating the Cut-off value was 0.14.Embodiment 6, indirect method detect sars coronavirus antibody
1) each 1 hole of blank, negative control and positive control is established in each test.Blank well does not add liquid; The positive and negative contrast need not dilution and directly gets in the 50 μ l adding positive and negative control wells; All the other detect the hole and add sample diluting liquid, and every hole 100 μ l add testing sample 10 μ l again.Fully mixing sticks sealing compound, puts 37 ℃ of incubations 30 minutes.
2) 50 times are concentrated wash plate liquid and be used to wash plate hole after with 50 times of dilutions of distilled water.
3) discard sample in each hole, washing lotion is filled it up with in every hole, discards after leaving standstill the several seconds, repeats 5 times, pats dry.
4) every hole adding enzyme is marked anti-human IgG working fluid 100 μ l, sticks sealing compound.Put 37 ℃ of incubations 20 minutes.
5) discard liquid in each hole,, pat dry with washing plate liquid cyclic washing 5 times.
6) every hole adds substrate solution A 50 μ l, adds substrate solution B 50 μ l again, pats mixing, puts 37 ℃ of lucifuge incubations 10 minutes.
7) after colour developing finished, every hole added stop buffer 50 μ l, pats mixing, immediately with the blank well zeroing, put and measured OD value (reference wavelength is 630nm) under the microplate reader 450nm wavelength.The composition of embodiment 7, sars coronavirus detection kit
The preferred sars coronavirus detection kit of the present invention is made up of following components:
Envelope antigen: sars coronavirus totivirus lysate;
Antigen coated microplate: be homemade 96 hole portable plates, according to embodiment 4 described bags by sars coronavirus totivirus lysate;
Enzyme labelled antibody: the second antibody of horseradish peroxidase-labeled (IgG or IgM);
Negative control and positive control serum: each 0.2 milliliter, contain 0.05% thimerosal, 0.01% Sodium azide;
The enzyme labelled antibody working fluid: with containing 20% sheep blood serum, the dilution of 0.05% thimerosal is diluted to suitable tiring with enzyme labelled antibody.
The 0.01M pH4.5 citrate buffer solution of substrate solution A:0.06% urea peroxide;
Substrate solution B:0.06%TMB0.01M pH4.5 citrate buffer solution;
Stop buffer: 1M sulfuric acid
Cleansing solution: 50 * PBS contains 0.1% Tween-20;
SARS coronary virus resistant positive serum OD is worthwhile more than 0.13 (IgG) negative control average.
SARS coronary virus resistant negative serum OD is worthwhile below 0.13 (IgG).
SARS coronary virus resistant positive serum OD is worthwhile more than 0.11 (IgM) negative control average.
SARS coronary virus resistant negative serum OD is worthwhile below 0.11 (IgM).
Indirect method detects sars coronavirus antibody kit instructions portion.
Claims (9)
1. method that detects sars coronavirus antibody, it is as follows to it is characterized in that specifically detecting step:
1) utilize the totivirus lysate bag of purifying by a kind of solid support;
2) biological sample to be detected is added on the solid support of bag quilt, hatch suitable formation under the condition of antigen-antibody immunity in conjunction with compound;
3) clean any unconjugated sars coronavirus antibody of removal;
4) adding is hatched under the condition that is fit to formation antigen-antibody immune complex through the second antibody at people's antibody of mark;
5) clean any unconjugated second antibody of removal;
6) detect in the mixtures incubated antigen-antibody immunity in conjunction with the existence of compound.
2. a kind of method that detects sars coronavirus antibody according to claim 1 is characterized in that said biological sample is meant blood, serum, blood plasma, urine sample, body fluid, saliva and other secretion or excreta and tissue or the cell extract from the experimenter.
3. a kind of method that detects sars coronavirus antibody according to claim 1 is characterized in that said solid support comprises organic and inorganic polymer.
4. a kind of method that detects sars coronavirus antibody according to claim 1, the condition that it is characterized in that said suitable formation antigen-antibody immune complex, be meant the suitable temperature and time condition of the potpourri of the sample that can hatch antigen and contain antibody, wherein temperature is 0-50 degree centigrade, about 20 minutes to about 48 hours time.
5. a kind of method that detects sars coronavirus antibody according to claim 4 is characterized in that the condition described in the said claim 4, and wherein temperature is 4-30 degree centigrade, and the time is 20 minutes to 4 hours.
6. a kind of method that detects sars coronavirus antibody according to claim 1 is characterized in that said second antibody is anti-IgG antibody and/or anti-IgM or IgA antibody.
7. a kind of method that detects sars coronavirus antibody according to claim 1, it is characterized in that said second antibody labelled with radioisotope, or carry out mark with horseradish peroxidase, alkaline phosphatase, beta galactosidase or acetylcholinesterase.
8. kit that detects sars coronavirus antibody, it is characterized in that: its kit consists of:
1) envelope antigen: sars coronavirus totivirus lysate;
2) antigen coated microplate: be adopted as homemade 96 hole portable plates, and wrap by sars coronavirus totivirus lysate:
3) enzyme labelled antibody: the second antibody of horseradish peroxidase-labeled (IgG or IgM or IgA);
4) negative control and positive control serum: contain 0.05% thimerosal, 0.01% Sodium azide;
5) enzyme labelled antibody working fluid: with containing 20% sheep blood serum, the dilution of 0.05% thimerosal is diluted to suitable tiring with enzyme labelled antibody;
6) the 0.01M pH4.5 citrate buffer solution of substrate solution A:0.06% urea peroxide;
Substrate solution B:0.06%TMB0.01M pH4.5 citrate buffer solution;
7) stop buffer: 1M sulfuric acid;
8) cleansing solution: PBS contains 0.1% Tween-20;
9) indirect method detects sars coronavirus antibody kit instructions portion.
9. a kind of kit that detects sars coronavirus antibody according to claim 8 is characterized in that said sars coronavirus totivirus lysate is to cultivate after adopting Vero-E6 cell inoculation virus, obtains through the ultrasonic degradation purifying.
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