CN1368638A - Reagent kit for enzyme immunoassay of hepatitis B virus proantigen S1 and its preparing process - Google Patents
Reagent kit for enzyme immunoassay of hepatitis B virus proantigen S1 and its preparing process Download PDFInfo
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Abstract
The ivnention discloses a reagent kit for enzyme-free determination of the preceding S1 antibody of B-type hepatitis virus and its preparing method. Higher position rate of preceding S1 antibody is obtained in detecting blood serum of B-type hepatitis virus by use of the invented reagent. The preceding S1 antibody detected is consistent with recovery state of B-type hepatitis, so it provides a indicator for whether the illness of B-type hepatitis can be recovered in time or not. The invented reagent has high specificity and high-application value in detecting hepatitis.
Description
The present invention relates to biotechnology reagent, be specifically related to a kind of hepatitis B virus pro-S
1Abzyme exempts to measure kit and preparation method.
The dna sequence analysis of hepatitis type B virus (HBV) shows that the S district of HBV gene is by S, preceding S1, preceding S2 forms, its encoded protein has major protein (HBsAg), middle molecule protein (HBsAg adds preceding S2) and high molecular weight protein (HBsAg, preceding S2 and preceding S1), because of pre-s1 protein is present in the high molecular weight protein, so the pre-s1 protein that infects in the blood all is present in the HBsAg positive serum, pre-s1 protein is made up of 108 or 119 amino acid, its length is different because of hypotype, the ayw hypotype is 108aa, the adw hypotype has 119 aa, and pre-s1 protein is positioned at the surface of virion, thereby it has the immunogenicity of height.Milich is with the high molecular weight protein immune mouse, the T cell proliferation test of the synthetic peptide of each section of S1 before doing then, experiment shows, the P1-21 of preceding S1 peptide chain, the T cell had significant proliferation that three sections polypeptide of 12-32 and 94-117 can cause illustrates that these three sections amino acid sequences have immunogenicity, is the immunologic determinants district, and P32-53 and P74-89 peptide section can not make T cell proliferation external, point out this two sections sequence non-immunogenicities.Further research to P1-21 and 12-32 peptide section, after with P12-32 peptide section immune mouse, can cause identical T cell proliferation with 1-21 and 12-32 again, the common sequences of pointing out this dipeptides section is that P12-21 is important antigenic determinant district, has played main activation.Show again with the experimental result of short peptide chain more, the immunocompetence of P12-19 only reaches 1.6% of P12-21, the activity of P14-21 only reaches 0.4% of P12-21, also promptly remove the amino acid of P12-21 amino acid end or c-terminus, just extremely influenced the immunogenicity of P12-21, confirmed to cause that the T cell is 10 peptides to the minimum amino acid no of immunogenicity identification.In the P12-21 peptide, substitute wherein the 14th, 18 and 19 respectively, immunogenicity only is equivalent to 66.6%, 0.7% and 1.6% of peptide chain respectively, illustrates that in 10 peptides of P12-21, the 18th and 19 amino acids are the most important to immunogenicity.This has higher reference value to S1 polypeptide antigen before synthetic.And Alberti P21-32, the S1 polypeptide all can detect the corresponding antibodies among the acute hepatitis b patients serum before three sections of 32-47 and the 94-117, illustrate that P32-53 does not have immunogenicity, and analyze that the former S 1 immune body of anti-P32-47 has even more important effect from the dynamics situation of former S 1 immune body.P32-47 antibody appears earlier in most acute hepatitis b patients in early days, occurs P21-32 and P94-117 antibody then, and P32-47 antibody only appears in the patient who has, and two kinds of antibody are negative all the time in addition.In recovering smoothly acute hepatitis b, P94-117 antibody all exists simultaneously with P32-47 antibody, the situation that does not have independent P94-117 antibody positive, and in chronic hepatitis B, often there are not two kinds of antibody in addition with regard to having only P94-117 antibody, this shows that P32-47 and P21-32 antibody majority come across prognosis preferably in the acute hepatitis b, and the P94-117 antibody positive is generally chronic hepatitis separately.
The infection experiment of chimpanzee proves, serum pre S 1 antigen albumen can detect from electrophoresis band in metainfective the 5th week the earliest, anti-former S 1 immune body occurs to the 8th all serum, pre S 1 antigen disappears during 18 weeks, at the whole viewing duration until 36 weeks, former S 1 immune body is positive all the time, and anti-HBs just occurred until 33 weeks, because be 6 week-6 month the latent period of hepatitis B, so when most of acute hepatitis b is fallen ill, anti-former S 1 immune body just can present positive findings, and it is the reliability index of early diagnosis of hepatitis B.
Former S 1 immune body generally comes across in the acute self limiting hepatitis B, and the acute hepatitis b and the long-term carrier of HBsAg of chronicity development, the recall rate of this antibody is all very low.All rehabilitations smoothly of 4 routine acute hepatitis bs of the former S 1 immune body positive in serum when Theilmanm report is admitted to hospital, and 6 the failing rehabilitation of routine former S 1 immune body feminine gender and transfer to chronic, 10 these antibody total negatives of routine HBsAg carrier.Illustrate and occur the early signal that former S 1 immune body is state of an illness prognosis bona in the serum.Budkowska points out that also the patient of the smooth rehabilitation of acute hepatitis b all has former S 1 immune body, and all detects less than former S 1 immune body in the chronic hepatitis of acute hepatitis b chronicity and pre S 1 antigen lasting masculin.Former S 1 immune body promotes that the mechanism of rehabilitation may be the competitive inhibitory effect that former S 1 immune body has been brought into play PHSA, antibody has sealed the PHSA acceptor on pre S 1 antigen and the liver cell, make PHSA can not bring into play " bridging " effect, enter hepatocellular approach thereby blocked HBV.In addition, former S 1 immune body with after HBV goes up pre S 1 antigen and combine, has been brought into play the effect of removing virus by other immunization route specifically, and therefore, former S 1 immune body is the interior neutrality antibody of body.Closely during the last ten years, numerous foreign study reports show, the former S 1 immune body of hepatitis B is a neutrality antibody, has the effect that stops virus replication and remove virus, and it is that hepatitis B diseases presents the very early time signal that recovers trend, and is more direct and timely than two half projects aspect the understanding state of an illness and judging prognosis.Therefore, it is the early recovery index of hepatitis B diseases, and anti-former S 1 immune body almost comes across in the serum simultaneously with HBsAg and anti-HBc, so it is again hepatitis B infected early diagnosis index.
According to Alberti A, Caralletto D, Chemello L.et al:Fine specificity of humanantibody response to the preS1 domain of hepatitis B Virus.Hepatology1990; 12 (2): 199-203. and TheilmannL, Klinkert MQ, Gmelin K, et al:Detection ofantibodies against pre-S1 proteins in sera of patients with hepatitis Bvirus (HBV) infection, J hepato 1987; Reports such as 4:22-28, when the acute hepatitis b patient is admitted to hospital, most of patients can detect the positive findings of indexs such as pre S 1 antigen, former S 1 immune body, HBsAg, HBV-DNA and anti-HBc simultaneously, though from animal experiment study, recognize in the middle of the latent period of infecting, index such as pre S 1 antigen and HBsAg does sth. in advance to come across in the blood in 2-4 week than former S 1 immune body, but this does not influence its effect as clinical early diagnosis index.
In hepatitis B two and half indexs; having only anti-HBs is protection antibody; its sun in blood changes the removing of expression virus; the rehabilitation of disease; but anti-HBs generally will just occur after HBsAg turns out cloudy, and the time that it occurs is later, to general acute hepatitis b patient; the positive of anti-HBs comes across 4-5 month after the morbidity, so its just basic no early stage prognosis effect.And the result that just is positive in the serum of anti-former S 1 immune body majority a few days after morbidity, it can with anti-HBc, HBsAg, early diagnosis indexs such as HBeAg are present in the serum simultaneously.Though anti-HBc also early occurs behind HBV, it is an infectious index, and anti-HBc is not a neutralizing antibody, there is no much effects to removing virus and state of an illness prognosis.And tangible indicative function is arranged in this respect with the early stage simultaneously anti-former S 1 immune body that occurs of anti-HBc, many-sided research has shown that all anti-former S 1 immune body has the effect that stops hepatitis B to enter the liver cell breeding and remove virus to this antibody, its sun in serum changes infectants such as indication HBsAg and pre S 1 antigen and all can turn out cloudy smoothly, disease is rehabilitation morning, it is unique that its early stage prognosis acts in all indexs of hepatitis B, so it has very big using value in the laboratory diagnosis of hepatitis B.
The objective of the invention is to develop a kind of hepatitis B virus pro-S
1Abzyme exempts to measure kit.
The invention provides a kind of hepatitis B virus pro-S
1Abzyme exempts to measure kit, and this kit is made up of by reaction bar, enzyme conjugates working fluid, positive control, negative control thing, sample diluting liquid, dense cleansing solution, developer A, developer B and stop buffer pre-bag.
Of the present invention dose of agent box is as follows in the clinical use result of first affiliated hospital of The 2nd Army Medical College:
Being subjected to Shanghai Xinxin Medical Biology Engineering Co.'s trust that the anti-preceding S1 elisa kit of the hepatitis B of its production has been carried out clinical use examines, the case blood serum sample comes from the court's contagious department and clinical laboratory, the strict by specification of experimental implementation carries out, and clinical use result and evaluation are as follows:
1, the preceding S of hepatitis B in the hepatitis B different phase
1Antibody positive rate
S before table 1 hepatitis B
1S before the positive rate serum mark serum specimen of antibody in hepatitis B is anti-
1Positive anti-preceding S
1Negative positive rate (%) HbsAg (+) anti-HBc (+) the 254 107 147 42.1 anti-HBc of anti-HBs (-) HbsAg (-) (+) the 127 35 92 27.6 anti-HBc of anti-HBs (-) HbsAg (-) (+) 175 105 70 60 anti-HBs (+) add up to 556 247 309 44.4
Anti-preceding S in the table 1
1Total positives rate in hepatitis B serum is 44.4%, this antibody can come across in any phase hepatitis B serum of HbsAg positive or negative, especially it should be noted that at HbsAg and turn out cloudy, and in the anti-HbsAg serum that sun does not change as yet, the positive rate of former S 1 immune body also reaches 27.6%, this has shown that this antibody occurs Zao than anti-HBs, also point out the sun commentaries on classics of this antibody to indicate that disease develops to convalescence, the positive rate of this antibody is 60% in anti-HBs (+) serum, show that in convalescence the life period of former S 1 immune body in serum is shorter than anti-HBs.
2, the time relationship of anti-preceding S1 and the commentaries on classics of anti-HBs sun
To the analysis of cases that successively occurs the former S 1 immune body positive in the 56 routine hepatitis B courses of disease time relationship of this antibody and anti-HBs antibody male rotary, the results are shown in Table 2.
The time relationship sun refunding example that S1 and anti-HBs sun turned to before table 2 was anti-adds up to the 512 34-5 months 5123 of the 71112 53-4 months, the 10 3321 92-3 months, the 17 17414 171-2 months of 12 2721 122-4 week in＜2 weeks to add up to 56 3 14 10 697 49 the number 2-3 month 3-4 month 4-5 month 5-6 month 6-7 month 7-8 month
From table 2, can find out, the time of occurrence of former S 1 immune body in serum occurred than anti-HBs in 1 month at least ahead of time, generally can do sth. in advance 12-16 week, before 56 examples are anti-in S1 (+) serum, in 8 months observation period, anti-at last HBs positive person 49 examples account for 87.5%, and the rehabilitation of hepatitis B is described because of the appearance of anti-HBs, so anti-former S 1 immune body is a hepatitis B diseases rehabilitation index the earliest, and good practical value is arranged.3, detect 252 parts of non-hepatitis B serum (and hepatitis B two half indexs are negative entirely), S before the anti-hepatitis B between assessment period altogether
14 parts of antibody (+), negative 248 parts, specificity 98.4%, false positive 1.6%, the specificity height of this reagent.4. because former S 1 immune body is not 100% positive in the hepatitis B, be 44.4% as the positive rate of this antibody in hepatitis B in the table 1, still unmatched both at home and abroad again S1 antibody kit can be done contrast therewith and measure, so do not do the test of reagent True Positive Rate in this examination.5. coefficient of variation experiment
Get 3 parts of hepatitis B former S 1 immune body positives, every part of serum is done 12 multiple holes simultaneously and is measured, and average as calculated variation within batch coefficient is 7.53%.1 2 3 4 5 6 7 8 9 10 11 12 X S CV1?0.962?0.947?1.034?0.857?1.086?0.953?0.893?0.895?0.934?0.955?0.959?0.890?0.947?0.064?6.7％2?0.524?0.549?0.493?0.555?0.581?0.487?0.590?0.605?0.531?0.524?0.510?0.587?0.545?0.040?7.3％3?0.410?0.443?0.398?0.384?0.410?0.459?0.493?0.395?0.476?0.447?0.486?0.419?0.435?0.038?8.6％
These 3 parts of positive serums are not being measured 5 times in the same date altogether, and average interassay coefficient of variation is 8.8%.1 2 3 4 5 X S CV1?0.895?1.056?0.927?0.976?1.031?0.977?0.068 6.9％2?0.474?0.536?0.613?0.492?0.557?0.534?0.055 10.3％3?0.390?0.376?0.470?0.413?0.387?0.407?0.038 9.2％
Estimate: this reagent detects in hepatitis B serum that the positive rate of former S 1 immune body is higher, and the former S 1 immune body that detects is consistent with the recovery of the hepatitis B state of an illness, and rehabilitation in time has early stage indication effect to hepatitis B diseases.The specificity of reagent is very high, and false positive is very low, and the coefficient of variation is in critical field, and the precision test meets the requirements, and therefore, we think this kit reliable in quality has higher using value in hepatitis B detects.
Kit of the present invention is as follows in the clinical use result of Xinhua Hospital Attached to Shanghai No.2 Medical Univ: the hepatitis B former S 1 immune body ELISA kit that uses Shanghai Xinxin Medical Biology Engineering Co.'s development is counted 368 examples to the first and second the third penta 4 viroid hepatitis patients serums, 1786 parts of blood samples are made former S 1 immune body and are measured, and the result is as follows:
One, the anti-preceding positive rate of S1 in every virus hepatitis
S1 (-) positive rate (%) first type 74 anti-HAV IgM (+) 72 0 72 0 before S1 (+) resisted before table 1 hepatitis B former S 1 immune body positive rate hepatitis type example number serum mark example number was anti-
Anti-HAV IgM (+) companion 2020
HBsAg (+) B-mode 225 is HBsAg (+) 225 139 86 61.7 at least
Third type, 48 anti-HCV (+) 37 0 37 0 more than one
Anti-HCV (+) companion HBsAg (+) 11 38 27.3 penta types 21 anti-HEV IgM (+) 18 0 18 0
Anti-HEV (+) accompanies hepatitis B 312 33.3
Serum mark adds up to 368 368 143 225 38.9
* the hepatitis B serum mark means HBsAg, anti-HBs, and HBeAg, anti-HBe a positive person occurs in five signs of anti-HBc at least.
Anti-former S 1 immune body (+) totally 143 examples in four viroid hepatitis, 368 examples in the table 1, the total positives rate is 38.9%, wherein mainly come across in the hepatitis B patient, anti-preceding S1 (+) person in the hepatitis B observation course of disease, occurs and account for 139/225, positive rate is 61.7%, do not have in first, the third penta 3 class hepatitis that merge hepatitis B, anti-former S 1 immune body is (-) all, and it measures characteristic is 100%.The three class hepatitis that are associated with hepatitis B are totally 16 examples, anti-before S1 (+) 4 examples, positive rate 25% is analyzed by statistics, in the simple hepatitis B anti-before S1 (+) rate and hepatitis B merge anti-in other hepatitis before S1 (+) rate compare X2=8.37, P＜0.01, both difference highly significants.
The anti-preceding S1 of anti-preceding S1 positive rate hepatitis type example number serum umber in 1786 parts of hepatitis serum of table 2, S1 before (+) is anti-, (-) positive rate, (%) first type 74 259 0 259 0 B-mode 225 1,192 572 620 48 third types, 48 235 7 228 2.9 penta types 21 100 4 96 4 add up to 368 1,786 583 1,203 32.6
Table 2 has shown the overall positive rate of anti-former S 1 immune body in virus hepatitis, and in all detection serum of hepatitis B, this antibody positive rate is 48%, is lower than 61.7% positive value in the table 1.Anti-preceding S1 positive rate in third type in this table and the Hepatitis E is that each calculates with total detection serum, and its value is respectively 2.9% and 4%.Third liver merges 11 examples that have of hepatitis B, totally 51 parts of anti-preceding S1 serum, and positive serum is 7 parts, positive rate is 13.7%, and viral hepatitis type E merges 3 examples of hepatitis B, and the serum that detects anti-preceding S1 has 17 parts, occur positive totally 4 parts, positive rate is 23.5%, and these two numerical value also all are lower than in the table 1 27.3% and 33.3% analog value, and the sun of S1 changeed relevant with stadium before this prompting was anti-, also illustrated in the simple hepatitis B, the S1 sun changes early before anti-, and after having merged third liver or viral hepatitis type E, the sun of having postponed antibody changes the time.
Two, the sun of anti-former S 1 immune body changes the time
The anti-former S 1 immune body sun of table 3 changes and the routine number of the relationship type of stadium＜2 all 2-4 week 4-12 week 12-16 week 16-20 week hepatitis B 139 62 47 17 58 third viral hepatitis type Es merging hepatitis B 4112
Table 3 is presented in the 139 routine hepatitis B, the anti-former S 1 immune body of 62 examples sun in 2 weeks of morbidity changes, account for 44.6%, positive revolution has reached 109 examples in 4 weeks, accounts for 78.4% of total routine number, illustrates that former S 1 immune body occurs in the hepatitis B course of disease early, the certain diagnosis and the value of judging prognosis are arranged, but after being associated with other hepatitis, the sun of this antibody changes time retardation, has influenced the recovery of the state of an illness.
Use the detection practice of Shanghai Xinxin Medical Biology Engineering Co.'s former S 1 immune body kit in virus hepatitis, think that the advantage of this kit is 1. high specificities, in 127 other type hepatitis of example of nonjoinder hepatitis B, none this antibody positive of example.2. there is early diagnosis to be worth.The positive rate of anti-former S 1 immune body is 61.7% in simple hepatitis B, and nearly half antibody male rotary in 2 weeks of morbidity.3. the recall rate of former S 1 immune body is relevant with the state of an illness, former S 1 immune body positive rate 61.7% in the simple hepatitis B, and the positive rate that is associated with this antibody in the hepatitis B of other hepatitis is 25%, both difference highly significants.This prompting latter's the often more difficult recovery of hepatitis B is low relevant with the positive rate of former S 1 immune body, in simple hepatitis B, the recovery that the sun of former S 1 immune body changes time and disease also has relation, and the positive commentaries on classics time early, the general state of an illness recovers more smooth, illustrates that former S 1 immune body works in the rehabilitation of hepatitis B.4. kit is stable, and easy to operate, testing result is correct.
Kit of the present invention is as follows in the clinical use result of Shuguang Hospital: 1. hepatitis B former S 1 immune body positive rate
Table 1 has compared the positive detection rate of former S 1 immune body in hepatitis B and non-hepatitis B patient serum.It is standard that and hepatitis B any one positive occurs with two half five indices, and and non-hepatitis B is two half five indices total negatives, still has the part hepatitis B patient to be associated with hepatitis C, and also classification takes statistics.Former S 1 immune body positive rate in the hepatitis B is 40.9% as can be seen from Table 1; be associated with this antibody positive rate of third liver apparently higher than hepatitis B; because former S 1 immune body is a protection antibody; can promote the recovery of the state of an illness, thus this statistical description be associated with third liver hepatitis B cause more difficult rehabilitation because of the former S 1 immune body positive rate is low.The positive rate 2.3% of serum former S 1 immune body in the non-hepatitis B group, this is the false positive rate of this reagent, specificity is 97.7%, illustrates that the specificity of this preceding S1 detection kit is higher.
Table 1 hepatitis B former S 1 immune body positive rate group serum umber former S 1 immune body (+) former S 1 immune body (-) positive rate (%) hepatitis B 584 239 345 40.9 hepatitis B merge third liver, 53 11 42 20.7 non-hepatitis B 178 4 174 2.32, hepatitis B former S 1 immune body and HbsAg negative conversion rate
Table 2 has been added up the negative conversion rate of HBsAg in the serum of hepatitis B of the positive and 63 routine former S 1 immune body feminine genders of 50 routine former S 1 immune bodies, the result shows that the HBsAg negative conversion rate of antibody positive group is apparently higher than the negative antibody group, the statistical discrepancy highly significant, illustrate that former S 1 immune body can promote that HBsAg turns out cloudy, so this detection of antibodies is to judge an early stage index of hepatitis B prognosis.
Table 2 former S 1 immune body and the HBsAg negative conversion rate group example number HBsAg several negative conversion rate former S 1 immune bodies (+) 50 41 82.0 of turning out cloudy
*The precision test of former S 1 immune body (-) 63 24 38.1P＜0.013. kit
(1) variation within batch coefficient
Get 2 parts of positive serums and respectively do 12 holes again, record the average batch of interior coefficient of interior change and be 6.85%.
1 2 3 4 5 6 7 8 9 10 11 12 X S CVA450?0.902?0.804?0.871?0.822?0.810?0.912?0.881?0.854?0.894?0.871?0.842?0.852?0.859?0.035?4.1％
0.595?0.617?0.682?0.553?0.586?0.708?0.523?0.681?0.680?0.596?0.572?0.588?0.615?0.059?9.6％
(2) interassay coefficient of variation
2 parts of hepatitis B former S 1 immune body positive serums are respectively measured 6 times at different time, and recording average coefficient of variation is 7.8%.
1 2 3 4 5 6 X S CVA450?0.871?0.903?0.825?0.897?0.817?0.917?0.871?0.042?4.8％
0.572?0.619?0.687?0.523?0.697?0.640?0.623?0.067?10.8％
Use is the result show, this reagent stability is good, easy and simple to handle, negative background is clear, and the yin and yang attribute contrast is obvious, and experimental result is more easily judged, the positive rate and the bibliographical information of former S 1 immune body are approaching, the sun of this index time of changeing is obviously changeed the time early than the moon of HBsAg in serum, and therefore, its detection judges that to the prognosis of the hepatitis B state of an illness higher reference significance is arranged.
Another object of the present invention has provided above-mentioned hepatitis B virus pro-S
1Abzyme exempts to measure the preparation method of kit, and this method is the preceding S1 dna sequence dna 1 10 20 30 40 50 60MGGWSSKPRKEMGTMLSYPNPLGFFPDHQLDPAFGANSNNPDWDFNPVKDDWPA ANQVGVGA of the preparation (1) of (one) genetic engineering pre S 1 antigen
70 80 90 100 110FGPRLTPPNGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDS HPQA (2) material: HBV are provided by 5 years four Room of institute of section of army.PCRKit, restriction enzyme, the T4DNA ligase is available from the magnificent company in Shanghai.GST, reduced form 4B be available from Sigma company, the synthetic upstream primer 5 of last sea base Kanggong department '-CGGATC-CATGGGAGGTTGGTCTTCCA, downstream primer 5 '-GC-GATTTCCTAGTGGAATGTTGTGGAGT.(3) method: be reflected on the PCR of the Progene company synthesizer and carry out, reaction conditions is 94 ℃ of 30s, 60C 30s, 72 ℃ of 30s, 35 circulations.The PCR product is by the order-checking of basic Kanggong department.(4) expression and purifying: the PCR product is cut with BamH I and EcoR I enzyme, and electrophoresis reclaims the fragment of 387 bp on 1% Ago-Gel, is connected with the carrier pGEX-4T-1 that same enzyme is cut, transformed into escherichia coli HB101 strain, induce and use IPTG, final concentration 1mmol/L, SDS-PAGE analysis result.Press Sepharose4B instructions purifying expressed fusion protein product.Adopt indirect elisa method to detect former S 1 immune body.(2) preparation of SPA-HRP (1) claims 5mg horseradish peroxidase (HRP) to add 0.5ml distilled water, adds in 15 ' 4 ℃ of the 0.5ml 60mM sodium periodate oxidations again.(2) add 4 ℃ 30 of 0.16M ethylene glycol 0.5ml blocking ' (3) and add the pure SPA0.5ml of 10mg/ml, pack is spent the night to sodium carbonate buffer.(4) take out liquid in the bag, add 5mg/ml potassium borohydride 0.2ml, 4 ℃ of 3h refill bag filter PH7.2 0.01M PBS dialysis 24h are changed liquid 4-5 time.(5) take out SPA-HRP work activity and titration (three) negative control sera and join (1), mix the serum of A450＜0.1 with 30 parts of this kit detection normal human serums.(2) above-mentioned pooled serum detects once after adding the equivalent calf serum again, determines A450＜0.1.(3) add 2/0,000 Sodium azides in the pooled serum, filter in the packing-20 ℃ and preserve.(4) positive control serum preparation (1) is got the anti-preceding S1 antiserum of rabbit and is done the titration of ELISA former S 1 immune body; Get 2 times of positive control serums of concentration that the titration of A450＞2.0 is tired.(2) get this concentration antiserum and add the equivalent calf serum and be ELISA again and measure in A450＞1.5 (3) above-mentioned positive serums and add 2/0,000 Sodium azides, filter packing, preserve in-20 ℃.(5) substrate buffer solution preparation 1, first liquid (1) respectively claim sodium hydrogen phosphate 18.4 grams, citric acid 5 grams, and PM50mg puts into volumetric flask and adds distilled water to 1000ml, adds 30% hydrogen peroxide 400ul after the stirring and dissolving again.(2) packing bottle 2, second liquid (1) claim EDTA 150mg to add in the 1000ml distilled water heating for dissolving after the filtration sterilization.(2) go up but back adding citric acid 1.05 grams of liquid cooling, add TMB250mg again with the dimethyl sulfoxide dissolving.(3) divide in the black bottle of packing into.(6) foundation of hepatitis B preS1 antibody ELISA method of immunity of the present invention.(1) be 5ug/ml concentration with 20mM Tris-Hcl liquid with pure pre S 1 antigen dilution, add 0.1ml in each reacting hole, put 24h in 4 ℃, abandoning supernatant pats dry.(2) in test tube, serum to be checked is done dilution in 1: 25 with distilled water (or physiological saline).(3) drip 1 sample diluting liquid in each hole, add the serum 50ul to be checked of dilution in 1: 25 then respectively, do positive control hole and negative control hole simultaneously.(4) put in 37 ℃ of incubators reaction after 25 minutes, get rid of liquid earlier under toilet paper arsis 2-3 in the hole in, wash 5 times with cleansing solution then, each cylindrical void stops 30 seconds kinds after filling it up with cleansing solution, gets rid of in the hole all will pat dry on toilet paper behind the liquid, so that thoroughly wash at every turn.(5) each hole adds 2ug/mlSPA-HRP bond 50ul, and 37 ℃ were reacted 25 minutes.(6) the same thorough washing pats dry.(7) each hole drips each 1 of substrate buffer solution first, second, puts in 37 ℃ and reacts 5 minutes, and every again hole drips 1 of stop buffer, surveys the A450 value.(8) result judges
Negative control A value * 2.5=critical value, all specimen hole A value＞critical values to be checked are promptly positive, and negative control calculated by 0.05 less than 0.05 o'clock.(7) sensitivity, specificity and precision test.1, the definite value of sensitivity determination (1) positive serum concentration
In detecting the pre S 1 antigen experimental implementation, in the energy and the antibody of the high dilution serum of 1ng/ml pre S 1 antigen be decided to be 1u/ml.During practical operation pre S 1 antigen is made into 20ng/ml concentration.(2) sensitivity experiment
With titration in the above-mentioned experiment is 1280u/ml, and 3 parts of positive serums of 160u/ml and 40u/ml are made sensitivity determination by the experimental implementation method after doing the variable concentrations dilution with sample diluting liquid, and the result is as follows:
Serum antibody titer (u/ml)
Serum
1280 640 320 160 80 40 20 10
No. 1＞2
*1.65 1.46 0.98 0.74 0.40 0.24 0.17
No. 2 1.15 0.70 0.46 0.27 0.15 0.06
No. 3 0.45 0.27 0.10
*A450
Measure 6 parts of negative serums, A450 is respectively 0.14,0.06,0.11,0.07,0.07,0.08, negative average A 450=0.088, positive critical value=0.088 * 2.5=0.22, then this detection method is respectively 20u/ml to the detection sensitivity of 3 parts of positive serums, 40u/ml and 20u/ml average out to 26.7u/ml.2, specific assay
And hepatitis B two half kits of producing with Shanghai Long March medical science company limited are detected totally 30 parts of the serum of five indices total negative, measure by this experimental technique former S 1 immune body, the result is as follows: 123456789 10 11 121 0.06 0.05 0.06 0.05 0.08 0.07 0.10 0.05 0.04 0.11 0.09 0.072 0.10 0.12 0.10 0.07 0.18 0.11 0.13 0.07 0.06 0.10 0.12 0.143 0.12 0.10 0.13 0.09 0.08 0.07
The A value of measuring by the experimental implementation method with 6 parts of calf serums is respectively 0.05,0.05,0.08,0.07 simultaneously, 0.06,0.09, mean value is 0.067, critical value=0.067 * 2.5=0.168, then in 30 parts of serum 1 part of serum being arranged is probable positive, specificity=29/30=96.7%.3, precision is measured
Get 3 parts of positive serums that the experiment absorbance is respectively high, normal, basic A value and do the precision test, test is done 12 multiple holes simultaneously for every part of serum in batch, and test was tested 1 time at interval for every part of serum in 2-3 days between batch, measured altogether 6 times, and the result is as follows:
Test in batch: 123456789 10 11 12 X S CV (%) 1 1.42 1.37 1.40 1.35 1.37 1.45 1.32 1.40 1.32 1.48 1.30 1.27 1.37 0.0625 4.62 0.74 0.86 0.70 0.81 0.83 0.80 0.72 0.82 0.70 0.85 0.82 0.84 0.79 0.059 7.53 0.26 0.25 0.31 0.27 0.24 0.32 0.25 0.26 0.25 0.25 0.28 0.29 0.27 0.0257 9.5
CV=7.2% in average crowd
Test between batch
1 2 3 4 5 6 X S CV(％)
1?1.45?1.30?1.41?1.38?1.28?1.29?1.35 0.071 5.3
2?0.72?0.86?0.70?0.75?0.84?0.77?0.773?0.0644?8.3
3?0.26?0.21?0.31?0.25?0.28?0.26?0.262?0.033 12.7
CV=8.77% between average crowd
Example 1, preparation hepatitis B virus pro-S
1Abzyme exempts to measure kit one, main material
1. genetic engineering pre S 1 antigen our company preparation
2. pure SPA Shanghai Vaccine and Serum Institute product
3. horseradish peroxidase U.S. Sigma company
4. goat-anti people IgM (u) U.S. Sigma company
5. the new isolation technics of enzyme reaction lath East China University of Science research department
6. calf serum Hangzhou Chinese holly biomaterial engineering corporation
7. glycerine Shanghai the May 4th pharmaceutical factory
8.Nacl Shanghai reagent one factory
9.Na2HPO4.12H2O Shanghai Xinhua chemical plant
10.KH2PO4 Tianjin chemical reagent six factories
11.Na2CO3 last marine rainbow photoinitiator chemical factory
12.NaHCO3 last marine rainbow photoinitiator chemical factory
13. potassium borohydride Shanghai reagent one factory
14. ethylene glycol Shanghai chemical reagent head factory
15. sodium metaperiodate Beijing Chemical Plant
16. ethylenediamine tetraacetic acid Shanghai reagent one factory
17.TMB Shanghai east wind chemical reagent work
18. citric acid Shanghai reagent one factory two, method: be reflected on the PCR of the Progene company synthesizer and carry out, reaction conditions is 94 ℃ of 30s, 60C 30s, 72 ℃ of 30s, 35 circulations.The PCR product is by the order-checking of basic Kanggong department.Three, expression and purifying: the PCR product is cut with BamH I and EcoR I enzyme, and electrophoresis reclaims the fragment of 387bp on 1% Ago-Gel, is connected with the carrier pGEX-4T-1 that same enzyme is cut, transformed into escherichia coli HB101 strain, induce and use IPTG, final concentration 1mmol/L, SDS-PAGE analysis result.Press Sepharose 4B instructions purifying expressed fusion protein product.Adopt indirect elisa method to detect former S 1 immune body.Four, the preparation of SPA-HRP (1) claims 5mg horseradish peroxidase (HRP) to add 0.5ml distilled water, adds in 15 ' 4 ℃ of the 0.5ml 60mM sodium periodate oxidations again.(2) add 4 ℃ 30 of 0.16M ethylene glycol 0.5ml blocking ' (3) and add the pure SPA0.5ml of 10mg/ml, pack is spent the night to sodium carbonate buffer.(4) take out liquid in the bag, add 5mg/ml potassium borohydride 0.2ml, 4 ℃ of 3h refill bag filter PH7.2 0.01M PBS dialysis 24h are changed liquid 4-5 time.(5) take out SPA-HRP work activity and titration five, negative control sera and join (1), mix the serum of A450＜0.1 with 30 parts of this kit detection normal human serums.(2) above-mentioned pooled serum detects once after adding the equivalent calf serum again, determines A450＜0.1.(3) add 2/0,000 Sodium azides in the pooled serum, filter in the packing-20 ℃ and preserve.Six, positive control serum preparation (1) is got the anti-preceding S1 antiserum of rabbit and is done the titration of ELISA former S 1 immune body; Get 2 times of positive control serums of concentration that the titration of A450＞2.0 is tired.(2) get this concentration antiserum and add the equivalent calf serum and be ELISA again and measure in A450＞1.5 (3) above-mentioned positive serums and add 2/0,000 Sodium azides, filter packing, preserve in-20 ℃.Seven, substrate buffer solution preparation 1, first liquid (1) respectively claim sodium hydrogen phosphate 18.4 grams, citric acid 5 grams, and PM50mg puts into volumetric flask and adds distilled water to 1000ml, adds 30% hydrogen peroxide 400ul after the stirring and dissolving again.(2) packing bottle 2, second liquid (1) claim EDTA 150mg to add in the 1000ml distilled water heating for dissolving after the filtration sterilization.(2) go up but back adding citric acid 1.05 grams of liquid cooling, add TMB250mg again with the dimethyl sulfoxide dissolving.(3) divide in the black bottle of packing into.
Example 2, one, purposes: this kit adopts the ELISA indirect method, and donor detects hepatitis B pre-s1 protein antibody (anti-preS1) usefulness in the human serum outward, has quick, sensitive, special and characteristics easily.Two, kit is formed: 1. pre-bag is reacted 1 bottle of 3ml three of bar 6. 20 times of dense cleansing solutions of 1 bottle of 3ml of 1 bottle of 3ml3. positive control of 48 holes, 2. enzyme conjugates working fluids, 1 200ul5. sample diluting liquid of 1 200ul, 4. negative controls 1 bottle of 13ml7. developer A1 bottle 3ml 8. developer B1 bottle 3ml 9. stop buffers, operation steps:
1. take out and wrapped, insert on the support, use immobilization with adhesive tape, with slip-off preventing by cylindrical void.
In test tube with serum to be checked, do dilution in 1: 25 with distilled water (or physiological saline).
3. drip 1 sample diluting liquid (50ul) in each hole, add the serum 50ul to be checked of dilution in 1: 25 then respectively, do positive control 1 hole simultaneously, negative control 1 hole (respectively adding 50ul), blank 1 hole (hole contains 2 of sample diluting liquids).
4. put in 37 ℃ of incubators reaction after 25 minutes, get rid of liquid earlier under toilet paper arsis 2-3 in the hole in, wash 5 times with cleansing solution then, each cylindrical void stops 30 seconds kinds after filling it up with cleansing solution, all will pat dry on toilet paper after getting rid of cleansing solution, so that washing is thorough at every turn.(20 times of dense cleansing solution 1ml+19ml distilled water are the work cleansing solution)
5. except that the blank hole, every hole adds 1 of enzyme conjugates working fluid (50ul), and reaction is 25 minutes in 37 ℃.
6. the same thorough washing pats dry.
7. each hole drips each 1 of developer A, B, puts that 5-10 minute every again hole of reaction drips 1 of stop buffer in 37 ℃, puts 450nm wavelength readings in the microplate reader.Four, the result judges:
Negative control A value * 2.5=critical value, all specimen hole A values to be checked are promptly positive greater than critical value.Negative control calculates by 0.05 less than 0.05.Five, points for attention:
1, kit is deposited in 4 ℃-8 ℃, the term of validity 12 months.
2, incubation reaction plate temperature and time must strictly be controlled, and washing must be thoroughly.
3, different lot number reagent must not be used with.
Claims (7)
1, a kind of hepatitis B virus pro-S
1Abzyme exempts to measure kit, it is characterized in that this kit is reacted bar by pre-bag, and enzyme conjugates working fluid, positive control, negative control thing, sample diluting liquid, dense cleansing solution, developer A, developer B and stop buffer are formed.
2, an a kind of hepatitis B virus pro-S as claimed in claim 1
1Abzyme exempts to measure the preparation of kit, it is characterized in that this method is: the preceding S1 dna sequence dna 1 10 20 30 40 50 60 MGGWSSKPRKEMGTMLSYPNPLGFFPDHQLDPAFGANSNNPDWDFNPVKDDWPAAN QVGVGA of the preparation (1) of (one) genetic engineering pre S 1 antigen
70 80 90 100 110 FGPRLTPPNGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQ A (2) material: HBV are provided by 5 years four Room of institute of section of army, PCRKit, restriction enzyme, the T4DNA ligase is available from the magnificent company in Shanghai, GST, reduced form 4B is available from Sigma company, the synthetic upstream primer 5 of last sea base Kanggong department '-CGGATC-CATGGGAGGTTGGTCTTCCA, downstream primer 5 '-GC-GATTTCCTAGTGGAATGTTGTGGAGT, (3) method: be reflected on the PCR of the Progene company synthesizer and carry out, reaction conditions is 94 ℃ of 30s, 60C 30s, 72 ℃ of 30s, 35 circulations, the PCR product is by the order-checking of basic Kanggong department, (4) expression and purifying: the PCR product is cut with BamH I and EcoR I enzyme, electrophoresis reclaims the fragment of 387bp on 1% Ago-Gel, be connected with the carrier pGEX-4T-1 that same enzyme is cut, transformed into escherichia coli HB101 strain, induce and use IPTG, final concentration 1mmol/L, the SDS-PAGE analysis result, press Sepharose 4B instructions purifying expressed fusion protein product, adopt indirect elisa method to detect former S 1 immune body, (2) preparation of SPA-HRP (1) claims 5mg horseradish peroxidase (HRP) to add 0.5ml distilled water, add again in 15 ' 4 ℃ of the 0.5ml 60mM sodium periodate oxidations, (2) add 4 ℃ 30 of 0.16M ethylene glycol 0.5ml blocking ' (3) and add the pure SPA0.5ml of 10mg/ml, pack is spent the night to sodium carbonate buffer, (4) take out liquid in the bag, add 5mg/ml potassium borohydride 0.2ml, 4 ℃ of 3h refill bag filter PH7.2 0.01M PBS dialysis 24h are changed liquid 4-5 time, (5) taking out SPA-HRP does active and titration (three) negative control sera is joined (1) with 30 parts of this kit detection normal human serums, the serum that mixes A450＜0.1, (2) above-mentioned pooled serum detects once after adding the equivalent calf serum again, determine A450＜0.1, (3) add 2/0,000 Sodium azides in the pooled serum, filter in the packing-20 ℃ and preserve, (four) positive control serum preparation (1) get rabbit anti-before the S1 antiserum do the titration of ELISA former S 1 immune body; Get 2 times of positive control serums of concentration that the titration of A450＞2.0 is tired, (2) getting this concentration antiserum adds the equivalent calf serum and is ELISA again and measures in A450＞1.5 (3) above-mentioned positive serums and add 2/0,000 Sodium azides, filter packing, preserve in-20 ℃, (5) the substrate buffer solution preparation 1, first liquid (1) respectively claims sodium hydrogen phosphate 18.4 grams, citric acid 5 grams, PM50mg, put into volumetric flask and add distilled water to 1000ml, add 30% hydrogen peroxide 400ul after the stirring and dissolving again, (2) packing bottle 2 after the filtration sterilization, second liquid (1) claims EDTA 150mg to add in the 1000ml distilled water, heating for dissolving, (2) go up but back adding citric acid 1.05 grams of liquid cooling, add TMB250mg again with the dimethyl sulfoxide dissolving, (3) divide in the black bottle of packing into, (6) foundation of hepatitis B preS1 antibody ELISA method of immunity of the present invention, (1) be 5ug/ml concentration with 20mM Tris-Hcl liquid with pure pre S 1 antigen dilution, add 0.1ml in each reacting hole, put 24h in 4 ℃, abandoning supernatant, pat dry, (2) in test tube, serum to be checked is done dilution in 1: 25 with distilled water (or physiological saline), (3) drip 1 sample diluting liquid in each hole, the serum 50ul to be checked that adds dilution in 1: 25 then respectively, do positive control hole and negative control hole simultaneously, (4) put in 37 ℃ of incubators reaction after 25 minutes, get rid of in the hole liquid earlier under toilet paper arsis 2-3, wash 5 times with cleansing solution then, each cylindrical void stops 30 seconds kinds after filling it up with cleansing solution, get rid of in the hole at every turn and all will on toilet paper, pat dry behind the liquid, so that thoroughly washing, (5) each hole adds 2ug/mlSPA-HRP bond 50ul, 37 ℃ were reacted 25 minutes, (6) the same thorough washing pats dry, (7) each hole drips the substrate buffer solution first, each 1 of second, put in 37 ℃ and reacted 5 minutes, every again hole drips 1 of stop buffer, surveys the A450 value, and (8) result judges
Negative control A value * 2.5=critical value, all specimen hole A value＞critical values to be checked are promptly positive, and negative control calculated by 0.05 less than 0.05 o'clock, (seven) sensitivity, specificity and precision test, 1, the definite value of sensitivity determination (1) positive serum concentration
In detecting the pre S 1 antigen experimental implementation, in the energy and the antibody of the high dilution serum of 1ng/ml pre S 1 antigen be decided to be 1u/ml, during practical operation pre S 1 antigen is made into 20ng/ml concentration, (2) sensitivity experiment
With titration in the above-mentioned experiment is 1280u/ml, and 3 parts of positive serums of 160u/ml and 40u/ml are made sensitivity determination by the experimental implementation method after doing the variable concentrations dilution with sample diluting liquid, and the result is as follows:
Serum antibody titer (u/ml)
Serum
1280 640 320 160 80 40 20 10
No. 1＞2
*1.65 1.46 0.98 0.74 0.40 0.24 0.17
No. 2 1.15 0.70 0.46 0.27 0.15 0.06
No. 3 0.45 0.27 0.10
*A450
Measure 6 parts of negative serums, A450 is respectively 0.14,0.06, and 0.11,0.07,0.07,0.08, negative average A 450=0.088, positive critical value=0.088 * 2.5=0.22, then this detection method is respectively 20u/ml to the detection sensitivity of 3 parts of positive serums, 40u/ml and 20u/ml average out to 26.7u/ml, 2, specific assay
And hepatitis B two half kits of producing with Shanghai Long March medical science company limited are detected totally 30 parts of the serum of five indices total negative, measure by this experimental technique former S 1 immune body, the result is as follows: 123456789 10 11 121 0.06 0.05 0.06 0.05 0.08 0.07 0.10 0.05 0.04 0.11 0.09 0.072 0.10 0.12 0.10 0.07 0.18 0.11 0.13 0.07 0.06 0.10 0.12 0.143 0.12 0.10 0.13 0.09 0.08 0.07
The A value of measuring by the experimental implementation method with 6 parts of calf serums is respectively 0.05 simultaneously, 0.05,0.08,0.07,0.06,0.09, mean value is 0.067, critical value=0.067 * 2.5=0.168, and then in 30 parts of serum 1 part of serum being arranged is probable positive, specificity=29/30=96.7%, 3, precision measures
Get 3 parts of positive serums that the experiment absorbance is respectively high, normal, basic A value and do the precision test, test is done 12 multiple holes simultaneously for every part of serum in batch, and test was tested 1 time at interval for every part of serum in 2-3 days between batch, measured altogether 6 times, and the result is as follows:
Test in batch: 123456789 10 11 12 X S CV (%) 1 1.42 1.37 1.40 1.35 1.37 1.45 1.32 1.40 1.32 1.48 1.30 1.27 1.37 0.0625 4.62 0.74 0.86 0.70 0.81 0.83 0.80 0.72 0.82 0.70 0.85 0.82 0.84 0.79 0.059 7.53 0.26 0.25 0.31 0.27 0.24 0.32 0.25 0.26 0.25 0.25 0.28 0.29 0.27 0.0257 9.5
CV=7.2% in average crowd
Test between batch
1 2 3 4 5 6 X S CV(％)
1?1.45?1.30?1.41?1.38?1.28?1.29?1.35 0.071 5.3
2?0.72?0.86?0.70?0.75?0.84?0.77?0.773?0.0644?8.3
3?0.26?0.21?0.31?0.25?0.28?0.26?0.262?0.033 12.7
CV=8.77% between average crowd.
3, a kind of hepatitis B virus pro-S according to claim 1
1Abzyme exempts to measure kit, it is characterized in that wherein said bag is-48 holes by the reaction bar.
4, a kind of hepatitis B virus pro-S according to claim 1
1Abzyme exempts to measure kit, it is characterized in that the positive control serum of wherein said positive control, the negative control serum of negative control thing.
5, a kind of hepatitis B virus pro-S according to claim 1
1Abzyme exempts to measure kit, it is characterized in that its described sample diluting liquid is PBST.
6, a kind of hepatitis B virus pro-S according to claim 1
1Abzyme exempts to measure kit, it is characterized in that wherein said cleansing solution is 20 times of concentrated cleaning solutions.
7, a kind of hepatitis B virus pro-S according to claim 1
1Abzyme exempts to measure kit, it is characterized in that wherein said developer A is H
2O
2, developer B is TMB.
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| CNB011052937A CN100385237C (en) | 2001-02-01 | 2001-02-01 | Reagent kit for enzyme immunoassay of hepatitis B virus proantigen S1 and its preparing process |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100582781C (en) * | 2006-06-27 | 2010-01-20 | 厦门大学 | Method for joint investigating hepatitis B virus pro S1 antigen and nuclear antigen and diagnostic kit |
| CN104569402A (en) * | 2014-12-15 | 2015-04-29 | 新乡医学院 | Harmless positive reference substance for detection through indirect enzyme linked immunosorbent assay |
| CN104569447A (en) * | 2014-12-15 | 2015-04-29 | 新乡医学院 | Harmless positive reference substance for detecting animal pathogen |
| CN104569403A (en) * | 2014-12-15 | 2015-04-29 | 新乡医学院 | Harmless positive control for enzyme-linked immune diagnosis of hepatitis c |
| CN108872596A (en) * | 2018-07-05 | 2018-11-23 | 重庆巴而思生物科技有限公司 | A kind of ELISA detection kit of HPV16 L1 antibody |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1134552A (en) * | 1995-08-25 | 1996-10-30 | 中国科学院上海植物生理研究所 | Quick diagnostic reagent kit for gonorrhoea and its production process |
| CN1057609C (en) * | 1998-01-22 | 2000-10-18 | 上海晶莹生物技术有限公司 | Cytotoxin production pylorus spirillum antibody enzyme immune testing box and preparation process thereof |
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2001
- 2001-02-01 CN CNB011052937A patent/CN100385237C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100582781C (en) * | 2006-06-27 | 2010-01-20 | 厦门大学 | Method for joint investigating hepatitis B virus pro S1 antigen and nuclear antigen and diagnostic kit |
| CN104569402A (en) * | 2014-12-15 | 2015-04-29 | 新乡医学院 | Harmless positive reference substance for detection through indirect enzyme linked immunosorbent assay |
| CN104569447A (en) * | 2014-12-15 | 2015-04-29 | 新乡医学院 | Harmless positive reference substance for detecting animal pathogen |
| CN104569403A (en) * | 2014-12-15 | 2015-04-29 | 新乡医学院 | Harmless positive control for enzyme-linked immune diagnosis of hepatitis c |
| CN104569403B (en) * | 2014-12-15 | 2016-08-24 | 新乡医学院 | Innoxious positive control for hepatitis C enzyme-linked immunologic diagnosis |
| CN108872596A (en) * | 2018-07-05 | 2018-11-23 | 重庆巴而思生物科技有限公司 | A kind of ELISA detection kit of HPV16 L1 antibody |
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| CN100385237C (en) | 2008-04-30 |
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