CN101735311B - Hepatitis E virus antigen, antigen composition thereof and kit comprising antigen or antigen composition and application thereof - Google Patents
Hepatitis E virus antigen, antigen composition thereof and kit comprising antigen or antigen composition and application thereof Download PDFInfo
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Abstract
The invention relates to a Hepatitis E virus antigen. The antigen has the following antigen fragments of: pS1-6, pS4-6, pS1-1 or pS4-1, wherein pS1-6 is a C tail end antigen of type 1 HEV ORF2 with a locus of 351-391 to 640-660aa; pS4-6 is the C tail end antigen of type 4 HEV ORF2 with a locus of 365-405 to 654-674aa; pS1-1 is an N tail end antigen of type 1 HEV ORF2 with a locus of 1-20 to 90-130aa; and pS4-1 is the N tail end antigen of type 4 HEV ORF2 with a locus of 1-20 to 104-144aa. The invention also relates to a composition comprising the antigen and a kit comprising the antigen or the antigen composition and application in detecting the Hepatitis E virus antigen. The Hepatitis E virus antigen provided by the invention has the advantages of strong antigen city and strong specificity. The kit provided by the invention and the application can detect an anti-HEV IgM antibody appearing in a sample as soon as possible so as to realize correct detection and diagnosis in the early period of HEV infection.
Description
Technical field
The present invention relates to a kind of hepatitis virus antigen, relate in particular to a kind of hepatitis E virus antigen, its antigen composition and contain test kit and the application of this antigen or antigen composition.
Background technology
HEV is a kind of nonencapsulated sub-thread positive chain RNA virus, and the about 7.2kb of genome total length has three opening reading frames (ORF), is followed successively by non-coding region (NCR), ORF1, ORF2, ORF3 and 3 ' end non-coding region and PolyA tail from 5 ' end.Wherein the N end parts amino acid of the disconnected amino acid in ORF3 and ORF1C end and ORF2 is overlapping.ORF1 1690 amino acid of encoding mainly are the Nonstructural Proteins relevant with virus replication.ORF2 660 amino acid of encoding are the structural protein of virus.ORF3 123 amino acid of encoding, function is still not clear.ICTV (ICTV) does not accurately classify to it temporarily at present.Lu (Lu L, Li CH, Hagedorn.2006.Phylogenetic analysis of global hepatitis E virus sequences:geneticdiversity, subtypes and zoonosis.Rev Med Virol 16:5-36.) etc. the nucleotide sequence of 421 HEV among the Genbank is analyzed, HEV mainly is divided into 4 genotype, and has tangible region.1 type mainly is distributed in developing countries such as Asia and Africa, and 2 types mainly are distributed in Mexico, and 3 types are mainly in developed countries such as Europe and America, and 4 types are mainly in the Asia.Namely there is 1 type in China, and the popular of 4 types also arranged.From animals such as pig, isolated at present the virus strain of a large amount of HEV 3 types and 4 types, thought that HEV 3 types and 4 types are infecting both domestic animals and humans, but whether 1 type and 2 types the are infecting both domestic animals and human sexually transmitted diseases further research of still needing.
Hepatitis E (HE) is a kind of viral hepatitis non-A non-B that is caused by hepatitis E virus (HEV).HE belongs to typical self-limiting disease, the acute hepatitis phase continue approximately one arrive around, generally can not develop into chronic hepatitis.Mainly propagate through excrement-mouth approach, existing distributing also has popularly, often causes that in the area of sanitary condition difference large-scale outbreak is popular, mainly caused by pollution of waterhead.Infection rate is the highest 15～40 years old person between twenty and fifty, and mortality ratio can reach 20% after the pregnant woman infected HEV, pregnant woman's lethality rate of especially conceived about three months even up to 25%, and the survivor also has higher abortion ratio and still birth rate.Probability contagious is lower, might have vertical transmission and blood propagation.HE takes place commonplace in the crowds of developing country such as Asia, Africa and America, can account for 50% of acute viral hepatitis in some areas, is to cause the important factor of falling ill and dying of illness.The HEV antibody positive rate reaches 1～5% in the blood donor of the popular country of some non-HE.The person of raising pigs in the popular district of non-HE with animal doctor that pig contacts closely in, anti-HEV antibody positive rate is far above common blood donor.
Serology and detection of nucleic acids are the main method of hepatitis E epidemiology and clinical diagnosis.Wherein the former mainly is the anti-HEV antibody that detects in the serum: IgM, IgG and IgA; Detection of nucleic acids mainly is by detection serum, the HEV RNA. in bile and the ight soil, and these detections can complementaryly be used for helping clinical difference to infect recently and previous infection, to reduce mistaken diagnosis.
Existing hepatitis E etiology detects and mainly contains following several method:
1. immunology detection
The method of immunology detection is primarily aimed at anti-HEV antibody, adopts enzyme-linked immune detection method, detects anti-HEV IgM, IgG and IgA antibody in the serum.
The morning that anti-HEV IgG occurs, in acute phase titre rising, longer duration, it detects the main indirect elisa method that adopts at present, and ORF2 and ORF3 antigen that utilization is recombinated detect.Use comparatively general commercialization reagent to be the detection kit of Genelab company and Abott company in the world wide.They all adopt HEV1 type and 2 type antigens for detecting antigen.But, because the anti-HEV IgG antibody time length is longer, so can't distinguish previous infection and recent infection.
Chau et al thinks that IgA antibody also can be used as HEV in the recent period or the sign antibody of acute infection, though the meaning in its positive time length and the HEV diagnosis is still waiting clinical and further confirmation epidemiological study.But in the diagnosis of acute HIV infection, the detection of IgA can be used as the ancillary test that IgM detects, the conclusive evidence diagnosis.
It has been generally acknowledged that IgM is more Zao than IgG as the most important significant antibody appearance of acute infection.A desirable IgM detection reagent should have following characteristics: sensitivity, and false positive rate is low, and the time length is short, can distinguish recent infection and previous infection.The commercialization reagent of domestic widespread use on the market at present is that Singapore Genelab company and the Wan Tai of Pekinese company produce.And the antigen that most of reagent place adopt all is the C end of ORF2 and the ORF3 antigen of part or total length.
In addition, Zhang et al has also reported and has utilized the method for the HEV antigen in the monoclonal antibody detection serum that makes up to come diagnosing acute to infect.What its ELISA used is the ORF2 antigen that comes from 1 type and 4 types.
2. molecular Biological Detection
Molecular Biological Detection refers to use the RT-PCR method to detect serum, ight soil, bile and the HEV RNA. that organizes in the equal samples.Yet, the HEV viremia time length is very short, missed viremia during many hospitalizes or near latter stage, viral RNA copy number in the blood is very low, be difficult to detect by RT-PCR, this situation is especially outstanding in the underdeveloped developing country of medical and health condition, though the ight soil toxin expelling time length slightly is longer than viremia, but may there be the material that influences PCR in the sample collecting difficulty in vulnerable to pollution and the ight soil.
At present there is very big difference in the positive rate that detects in patient HE of the RT-PCR of report, and from 30% to 80% does not wait, this may with the sample collecting time, the preservation situation, the primer coupling, the PCR condition is relevant with other uncertain factors.Therefore, the RT-PCR diagnostic method that detects HEV RNA at present only is used for laboratory study, does not also have the commercialization reagent in clinical wide popularization and application.
To sum up, be badly in need of a kind of stablizing at present, fast, the reagent of highly sensitive and specific HEV diagnosis of infection reagent and gene type.For this reason, the inventor carries out partitioned representation by ORF2 and the ORF3 to HEV, and the antigenicity of obstructed antigen fragment has filtered out the antigen fragment combination of the anti-HEV IgM of best detection after testing, obtained highly sensitive, high specificity, the test kit of the anti-HEV IgM antibody that occurs in the test sample as early as possible, thus realized accurately detecting diagnosis in early days what HEV infected.
Summary of the invention
The object of the present invention is to provide a kind of hepatitis E virus antigen, the antigenicity of this antigen is preferable, high specificity.
For realizing purpose of the present invention, adopt following technical scheme:
A kind of hepatitis E virus antigen, wherein:
The antigen fragment of described antigen is pS1-6, pS4-6, pS1-1 or pS4-1;
Described pS1-6 is the C-terminal antigen of 1 type HEV ORF2, and its site is 351～391 to 640～660aa;
Described pS4-6 is the C-terminal antigen of 4 type HEV ORF2, and its site is 365～405 to 654～674aa;
Described pS1-1 is the N-terminal antigen of 1 type HEV ORF2, and its site is 1～20 to 90～130aa;
Described pS4-1 is the N-terminal antigen of 4 type HEV ORF2, and its site is 1～20 to 104～144aa.
Be preferably as follows antigen fragment:
Described pS1-6 is the C-terminal antigen of 1 type HEV ORF2, and its site is 371-660aa;
Described pS4-6 is the C-terminal antigen of 4 type HEV ORF2, and its site is 385-674aa;
Described pS1-1 is the N-terminal antigen of 1 type HEV ORF2, and its site is 1-110aa;
Described pS4-1 is the N-terminal antigen of 4 type HEV ORF2, and its site is 1-124aa.
Among the present invention, described 1 type HEV ORF2 and 4 type HEV ORF2 are referring to " 1 type and 4 type hepatitis E virus ORF2 antigenicities are relatively " (" 1 type and 4 type hepatitis E virus ORF2 antigenicities are relatively " of He Peng, Zhang Huayuan, Wang Youchun and Li Zhuo, China's microbiology and Journal of Immunology the 26th the 3rd phase of volume of March in 2006: 210-214), 660 amino acid of 1 type HEV ORF2 coding wherein, 4 type HEV ORF2 674 amino acid of encoding.
Specific as follows as above-mentioned preferred antigen fragment:
The fragment of pS1-6 for getting 371-660aa among 660 amino acid whose 1 type HEV ORF2 of coding;
The fragment of pS4-6 for getting 385-674aa among 674 amino acid whose 4 type HEV ORF2 of coding;
The fragment of pS1-1 for getting 1-110aa among 660 amino acid whose 1 type HEV ORF2 of coding;
The fragment of pS4-1 for getting 1-124aa among 674 amino acid whose 4 type HEV ORF2 of coding.
Another object of the present invention is to provide a kind of hepatitis E virus antigen composition, this antigen composition antigenicity is strong, high specificity, and detection time early is more suitable for clinical application.
Antigen composition of the present invention is that described antigen composition makes up or the N-terminal of pS1-1 and the C-terminal combination of pS4-6 for C-terminal and the N-terminal combination of pS1-1, C-terminal and the N-terminal combination of pS4-1, the C-terminal of pS1-6 and the N-terminal of pS4-1 of pS4-6 with pS1-6.
Preferably, antigen composition provided by the present invention is that the C-terminal of pS1-6 and the N-terminal of pS1-1 are made up or the C-terminal of pS4-6 and the N-terminal of pS4-1 are combined.
The present invention also provides a kind of test kit that detects hepatitis E virus IgM antibody, and this test kit has been realized the purpose that accurately detects diagnosis in early days in the HEV infection, is used for indirect ELISA method and detects HEV IgM antibody
Test kit provided by the present invention contains arbitrary antigen of the present invention or antigen composition.
The present invention also provides described antigen or the application of antigen composition in detecting hepatitis E virus antibody, namely utilizes indirect ELISA method to detect the in vitro method of HEV IgM antibody in the sample.
It below is detailed description of the present invention.
Existing confirmed HEV genome co-exists in 3 opening code-reading frames (ORF1-3).ORF1(28-5106nt)
The maximum albumen of coding HEV virus, the about 186kDa of this albumen comprises at least 4 structural areas.Up to the present have only the ORF1 albumen that laboratory seldom can The expressed, the ORF1 albumen of partitioned representation is used for function assessment research more, and the ORF1 fragment is only used in HEV antibody Western trace detection reagent at present, seldom uses in the ELISA detection reagent.
(about 5147-7126nt), the coding molecule amount is about the viral capsid proteins of 72kDa to ORF2 gene length 1980bp.ORF2 albumen has 111 amino acid whose signal sequences and three potential carbonylation sites that may be used for secreting, expressing.The ORF2 protein antibodies has great importance in HEV infects, though the infection of HEV is done one's utmost not illustrate yet, but as the ORF2 albumen of HEV capsid protein in course of infection, playing the part of probably with host cell membrane on receptors bind and bring out viral capsid and the role that enters cell, all is anti-ORF2 protein antibodies once most neutralizing antibodies of finding at present therefore.
The ORF2 albumen that is used for antibody test at present is mainly derived from the complete ORF2 albumen of insect cell system expression and the ORF2 protein fragments of intestinal bacteria system expression, but different antigen shows different antigen binding capacities, can be more effective in conjunction with the anti-HEV antibody in convalescent's serum than ORF2 albumen total length such as the ORF2 protein fragments.The antigen of employed reagent use mostly is the C end fragment of ORF2 in the market, does not almost use the reagent of its N end antigen.And its gene source mostly is 1 type and 2 types.But, in existing 1 type of China, 4 types are arranged also, and the ORF2 albumen of 4 types is than long 14 amino acid of the ORF2 albumen of 1 type.
A little phosphorylated protein of ORF3 coding HEV virus, molecular weight is about 13.5kDa.It interacts with the liver cell skelemin that is become AMBP.ORF3 albumen is less, its epitope that contains is also less relatively, its C end, especially 105-122aa it is said that antigenicity is higher, antibody positive rate at this epi-position among patient HE is higher, comprise the part fragment of ORF3 albumen at present in the antibody diagnosing reagent, the same ORF2 of its gene source also mostly is 1 type and 2 types more.And Chinese popular its ORF3 albumen of 4 type HEV is than short 9 amino acid of the ORF3 albumen of 1 type.
In order to obtain to detect the high-sensitivity detecting method of HEV IgM antibody, the inventor angles out two strain virus genes of 1 type and 4 types from patient's faecal samples, with its partitioned representation in intestinal bacteria, the antigenicity of obstructed regional antigen fragment in unified zone and the genotype between two genotype of comparison, filter out best antigen, carry out ELISA and detect.
What unless otherwise defined, all technology and scientific term all expressed here is to the present invention relates to the common implication that those of skill in the art understand in the field.Here used name and in cell cultures, molecular genetics, nucleic acid chemistry, the Immunology Lab operation steps is widely used conventional steps in the corresponding field.
An aspect of of the present present invention relates to C-terminal and the N-terminal antigen site of two genotype HEV ORF2 antigens, and it is:
The antigen fragment of described antigen is pS1-6, pS4-6, pS1-1 or pS4-1;
Described pS1-6 is the C-terminal antigen of 1 type HEV ORF2, and its site is 351～391 to 640～660aa;
Described pS4-6 is the C-terminal antigen of 4 type HEV ORF2, and its site is 365～405 to 654～674aa;
Described pS1-1 is the N-terminal antigen of 1 type HEV ORF2, and its site is 1～20 to 90～130aa;
Described pS4-1 is the N-terminal antigen of 4 type HEV ORF2, and its site is 1～20 to 104～144aa.
Be preferably as follows antigen fragment:
Described pS1-6 is the C-terminal antigen of 1 type HEV ORF2, and its site is 371-660aa;
Described pS4-6 is the C-terminal antigen of 4 type HEV ORF2, and its site is 385-674aa;
Described pS1-1 is the N-terminal antigen of 1 type HEV ORF2, and its site is 1-110aa;
Described pS4-1 is the N-terminal antigen of 4 type HEV ORF2, and its site is 1-124aa.
Another aspect of the present invention relates to antigen composition, the two ends combination of a type of hepatitis E and the ORF2 antigen of four types, the i.e. N of corresponding hypotype end and the combination of C end.Be specially described antigen composition is made up or the N-terminal of pS1-1 and the C-terminal combination of pS4-6 for C-terminal and the N-terminal combination of pS1-1, C-terminal and the N-terminal combination of pS4-1, the C-terminal of pS1-6 and the N-terminal of pS4-1 of pS4-6 with pS1-6.Preferably with the N-terminal combination of the C-terminal of pS1-6 and pS1-1 or the N-terminal of the C-terminal of pS4-6 and pS4-1 combined.
One side more of the present invention relates to a kind of aforementioned arbitrary antigen composition and any antigen wherein of utilizing and is used for the test kit that indirect ELISA method detects HEV IgM antibody.
The present invention relates to a kind of in vitro method that utilizes indirect ELISA method to detect HEV IgM antibody in the sample.
Compared with prior art, the present invention has following advantage:
1) strong, the high specificity of antigen provided by the present invention or antigen composition antigenicity;
2) test kit provided by the present invention is highly sensitive, high specificity, and the test kit of the anti-HEV IgM antibody that occurs in the test sample as early as possible, thus realized accurately detecting diagnosis in early days what HEV infected;
3) antigen provided by the present invention or antigen composition can be applicable to and detect in the hepatitis E virus antibody, thereby utilize indirect ELISA method to detect the in vitro method of HEV IgM antibody in the sample.
Description of drawings
Fig. 1-A is that the SDS-PAGE of fusion polypeptide pS1-1 to 6 behind the purifying detects;
Fig. 1-B is that the SDS-PAGE of fusion polypeptide pS4-1 to 6 behind the purifying detects;
Fig. 1-C is the fusion polypeptide pS1-9 behind the purifying, pS1-7, and pS1-8, pS4-9, the SDS-PAGE of pS4-7 and pS4-8 detects, and wherein M represents pre-dsred protein marker;
Fig. 2-A is the immunoblotting assay anti-HEV IgM antibody response of the fusion polypeptide pS4-1 to 9 of purifying, and M represents pre-dsred protein marker among the figure;
Fig. 2-B is the immunoblotting assay anti-HEV IgM antibody response of the fusion polypeptide pS1-9 to 1 of purifying, and wherein M represents pre-dsred protein marker, and primary antibodie is the monkey immune serum of the anti-HEV IgM antibody response positive;
Fig. 3-A, Fig. 3-B, Fig. 3-C and Fig. 3-D have inoculated the HEV IgM reaction that the serial serum of the monkey that the 1 type HEV in people source infects is surveyed polypeptide;
Fig. 3-E and Fig. 3-F have inoculated the HEVIgM reaction that the serial serum of the monkey that the 4 type HEV in people source infect is surveyed polypeptide;
Fig. 3-G, Fig. 3-H, Fig. 3-I and Fig. 3-J have inoculated the HEV IgM reaction that the serial serum of the monkey that the 4 type HEV in pig source infect is surveyed polypeptide;
Wherein 0 week took a blood sample weekly after the inoculation successively for before inoculating among Fig. 3-A-J.Its anti-HEV IgM antibody detects with the ELISA method, wraps by following antigen: the pS1-1 of 1 type, pS1-6 and pS1-8, the pS4-1 of 4 types, pS4-6 and pS4-8.Its value uses S/CO to represent, 1 as positive critical.
Fig. 4-A-J uses combined antigen pS1-1+pS1-6 and the pS4-1+pS4-6 of 1 type and 4 types respectively, uses the anti-HEV IgM reaction in the serial blood among ELISA method detection Fig. 3-A-J.
Fig. 5-A-C detects the IgM antibody in the acute hepatitis patient series serum.Detection method is ELISA, and envelope antigen is single antigen or the combined antigen of 1 type and 4 types.
Embodiment
Below be the specific embodiment of the present invention, described embodiment is in order to further describe the present invention, rather than restriction the present invention.
Present embodiment relates to C-terminal and the N-terminal antigen site of two genotype HEV ORF2 antigens, and it is:
Described pS1-6 is the C-terminal antigen of 1 type HEV ORF2, and its site is 351～391 to 640～660aa;
Described pS4-6 is the C-terminal antigen of 4 type HEV ORF2, and its site is 365～405 to 654～674aa;
Described pS1-1 is the N-terminal antigen of 1 type HEV ORF2, and its site is 1～20 to 90～130aa;
Described pS4-1 is the N-terminal antigen of 4 type HEV ORF2, and its site is 1～20 to 104～144aa.
The specific embodiments of present embodiment is:
At first from the clinical patients fecal sample, angle out gene, belong to 1 type and 4 types respectively through sequencing analysis.Analyze its iso-electric point and wetting ability through Biosun biosoftware (Military Medical Science Institute's development), and according to document (Panda SK, Thakral D, Rehman is E Virus.Rev.Med.Virol17:151-180. S.2007.Hepatitis) report, principle (except a pair of antigen of N end, other home position is identical) according to two genotype correspondences is divided into six sections with ORF2 albumen; In like manner, ORF3 albumen is divided into three sections, its concrete segmentation position and name see Table 1.
Table 1. reorganization HEV-pBVIL1 fusion rotein and antagonism HEV1 type and the sero-fast immune response of 4 type hepatitis E viruss
aThe anti-HEV IgM antibody response positive
bAnti-HEV IgM antibody response negative or detect less than
cAnti-HEV IgM antibody response strong positive
d1 type HEV is than short 14 amino acid of 4 type HEV ORF2 total lengths, and simultaneously, ORF3 is than long 9 amino acid of the latter.
All antigen fragments are all cloned into prokaryotic expression carrier pBVIL1 (Military Medical Science Institute makes up, referring to ZL00100695.9) and are successfully expressed (except pS4-2, advancing the pThioHis successful expression through time cloning again).Through ion exchange chromatography purifying and freeze-drying commonly used.Commercialization BCA reagent is used in its concentration determination, and (China), purity is determined through conventional thin layer scanning for VigorousBiotechnology Beijing Co.Ltd, Beijing.The albumen of freeze-drying is dissolved with the concentration of 0.5mg/ml with carbonate buffer solution, get 8 μ l and carry out the SDS-PAGE electrophoresis, carry out coomassie brilliant blue staining and immunoblotting assay respectively.Wherein the former result show all antigens all present clear band (Fig. 1-A-C), its molecular weight size with expect consistently, band is single, namely the albumen behind the successful expression and purification does not have other foreign proteins substantially.Latter Western Blot detects, and its operating process is routine operation, and albumen is transferred to nitrocellulose filter (Hybond-C Extra, 0.45 μ m; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK), and 5% skimmed milk, the PBS sealing 1.5h of pH7.4, primary antibodie is the mixture of the monkey positive serum of experimental infection, the 1:500 dilution.Two anti-for the anti-human IgM antibody of goat of alkali phosphatase enzyme mark (Santa Cruz Biotechnology, Inc.Santa Cruz, CA).All Incubating Solutions are the PBST that contains 1% milk.Last NBT/BCIP (Dingguo, beijing, China) colour developing.Interpretation of result shows as Fig. 2-A-B and table 1.The immunoblotting result shows that the binding ability of each section antigen and antibody is visibly different: pS1-1, pS1-6, pS1-8, pS1-9, pS4-1, pS4-6, pS4-8 and pS4-9 are correspondence position binding ability more intense antigen fragments in two genotype.Other antigen fragments or not reaction, perhaps reaction is very weak, and is perhaps inconsistent between two genotype.
Further with all antigen fragment bags by elisa plate.The serial serum that detects derives from the monkey of ten experimental infections, wherein is numbered JE11, JE12, JE47, the 1 type HEV in the attack strain behaviour source of the monkey of JE48; JE13, the 4 type HEV in the attack strain behaviour source of JE14; JE15, JE16, JE51, the attack strain of JE52 are the 4 type HEV in pig source.Its result's demonstration (Fig. 3-A-J): 1) antigen fragment of the corresponding zone of the ORF2 of two different genotype does not have significant difference when detecting serial blood; 2) the different antigen fragments of same genotypic ORF2 have different antigenicities, the pS1 at two ends-1, pS4-1, pS1-6, antigenicity the best of pS4-6 antigen; 3) N of ORF2 end and C end show complementarity; 4) antigen fragment of the corresponding zone of the ORF3 of two different genotype demonstrates genotypic specificity; 5) pS1 of ORF3-8, pS4-8 antigen and IgM antibody response are best; 6) time of in serial serum, occurring of the antibody of anti-ORF2 antigen more Zao than the antibody of anti-ORF3 antigen, and the time length is short; 7) anti-pS1-1 in serial serum, pS4-1, pS1-6, the antibody time of occurrence of pS4-6 antigen is early than the rising of transaminase.To sum up, the inventor selects pS1-1, pS4-1, and pS1-6, pS4-6 antigen is as the best antigen that detects anti-HEV IgM antibody.
Present embodiment relates to antigen composition, the two ends combination of a type of hepatitis E and the ORF2 antigen of four types, the i.e. N of corresponding hypotype end and the combination of C end.Be specially the N-terminal combination of the C-terminal of pS1-6 and pS1-1 or the N-terminal of the C-terminal of pS4-6 and pS4-1 combined.
In the test of embodiment 1, the inventor finds that the N end of ORF2 albumen and C end antigen have complementarity when the anti-HEVIgM antibody of detection, with pS1-1 and pS1-6, pS4-1 and pS4-6 makes up the serial serum of detected above-mentioned ten monkeys of bag respectively, result's demonstration (Fig. 4-A-J), the detection time of combined antigen sees that with single the detection time of antigen is almost consistent, but the reagent susceptibility that makes up behind the envelope antigen strengthens, and it detects the adding up of verification and measurement ratio that positive rate is two each and every one isoantigens.For example, pS1-1 and pS1-6 combination back can detect the antibody in other all serial blood except JE16, by contrast, but based on three parts of seronegativities of reagent of the single antigen of pS1-1, and only based on the reagent of pS1-6 two parts of seronegativities are arranged also.The portfolio ratio of antigen is based on the concentration 1:1 that wraps quilt separately separately.
Present embodiment relates to a kind of aforementioned antigen composition and any antigen wherein of utilizing and is used for the test kit that indirect ELISA method detects HEV IgM antibody.
Indirect elisa method is adopted in test, uses 96 hole polystyrene plates to wrap quilt, uses the carbonate buffer solution of pH9.6 that antigen diluent is become 0.5mg/ml.Adopt the gradient dilution method to select best bag by concentration, be about to desire bag antigen gradient dilution and wrap quilt, choose the positive and negative sample of conclusive evidence respectively as the positive and negative control, along with reducing of antigen coated concentration, its P/N value can become big gradually, reach a plateau, the inventor selects the antigen concentration of this moment as the antigen coated concentration of ELISA.The antigen pS1-1 that selects, pS1-6, pS4-1, pS4-6, the suitableeest bag of pS1-1+pS1-6 and pS4-1+pS4-6 is respectively by concentration: 0.5,2.0,0.5,2.0,0.5+2.0 and 0.5+2.0 μ g/ml, every hole adds 100 μ l solution, 37 ℃, 2h, 4 ℃ are spent the night then, and after the PBST of pH7.4 washed three times, every hole added the bag that contains 20% bovine serum and is cushioned liquid 200 μ l, 37 ℃ of sealing 2h remove confining liquid, be stored in 4 ℃ standby.
Use the source of the donating blood serum of 100 parts of anti-HEV IgG and IgM jack to jack adapter to carry out determining of CUT-OFF value, calculate its mean value and standard deviation.PS1-1, pS1-6, pS4-1, it is not 0.241 ± 0.071,0.359 ± 0.123 that pS4-6, the mean value of pS1-1+pS1-6 and pS4-1+pS4-6 antigen coated microplate add the standard difference, 0.345 ± 0.097forpS4-1,0.366 ± 0.127,0.174-/+0.055 and 0.251-/+0.029.When the CUT-OFF value be made as mean value when adding 3 times standard deviation all negative samples be negative.The mean value that its value approximates three negative controls of each plank greatly adds 0.21,0.36,0.29,0.38 successively, 0.16 and 0.09.
When carrying out sample detection, use the PBST that contains 5% bovine serum with testing sample 1:10 dilution, the adding of 100ul/ hole is coated with in the microwell plate of antigen, hatch 30min for 37 ℃, PBST washing 5 times, the concentration of every hole adding 100ul HRP mark is the anti-human IgM antibody of 0.1 μ g/ml then, except the blank well, 37 ℃, after 30min is hatched, remove liquid, and with PBST washing 5 times, add tmb substrate, 37 ℃ of colour developing 15min read each empty A value at wavelength 450nm and 630nm place.
This test example relates to the C-terminal antigen that utilizes 1 type HEV ORF2, HEV IgM antibody is detected in the site for the pS1-6 indirect ELISA method of 371-660aa test kit.
The method that present embodiment adopts is identical with embodiment 3, and the result shows that its antigenicity is preferable, and the reagent susceptibility strengthens.
Be that other pS1-6 antigen fragment of 351～391 to 640～660aa has carried out similar test for C-terminal antigen, the site of 1 type HEV ORF2, its result is consistent with the result of this embodiment.
This test example relates to the N-terminal antigen that utilizes 4 type HEV ORF2, HEV IgM antibody is detected in the site for the pS4-1 indirect ELISA method of 1-124aa test kit.
The method that present embodiment adopts is identical with embodiment 3, and the result shows that its antigenicity is preferable, and the reagent susceptibility strengthens.
Be that other pS4-1 antigen fragment of 1～20 to 104～144aa has carried out similar test for N-terminal antigen, the site of 4 type HEV ORF2, its result is consistent with the result of this embodiment.
Present embodiment relates to a kind of in vitro method that utilizes indirect ELISA method to detect HEV IgM antibody in the sample.
The inventor uses the ELISA method of the combined antigen bag quilt of setting up to detect three parts of clinical patients series serum, and it the results are shown in Figure 5-A-C.1028 to be example, along with transaminase is tending towards normally gradually, the antibody of anti-ORF3 raises gradually, reduce after reaching peak value, and anti-ORF2 combined antigen IgM antibody reduces gradually along with the reduction of transaminase in the serial serum, when transaminase is recovered just often, antibody also detect substantially less than.By the end of last duplicate samples that detects, anti-ORF2 antibody and transaminase all recover normal substantially among the patients serum, and anti-ORF3 antibody is also very strong.So, resist the IgM antibody of ORF3 mutually, the time that the antibody of anti-ORF2 continues is short.
Claims (3)
1. hepatitis E virus antigen is characterized in that:
The antigen fragment of described antigen is pS4-6 or pS4-1;
Described pS4-6 is the C-terminal antigen of 4 type HEV ORF2, and its site is 385～674aa;
Described pS4-1 is the N-terminal antigen of 4 type HEV ORF2, and its site is 1～124aa.
2. hepatitis E virus antigen composition is characterized in that: described antigen composition is for pS4-6 and pS4-1 combination; Wherein:
Described pS4-6 is the C-terminal antigen of 4 type HEV ORF2, and its site is 385～674aa;
Described pS4-1 is the N-terminal antigen of 4 type HEV ORF2, and its site is 1～124aa.
3. test kit that detects hepatitis E virus IgM antibody, it is characterized in that: described test kit contains the described arbitrary antigen of claim 1 or the described antigen composition of claim 2.
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