CN1908666A - Enzyme linked immunity diagnose reagent kit for HB core antigen detecting in two sandwich method and application thereof - Google Patents
Enzyme linked immunity diagnose reagent kit for HB core antigen detecting in two sandwich method and application thereof Download PDFInfo
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Abstract
The disclosed dual-sandwich enzyme-linked immunologic diagnosis agent box for hepatitis-B core antibody comprises: a micro-porous reaction plate composed by the hepatitis-B core antigen recombinant with purity more than 90% and 5*104~5*105u/mg titer, and the enzyme compound composed by the hepatitis-B core antigen recombinant marked by HRP. This invention has high specificity and sensitivity and convenient to qualitative or semi-qualitative detection.
Description
Technical field
The present invention relates to a kind of two sandwich method enzyme-linked immunologic diagnosis kits that detect hepatitis B core antibody, be applicable to the qualitative or half-quantitative detection of hepatitis B core antibody.
Background technology
Hepatitis B (HBV) is the pathogen that causes human hepatitis B, is to infect one of deadly virus the most widely at present in the world.According to statistics, it is carrier [Zuckerman J N, Zuckerman A J.Current topics in hepatitis B.J Infect, 2000 of chronic HBV that there is the crowd above 3,500,000,000 in the whole world, 41 (2): 130～136], wherein in state-owned 1.2 hundred million; And have a million people in the world approximately because of infecting the dead [Parkin of HBV every year, D.M, Pisani P, Munoz N and Ferlay J.The global healthburden of infection associated cancers.Cancer Surv, 1999,33:5～33.].
Owing to also do not treat at present the effective means of hepatitis B, so vaccine inoculation, early detection diagnosis are the emphasis of prevention hepatitis B.
The etiological diagnosis of hepatitis B, the most frequently used is the detection of hepatitis B blood serum designated object (HBVM).HBVM mainly comprises seven kinds of blood serum designated objects: hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), hepatitis B core antibody (anti-HBc), hepatitis B virus e antigen (HBeAg), hepatitis B e antibody (anti-HBe), HBV-DNA and DNA polymerase (DNA-p).Understand patient HBV infection, duplicate and state of an illness recovery situation by the multi-angle that detection HBVM can be indirect.
In HBVM, anti-HBc is comparatively unique: at first, anti-HBc is except that the initial period that HBV infects, and other all stages are the detected a kind of blood serum designated object of energy all; Acute HBV infection period and Chronic HBV the carry phase of this mark after HBsAg disappears all exists, even can preserve all the life.Therefore it is to reach the index that past attempts infects HBV now, to the detection of the anti-HBc information that can dynamically offer the best for the hepatitis B popularity of specific crowd.Secondly, anti-HBc be window phase (HBsAg can not detect and period that anti-HBs does not also occur) only, can detected blood serum designated object [Lee W M.Hepatitis B virus infection.NEngl J Med., 1997,337 (24): 1733～1745.].Because mainly being derived from, many blood products with infection HBV risk are in HBV carrier [the Schreiber G B that infects window phase, Busch M P, KleinmanS H, Korelitz J J.The risk of transfusion-transmitted viral infections.N Engl J Med, 1996,334 (26): 1685～1690.], so the screening of anti-HBc is infected prevention blood transfusion back HBV has great significance.In addition, people also find: some HBV variants show as feminine gender in HBsAg detects, but its anti-HBc but can be detected [Carman W F, Korula J, Wallace L.Fulminantreactivation of hepatitis B due to envelope protein mutant that escaped detection bymonoclonal HBsAg Elisa.Lancet, 1995,345:1406～1407.; Jongerius J M, WesterM, Cuypers H T M, van Oostendorp W R, Lelie P N, van der Poel C L, et al.Newhepatitis B virus mutant form in a blood donor that is undetectable in several hepatitisB surface antigen screening assays.Transfusion, 1998,38 (1): 56～59.].So, can reduce HBV the infected by the risk of omission to the detection of anti-HBc.In sum: the detection of anti-HBc is identified the tool significance to the diagnoses and treatment of hepatitis B, epidemiology survey and to the hepatitis B vaccine security.
At present, the detection method of hepatitis B core antibody (anti-HBc) mainly is divided into three major types: immunoadsorption hemagglutination test (IAHA), enzyme linked immunological absorption detect (ELISA) technology and radio-immunity detection (RAI) technology.Wherein elisa technique is highly sensitive, and price is relatively low, and domestic and international application is comparatively extensive.[Yao Zhen etc., molecule hepatitis type B virus related diseases.Chinese Medicine science and technology publishing house, 2003, p27～28.]
The ELISA detection method of normal employing is competitive inhibition method.The advantage of this detection method is: the purity requirement to envelope antigen is not high, and the interfering material in antigen-like material is difficult for removing, or when being difficult to obtain enough purifying antigens, available this method detection specificity antibody.But shortcoming also is clearly: sensitivity is not high, and the anti-HBc sample of low expression level is difficult to detect; Simultaneously, because the purity of envelope antigen is not high, thus may cause nonspecific reaction, thus the false positive testing result appears.Though bibliographical information is arranged in recent years both at home and abroad: adding certain reductive agent can increase specificity [the John A Weare et al.Improvement in the specificity of assays for detection of antibody to Hepatitis B coreantigen.J Clin Microbiol that competition inhibition method detects anti-HBc, 1991,29 (3): 600～604.].But the fact shows: this species specific raising is a cost to sacrifice sensitivity, can not tackle the problem at its root.Secondly, in testing process, add sample because of elder generation, enzyme-added again mark anti-HBc, so the anti-HBc that can cause sample is preferentially in conjunction with HBcAg, make sample anti-HBc and enzyme mark anti-HBc produce " unfair competition ", when sample determination in batch, the conference that adds of this time difference makes " unfair competition " aggravation, [Du Qing etc., the enzyme-added time is detected the influence of HBcAb to the problem that testing result differs before and after may producing to the competitive ELISA method.Jangsu Med J，2005，31(6)：466～468.]。In addition, traditional competition inhibition method detection is not easy to by visual weak positive and the negative sample distinguished.
Summary of the invention
The object of the present invention is to provide a kind of two sandwich method enzyme-linked immunologic diagnosis kits that detect hepatitis B core antibody, this dual-antigen sandwich method enzyme-linked immunologic diagnosis kit not only can use all types enzyme mark color comparator that exists at present on the market, the more important thing is and can detect more accurately hepatitis B core antibody apace.
To achieve these goals, the present invention will use following technical measures: a kind of two sandwich method enzyme-linked immunologic diagnosis kits that detect hepatitis B core antibody, this kit comprises micro reaction plate and enzyme conjugates, micro reaction plate be by 90% above purity, to tire be 5 * 10
4～5 * 10
5Hepatitis B virus core antigen recombinant protein (rHBcAg) bag of u/mg is formed; Enzyme conjugates is to be obtained by horseradish peroxidase (HRP) marking hepatitis B cAg recombinant protein.
The present invention's above-mentioned 90% above purity, to tire be 5 * 10
4～5 * 10
5After the hepatitis B virus core antigen recombinant protein of u/mg can contain the recombination engineering bacteria of hepatitis B virus core antigen protein gene by expression, obtain through ammonium sulfate precipitation and immunoaffinity chromatography separation and purification, wherein the eluent of immunoaffinity chromatography is the 0.1mol/mlpH11.5 triethylamine.
The working concentration of the hepatitis B virus core antigen recombinant protein of above-mentioned bag quilt is 1: 200～2000 (volume ratios); The working concentration of enzyme conjugates is 1: 500～2000 (volume ratios).
The working concentration of above-mentioned enzyme conjugates is 0.05% Tween-20 by the bovine serum albumin(BSA) that contains 10 grams per liters, volume ratio, and the 0.01mol/LpH7.4 phosphate buffer dilution of the amino pyrrole purine of the thimerosal of 0.1 grams per liter, 5 grams per liters obtains.
Of the present invention pair of sandwich method enzyme-linked immunologic diagnosis kit used in detecting hepatitis B core antibody.
The present invention compared with prior art has the following advantages and effect:
1, the specificity height of Jian Ceing;
That 2, detects is highly sensitive;
3, can eliminate the error that is produced because of the running time difference;
4, testing result is easy to visual judgement;
5, can carry out half-quantitative detection to sample simultaneously.
Description of drawings
Fig. 1 confirms to test for kit of the present invention is used to detect anti-HBc really;
Fig. 2 is used to detect the kinetic curve figure of anti-HBc for kit of the present invention.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that embodiment only to be used to the present invention is described and be not used in restriction the scope of protection of present invention.
Embodiment 1 detects two sandwich method enzyme-linked immunologic diagnosis kits of hepatitis B core antibody and forms and preparation
Horseradish peroxidase: available from Faizyme;
Recombination engineering bacteria: E.coli JM105 (pKK 223-3) [Marc Tordjeman et al.Specificdetection of anti-HBC antibodies with an enzyme immunoassay using recombinantHBcAg and monoclonal antibodies.J.Virol.Meth, 1993,43:21～30]; E.coli JM105 (pFS 14NSD) [Florian Schodel et al.Structure of hepatitis B Virus Core ande-Antigen:a single precore amino acid prevents nucleocapsid assembly.J Biol Chem, 1993,268 (2): 1332～1337]; [Zhou Fuyuan etc. use pQE30 system expression and purifying hepatitis B virus core antigen to E.coli M15 (pQE30).Chin J Biologicals，2000，13(1)：13～15]；
The hepatitis B virus core antigen recombinant protein: the recombination engineering bacteria 8000rpm that will express hepatitis B virus core antigen obtained bacterial sediment in centrifugal 10 minutes; The thalline of collecting is resuspended with 0.01mol/L pH7.4 phosphate buffer, and ice bath about the broken bacterium 3min of ultrasound wave, is clarified to suspension; Thereafter 10000rpm, centrifugal 20min collects supernatant; To go up immune affinity chromatographic column behind this supernatant usefulness 40wt% ammonium sulfate precipitation, with 0.1M pH11.5 triethylamine solution wash-out, obtaining 90% above purity, tiring is 5 * 10
4～5 * 10
5The hepatitis B virus core antigen recombinant protein of u/mg purifying;
Polystyrene micro-reaction plate: available from Greiner bio-one;
Coating buffer: 50mmol/L pH9.5Na
2CO
3-NaHCO
3Damping fluid;
Confining liquid: contain 50 grams per liter sucrose, 10 grams per liter bovine serum albumin(BSA)s, the 0.01mol/LpH7.4 phosphate buffer of 0.1 grams per liter thimerosal;
Sample diluting liquid: contain 10 grams per liter bovine serum albumin(BSA)s, volume ratio is 0.05%Tween-20, the 0.01mol/L pH7.4 phosphate buffer of 0.1 grams per liter thimerosal;
The enzyme dilution: contain 10 grams per liter bovine serum albumin(BSA)s, volume ratio is 0.05%Tween-20,0.1 grams per liter thimerosal, the 0.01mol/L pH7.4 phosphate buffer of the amino pyrrole purine of 5 grams per liters;
Cleansing solution: containing volume ratio is the 0.01mol/LpH7.4 phosphate buffer of 0.5%Tween-20;
Substrate buffer solution: contain the 0.51g/L citric acid, 1.46g/LNa
2HPO
4PH5.0 phosphoric acid-citrate buffer solution;
Substrate solution: contain 0.1g/L tetramethyl benzidine (TMB), volume ratio is 50% dimethyl formamide (DMF), and volume ratio is 0.3%H
2O
2Substrate buffer solution;
Stop buffer: 2mol/LH
2SO
4
Enzyme conjugates: adopt periodates improved method [Wilson M B, Nakane P K.Recentdevelopments in the periodate method of conjugating horseradish peroxidase toantibodies.Immunofluorescence and related staomomg techniques.North-HollandBiomedical Press, Amsterdam, 1978, pp215-224.; Nanci Donacki, PeroxidaseConjugation by Periodate Method.Protocol online.2004.] usefulness horseradish peroxidase (HRP) marking hepatitis B cAg recombinant protein (rHBcAg);
Micro reaction plate: with coating buffer with hepatitis B virus core antigen 1: 200 by volume～2000 dilution after,, put 4 ℃ and spend the night by 96 orifice plates with 100 μ l/ holes bag; Wash plate 5 times, each 5 minutes thereafter; Add 150 μ l/ hole confining liquids again in 96 orifice plates, put 37 ℃ of incubations 2 hours, wash oven dry behind the plate 3 times at last, seal up for safekeeping with sealing compound, standby in 4 ℃ of preservations.
The two sandwich method enzyme-linked immunologic diagnosis kits of embodiment 2 usefulness detect hepatitis B core antibody
1. add sample to be measured 100 μ l/ holes (sample to be measured is done dilution in 1: 30 with sample diluting liquid) at micro reaction plate, negative control, each two hole of positive control are set, and establish blank one hole; Shrouding is put 37 ℃ of incubation 1h;
2. wash plate 3 times, each 5min
3. add 1: 1200 enzyme-labelled antigen 100 μ l/ hole after the dilution, the only enzyme-added dilution in blank hole; Shrouding is put 37 ℃ of incubation 30min;
4. wash plate 3 times, each 5min
5. add substrate solution, shrouding is put 37 ℃ of incubation 15min;
6. add 50 μ l/ hole stop buffer cessation reactions;
7. detect: detect the absorbance value of each hole at 450nm, 630nm place with enzyme mark photometer; Its testing result is: sample OD
450=0.985, OD
630=0.041; Blank OD
450=0.065, OD
630=0.047; Negative control 1OD
450=0.084, OD
630=0.045; Negative control 2OD
450=0.088, OD
630=0.045; Positive control 1OD
450=0.794, OD
630=0.045; Positive control 2OD
450=0.820, OD
630=0.052.As calculated: sample OD=0.926; Negative control OD1=0.021, OD2=0.025; Positive control OD1=0.741, OD2=0.754.
8. the result judges: the average OD value of sample OD value/negative control 〉=2.1 are judged as the positive, otherwise negative; Wherein negative control OD value is lower than 0.05 calculating of 0.05 do, is higher than 0.05 by actual OD value calculating.Therefore, can judge: this sample is the hepatitis B core antibody positive.
3 pairs of sandwich method enzyme-linked immunologic diagnosis kits of embodiment are competed the comparison that inhibition method enzyme-linked immunologic diagnosis kit detects hepatitis B core antibody with tradition
1. our serum totally 943 examples (table 1) of having gathered different HBV states.Every part of blood serum sample all suppresses ELISA method diagnostic kit (available from Shanghai Kehua Bio-engineering Co., Ltd) with double antigens sandwich ELISA method and competition and detects simultaneously.If test result of samples is in rim condition and two kinds of testing results there are differences, then detect again one time with two kinds of trace routines.
The detected blood serum sample classification of table 1
| Sample type | Sample size | Shared number percent % |
| Past and the normal human serum that does not infect now HBV were injected the normal human serum HBsAg that hepatitis B vaccine or previous infection are crossed HBV +，HBcAb - HBsAg +，HBcAb +HBcAg only + | 90 260 5 583 5 | 9.54 27.6 0.53 61.8 0.53 |
| Add up to | 943 | 100 |
Testing result shows: have the testing result of 16 parts of blood serum samples inconsistent.Testing result with competition inhibition ELISA method detection kit is a reference value, and then the sensitivity of double antigens sandwich ELISA method can reach 99.8%, and specificity reaches 95.7%, and coincidence rate is 98.3% (table 2).
Two kinds of diagnostic kits of table 2 detect relatively complete list of hepatitis B core antibody result
| Dual-antigen sandwich method adds up to | Competition inhibition method | Add up to 612 331 943 | ||
| + - | + 597 1 a 598 | - 15 b 330 345 | ||
Sensitivity: 597/598=99.8%, specificity: 330/345=95.7%, coincidence rate: 927/943=98.3%; A:1 part serum HBsAg positive, b:3 part serum HBsAg positive, 7 parts of serum HBsAb positives, 5 parts of serum do not have the hepatitis B mark
2. specificity relatively
We carry out the detection of two sandwich method confirmatory tests (being neutralization test) to these 16 parts of inconsistent blood serum samples of testing result, with the specificity of conclusive evidence detection.Its testing result (Fig. 1) is: the neutralization test comparison of 15 parts of blood serum samples all wants much shallow according to the colour developing result of test and the test of adding normal human serum, explanation is in dual-antigen sandwich method detects, the testing result of this 15 duplicate samples is a true positives, is the specific detection result.And the blood serum sample of another part numbering 12.06.61, the colour developing of its neutralization test and control test shows that this is nonspecific reaction much at one.In sum: the present invention is detecting aspect the specificity of anti-HBc than existing competition inhibition ELISA diagnostic kit height.
3. the comparison of sensitivity:
Choose 7 parts of different positive serum samples and carry out doubling dilution, the greatest dilution of blood serum sample when titre reached home in record, thereby the relatively sensitivity of two kinds of detection methods, the result shows: the sensitivity of double antigens sandwich ELISA method is 32 to 256 times (table 3) of competition law.
Table 3 competition inhibition ELISA method and double fastener heart ELISA method remolding sensitivity are
| Sample | The terminal point titre | Relative sensitivity | |
| Competition inhibition ELISA method | Double antigens sandwich ELISA method | ||
| 1 2 3 4 5 6 7 | 240 480 1920 480 60 240 30 | 7680 15360 122880 15360 15360 61440 3840 | 32 32 64 32 256 256 128 |
In addition, [Yuan Hong, Xiong Yan etc., the dynamics comparative studies of four kinds of hepatitis B mark ELISA kits after deliberation.Med J West China, 2003,1 (1): 18～20.] to find: many domestic reagent boxes, the CO value of its anti-HBc approach the platform area and the linear zone intersection of kinetic curve figure, therefore are easy to cause the result to judge by accident in this zone.But be used to detect the S/CO kinetic curve figure (Fig. 2) of anti-HBc from kit of the present invention: on the one hand, the CO value of its anti-HBc is in the linear change district of kinetic curve figure, helps the qualitative interpretation to the blood serum sample testing result; On the other hand, its range of linearity broad, different dilution of sample curves are almost parallel, help hepatitis B core antibody in the blood serum sample is carried out semiquantitative detection, and this semiquantitative detection has great importance in clinical detection and diagnosis.
4. double fastener heart ELISA method repeatability is analyzed
Choose basic, normal, high 3 parts of different positive serum samples, use with a collection of double fastener heart method diagnostic kit and detect, every duplicate samples is parallel survey 8 holes respectively, carry out kit and criticize interior repeatability detection; In like manner choose basic, normal, high 3 parts of different positive serum samples, detect with the dual-antigen sandwich method diagnostic kit of 6 parts of different batches, the parallel survey of every duplicate samples 2 holes, carry out kit criticize between repeatability detect.
Testing result is: batch interior average coefficient of variation of this kit is 5.39% (table 4-1); Average coefficient of variation is 6.99% (table 4-2) between batch.Can determine thus: reagent used in the double fastener heart determination method is all more stable, this kit repeated satisfied.
Repeated testing result in the two sandwich method detection kit of table 4-1 are criticized
| The sample title | S/CO mean value | The S/CO intermediate value | The S/CO scope | The SD value | The CV% value |
| 1 2 3 | 8.326 6.411 2.194 | 8.238 6.448 2.200 | 7.743～9.038 5.933～6.657 1.924～2.267 | 0.459 0.262 0.144 | 5.50％ 4.09％ 6.57％ |
| Average CV% | 5. 39％ | ||||
Repeated testing result between the two sandwich method detection kit of table 4-2 are criticized
| The sample title | S/CO mean value | The S/CO intermediate value | The S/CO scope | The SD value | The CV% value |
| 1 2 3 | 7.321 12.066 0.288 | 7.352 12.248 2.800 | 6.648～7.848 10.952～ 13.100 2.70～3.50 | 0.406 0.774 0.259 | 5.55％ 6.41％ 9.00％ |
| Average CV% | 6.99％ | ||||
5. double fastener heart ELISA method stability analysis
Reagent storage is preserved in 37 ℃ of environment, regularly detect, till its quality index begins to descend.It has been generally acknowledged that at 37 ℃ and whenever stablized one day, can be equivalent to 4～10 ℃ and preserve one and a half months.Show according to measurement result (table 5): this kit was placed 5 days for 37 ℃, and its S/CO value changes the non-significant difference of genus; But at the 5th day, sample surpassed 15% in the coefficient of variation (CV%) of 4 ℃ and 37 ℃ two groups of data being measured.So infer: this dual-antigen sandwich method detection kit can be preserved 6 months at 4 ℃ at least, the stability of its kit better.
Total the above: two sandwich method enzyme-linked immunologic diagnosis kits are highly sensitive, specificity good, testing result accurately and reliably, its stability and repeatability meet the clinical detection requirement.
The Detection of Stability result of table 5 pair sandwich method diagnostic kit
| The sample title | The 3rd day | The 4th day | The 5th day | |||
| S/CO mean value ± SD value | ||||||
| 4℃ | 37℃ | 4℃ | 37℃ | 4℃ | 37℃ | |
| 1 2 3 | 0.786±0.055 1.276±0.045 1.065±0.088 | 0.898±0.017 1.491±0.045 1.215±0.001 | 0.824±0.001 1.376±0.005 1.216±0.088 | 0.79±0.071 1.37±0.028 1.12±0.014 | 0.886±0.021 1.377±0.030 1.171±0.066 | 0.639±0.040 1.000±0.053 0.88±0.027 |
| CV％ | 9.34～9.97 | 0.38～5.82 | 20.07～22.91 | |||
The t assay: third and fourth, the difference between five days 4 ℃ and 37 ℃ of two groups of measured data is non-significant difference
Claims (5)
1, a kind of two sandwich method enzyme-linked immunologic diagnosis kits that detect hepatitis B core antibody, this kit comprises micro reaction plate and enzyme conjugates; It is characterized in that: micro reaction plate be by 90% above purity, to tire be 5 * 10
4～5 * 10
5The hepatitis B virus core antigen recombinant protein bag of u/mg is formed; Enzyme conjugates is to be obtained by horseradish peroxidase-labeled hepatitis B virus core antigen recombinant protein.
2, according to claim 1 pair of sandwich method enzyme-linked immunologic diagnosis kit is characterized in that: 90% above purity, to tire be 5 * 10
4～5 * 10
5The hepatitis B virus core antigen recombinant protein of u/mg is after containing the recombination engineering bacteria of hepatitis B virus core antigen protein gene by expression, obtain through ammonium sulfate precipitation and immunoaffinity chromatography separation and purification, wherein the eluent of immunoaffinity chromatography is a 0.1mol/ml pH11.5 triethylamine.
3, according to claim 1 or 2 described pairs of sandwich method enzyme-linked immunologic diagnosis kits, it is characterized in that: the working concentration volume ratio of the hepatitis B virus core antigen recombinant protein of bag quilt is 1: 200～2000; The working concentration volume ratio of enzyme conjugates is 1: 500～2000.
4, according to claim 3 pair of sandwich method enzyme-linked immunologic diagnosis kit, it is characterized in that: the working concentration of enzyme conjugates is 0.05% Tween-20 by the bovine serum albumin(BSA) that contains 10 grams per liters, volume ratio, and the 0.01mol/L pH7.4 phosphate buffer dilution of the amino pyrrole purine of the thimerosal of 0.1 grams per liter, 5 grams per liters obtains.
5, described pair of sandwich method enzyme-linked immunologic diagnosis kit of claim 1 used in detecting hepatitis B core antibody.
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| CN102043050A (en) * | 2009-10-23 | 2011-05-04 | 上海荣盛生物药业有限公司 | In vitro detection method of hepatitis B virus C antibody |
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| CN103217533A (en) * | 2012-01-21 | 2013-07-24 | 厦门大学 | Anti-HBc quantitative determination method and application of anti-HBc quantitative determination method for monitoring disease progression of chronic hepatitis B patients and predicting therapeutic effect |
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| CN1132857A (en) * | 1995-10-31 | 1996-10-09 | 刘毅维 | Detection method of core antigen of hepatitis virus |
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| CN102043050A (en) * | 2009-10-23 | 2011-05-04 | 上海荣盛生物药业有限公司 | In vitro detection method of hepatitis B virus C antibody |
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| CN103616511A (en) * | 2013-12-06 | 2014-03-05 | 苏州长光华医生物医学工程有限公司 | Hepatitis B core antibody measurement kit and detection method thereof |
| CN106785256A (en) * | 2017-01-18 | 2017-05-31 | 苏州富电通讯有限公司 | A kind of medium comb filter |
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