CN101308138B

CN101308138B - Dengue virus IgM antibody ELISA diagnostic kit

Info

Publication number: CN101308138B
Application number: CN200710028004A
Authority: CN
Inventors: 江立敏; 周惠琼; 郑夔; 柯昌文
Original assignee: GUANGDONG PROV DISEASE PREVENTION CONTROL CENTRE
Current assignee: GUANGDONG PROV DISEASE PREVENTION CONTROL CENTRE
Priority date: 2007-05-15
Filing date: 2007-05-15
Publication date: 2012-09-19
Anticipated expiration: 2027-05-15
Also published as: CN101308138A

Abstract

The invention relates to a dengue virus antibody enzyme-linked immunity diagnostic kit. The kit includes an enzyme labeling board, a negative control serum, a positive control serum, an enzyme marker, a sample diluent, a concentrated washing liquid, a substrate chromogenic liquid, a stop solution and a sealing plate glue, wherein the enzyme marker contains a goat anti-human IgM-HRP; an envelope antigen on the enzyme labeling board includes: a recombinant dengue virus type 1 envelope protein specific antigen with the amino acid sequence of SEQ ID No.1; a recombinant dengue virus type 2 envelope protein specific antigen with the amino acid sequence of SEQ ID No.3; a recombinant dengue virus type 3 envelope protein specific antigen with the amino acid sequence of SEQ ID No.5; and a recombinant dengue virus type 4 envelope protein specific antigen with the amino acid sequences of SEQ ID No .7. An early diagnosis can be made to patients infected with dengue fever for the first time through detecting dengue virus IgM antibodies in serum.

Description

Dengue virus IgM antibody ELISA diagnostic kit

Technical field

The invention belongs to biological technical field, relate to a kind of enzyme-linked immunologic diagnosis kit, especially relate to a kind of dengue virus antibody ELISA diagnostic kit.

Background technology

(Dengue Fever is that (Dengue Virus, the acute mosquito matchmaker infectious disease that DV) causes is mainly bitten propagation through Aedes aegypti or aedes albopictus by 4 serotype dengue virus DF) to dengue fever.Dengue virus belongs to flaviviridae (Flaviviridae), Flavivirus (Flavivirus).DF distributes the extensivelyst, and morbidity at most endangers bigger a kind of arthropod borne viral disease, is widely current in more than 100 countries and regions of global tropical and semi-tropical Africa, America, Southeast Asia and Atlantic ocean region-west.Estimate that according to WHO the whole world has the health of 2,500,000,000 populations to be on the hazard approximately, has 5,000 ten thousand people to infect dengue virus every year.Along with global warming is on the rise, have the tendency to spread in the dengue fever geographic distribution, the order of severity of the incidence of disease and epidemic situation also increases to some extent, and dengue fever has become global serious public health problem.

Dengue fever is after more than 30 year repose period passed through by China, and Foshan generation suddenly in 1978 is popular, and since then, dengue fever is the infectious disease of Guangdong Province's keypoint control always.The dengue fever epidemic situation oncoming force is anxious, and diffusion is not easy control soon, and producing greatly to life and health generation grave danger of the people, to local economic development influences, thereby becomes a great public health problem.Making a definite diagnosis as early as possible of dengue fever suspected case can help to find epidemic situation early, takes measures as early as possible, controls epidemic situation at minimum zone, thereby minimizes loss.At present China mainly relies on the ELISA reagent of import to the laboratory diagnosis of dengue fever, and it costs an arm and a leg, in basic hospital and city, Disease Control and Prevention Center at county level is difficult to popularize; In addition; The import reagent buying arrival time is long, and the laboratory fails in time to take reagent sometimes, and delay diagnosis.First patient often owing to mistaken diagnosis, does not in time adopt an effective measure, and causes the dengue fever epidemic situation to spread.Every epidemic situation has been absorbed in very nervous and passive situation when it is found, and then spends huge manpower and materials and go to control.Therefore, develop easy, quick, economical, be suitable for dengue fever diagnostic reagent that grass-roots unit promotes the use of and become and be badly in need of the difficult problem that solves in the dengue fever control epidemic situation.

After the people had infected dengue virus, generally after the latent period through 3～10 days (common 4～5 days), it was the dengue fever of cardinal symptom that the sufferer can occur with heating, pain; Simultaneously; The immune system of body can produce a series of immune response, the primary infection dengue virus, and it is slower that body produces the speed of antibody; Titre is also lower, the antibody of IgM for replying at first.IgG just occurred at first weekend of morbidity, and titre is very low, and ascending velocity is also slow.According to PAHO's standard, by the 5th day of morbidity, 80% can find the IgM of detection level, 6-10 days of morbidity, and 93-99% can find the IgM of detection level, and the sustainable existence of IgM is more than 90 days.

Summary of the invention

The object of the present invention is to provide a kind of dengue virus IgM antibody ELISA diagnostic kit,, can carry out early diagnosis the patient of primary infection dengue fever through detecting dengue virus IgM antibody in the serum.

A kind of dengue virus IgM antibody ELISA diagnostic kit of the present invention; Comprise enzyme labeling plate, negative control sera, positive control serum, enzyme labeling thing, sample diluting liquid, concentrated cleaning solution, substrate colour developing liquid, stop buffer, shrouding glue; Said enzyme labeling thing comprises goat-anti people IgM-HRP, i.e. goat-anti people IgM-horseradish peroxidase; Envelope antigen on the said enzyme labeling plate is the recombinant expressed dengue virus envelope protein specific antigen of genetic engineering, and said antigen comprises: amino acid sequence is the reorganization 1 type dengue virus envelope protein specific antigen of SEQ ID No.1; Amino acid sequence is the reorganization 2 type dengue virus envelope protein specific antigens of SEQ IDNo.3; Amino acid sequence is the reorganization 3 type dengue virus envelope protein specific antigens of SEQ ID No.5; Amino acid sequence is the reorganization 4 type dengue virus envelope protein specific antigens of SEQ ID No.7.

Said reorganization 1 type dengue virus envelope protein specific antigen is coded by the dna sequence dna of SEQ IDNo.2; Said reorganization 2 type dengue virus envelope protein specific antigens are coded by the dna sequence dna of SEQ ID No.4; Said reorganization 3 type dengue virus envelope protein specific antigens are coded by the dna sequence dna of SEQ ID No.6; Said reorganization 4 type dengue virus envelope protein specific antigens are coded by the dna sequence dna of SEQ IDNo.8.

Described positive control serum is in the PBS of the pH7.4 of the 10mM that contains 20% lowlenthal serum and 0.02% Sodium azide, adds the positive serum of confirming concentration and processes, and the A value of positive control should be greater than 1.5; Negative control sera is in the PBS of the pH7.4 of the 10mM that contains 20% lowlenthal serum and 0.02% Sodium azide, and the ratio in 5% adds the negative serum that mixes to be processed, and the A value of negative control should be less than 0.1.

The preparation method of said enzyme labeling plate is: the dengue virus envelope protein specific antigen that described genetic engineering is recombinant expressed is after carbonate buffer solution dilutes by the concentration of confirming with the CB of the pH9.6 of 0.05M; Encapsulate in the microwell plate by the 0.1ml/ hole; 2 ℃～8 ℃ absorption 24～26 hours after; With 10mM PBST is that the tween phosphate buffer is washed plate by the 0.25ml/ hole, presses the 0.15ml/ hole at 2 ℃～8 ℃ sealings 18～20 hours, the confining liquid that reject is unnecessary with confining liquid again; After vacuum drying treatment,, put 2～8 ℃ of preservations with the aluminum foil bag sealing.

Consisting of of described confining liquid: the PBS of pH7.4 that contains the 10mM of 20% lowlenthal serum and 0.02% Sodium azide is a phosphate buffer.

Described enzyme labeling thing is with the goat-anti people IgM-HRP working fluid of enzyme mark dilution by the working concentration preparation; Consisting of of described enzyme mark dilution: the 10mM phosphate buffer of pH7.4 is PBS, wherein contains 10% lowlenthal serum, 0.2% Tween-20 and 0.02% Sodium azide.

Dengue virus is the sub-thread positive chain RNA virus, and length is about 11Kb, 3400 amino acid of encoding, and molecular weight is 4200KDa.Genome contains a long open reading frame, whole albumen of coding virus: comprise 3 kinds of structural proteins and 7 non-structures (NS) albumen.Wherein the E structural proteins are a kind of envelope glycoproteins, and subgroup is special all to be positioned on the envelope protein E with type specific antigen determinant and neutralization, the inhibiting major antigen epi-position of blood clotting, so the E structural proteins are significant to etiological diagnosis.

Dengue virus IgM antibody ELISA diagnostic kit of the present invention adopts the type specific antigen (about 120 amino acid) in reorganization dengue virus 1-4 type E protein B district, avoids producing cross reaction with yellow fever virus, japanese encephalitis virus antigen.External commercialization dengue virus antibody ELISA diagnostic reagent adopts the totivirus antigen of purifying more, and these reagent shortcomings are that background is high, have cross reaction with flaviviridae, and the most common person is japanese encephalitis virus and yellow fever virus.The kit of the present invention's development has overcome the shortcoming of external reagent.

Compare with similar products at home and abroad

Still there is not the enzyme that the detects dengue virus antibody agent of being excused from an examination at home.Abroad, commonly used dengue fever elisa diagnostic kit all adopts the totivirus antigen of purifying after the cellular incubation.The Dengue IgM ELISA diagnostic kit of producing like the Australian PanBio company of present unique acquisition drugs approved by FDA code; Its deficiency is that background is high; Thereby need mark color comparator by enzyme and detect; Threshold value (cut-off value) is 0.4-0.65, has cross reaction with flaviviridae such as japanese encephalitis virus.The dengue virus IgM antibody ELISA diagnostic reagent of the present invention's preparation is indirect method IgM-ELISA, and method is easy, easy to operate, and sensitivity is high, produces cross reaction hardly with yellow fever virus, japanese encephalitis virus antigen.

The successful development of dengue virus IgM antibody ELISA diagnostic kit of the present invention; Filled up the blank that China does not have the dengue fever diagnostic reagent; Finish the be excused from an examination situation of the long-term dependence on import of agent of China's dengue fever enzyme; It can substitute import reagent comprehensively, is used for the detection of the conventional dengue fever monitor sample of China.The present invention is for finding, early diagnose and early handle the morning of China's dengue fever suspected case from now on; In time the reply epidemic situation prevents the dengue virus propagation, ensures that the people's health can play a significant role; Relative import reagent box; The present invention has cheap advantage, ensures when China's dengue fever monitoring is carried out smoothly, and is that the saving of China disease control system detects cost.

Description of drawings

Fig. 1 is the structure synoptic diagram of expression plasmid.

Fig. 2 is for stepping on leather recombinant antigen purge process SDS-PAGE protein electrophoresis collection of illustrative plates; 1: protein standard P7708V; 2: the centrifugal supernatant in ultrasonic back; 3: the albumen of affinitive layer purification; 4: the albumen of cation exchange purification; 5: the albumen of molecular sieve purification.

Fig. 3 is a recombinant protein Western Blot qualification result; M: protein marker P7708V; The 1-3:WB reaction zone; 4: negative control.

Embodiment

Embodiment one: clone, expression and the purifying of stepping on leather antigen

(1) material

1, standard strain: step on leather 1 type (Hawaii strain), be called for short D1; Step on leather 2 types (NGC strain), be called for short D2; Step on leather 3 types (H87 strain), be called for short D3; Step on leather 4 types (H241 strain), be called for short D4; Available from Nat'l Pharmaceutical & Biological Products Control Institute, preserve by the Guangdong Prov. Disease Prevention-control Center.

2, plasmid: cloning vector BM13 plasmid is available from TaKaRa company, and expression plasmid pET22b (+) is the Novagen product.Preserve by the Guangdong Prov. Disease Prevention-control Center Micro biological Tests.

3, bacterial strain: recipient bacterium E.coli TOP10F ' and the e. coli bl21 Star that expresses usefulness ^TM(DE3), preserve by the Guangdong Prov. Disease Prevention-control Center Micro biological Tests available from Invitrogen company.

4, enzyme: restriction enzyme, T4DNA ligase, Taq archaeal dna polymerase and dNTP are respectively available from New-England, Invitrogen, BOEHRINGER MANNHEIM and Dalian treasured bioengineering company limited etc.

5, other reagent: Yeast Extract (dusty yeast), TRYPTONE (peptone) and IPTG derivant are the BBI products, available from Shanghai bioengineering company limited.

(2) method

1. the extraction of viral RNA and cDNA are synthetic

Adopt the QIAGEN QIAamp Viral RNA mini Kit of company.By the kit requirement, extract viral RNA, with the synthetic cDNA of random primer reverse transcription.

2.PCR amplification gene

With reference to the envelope protein gene sequence (from Genbank) of above-mentioned dengue virus 1-4 type strain, design primer, hold at 5 ' of primer to add the EcoRI restriction enzyme site, hold at 3 ' of primer to add the XhoI restriction enzyme site, be beneficial to be cloned into expression vector.

Primer to stepping on leather 1 type envelope protein gene is:

D1E-F：5’-GGAGGAATTCAAAATGGACAAACTGACTCT-3’

D1E-R：5’-CCCACCTCGAGACGACGTGCACCACGGGCAGTCGC-3’

Primer to stepping on leather 2 type envelope protein genes is:

D2E-F：5’-GGAGGAATTCCGTATGGACAAACTACAGCT-3’

D2E-R：5’-CCCACCTCGAGACGCTTCGCACCACGCATTGTTGT-3’

Primer to stepping on leather 3 type envelope protein genes is:

D3E-F：5’-GGAGGAATTCAAGATGGACAAATTGAAACT-3’

D3E-R：5’-CCCACCTCGAGACGACGTGCACCACGGGCAGTGGC-3’

Primer to stepping on leather 4 type envelope protein genes is:

D4E-F：5’-GGAGGAATTCCGTATGGAGAAATTGCGTATTAAGG-3’

D4E-F：5’-CCCACCTCGAGACGCTTTGCGCCACGGTATGTGGA-3’

Loop program is: with Invitrogen high-fidelity enzyme, and 95 ℃ of hot starts in 15 minutes, 94 ℃ of sex change 15s, 55 ℃ of annealing 30s, 68 ℃ are extended 1min, totally 22 circulations, 68 ℃ remake and use 5min, are cooled to 4 ℃.

3.PCR the purifying of fragment and recovery

Adopt QIAGEN Gel Extraction Kit purifying PCR fragment.

4. the clone of gene

Basic operation is carried out with reference to " Molecular Cloning " (Sambrook et al., 1989).Comprise that enzyme cuts and be connected; The evaluation of the conversion of DNA, the screening of recombinant plasmid and recombinant plasmid.The cloned genes fragment is confirmed through sequencing.The PCR fragment of purifying is cloned in survey fragment nucleotide sequence on the BM13 plasmid earlier; The recombinant expressed leather 1-4 type envelope protein antigen (E) of stepping on is expressed as D1E, D2E, D3E, D4E respectively, and wherein, the D1E-D3E sequence is 372 nucleotide; 124 amino acid of encoding; 369 nucleotide of D4E sequence, 123 amino acid of encoding, concrete sequence is following:

The nucleotide of D1E and amino acid sequence (372 nucleotide, 124 amino acid of encoding)

Nucleotide sequence (SEQ ID No.2)

AAAATGGACAAACTGACTCTAAAAGGGATGTCATATGTTATGTGCACAGGCTCATTCAAGCTAGAGAAAGAAGTGGCTGAGACCCAACATGGAACCGTTCTAGTGCAGATTAAATACGAAGGAACAGATGCACCATGCAAGATCCCTTTTTCGACCCAAGATGAAAGAGGAGTAACCCAGAACGGGAGATTAATAACAGCCAACCCTATAGTTACTGACAAAGAAAAACCAGTCAACATTGAGGCAGAACCACCTTTTGGTGAGAGTTACATCGTGATAGGAGCAGGTGAAAAAGCTTTGAAACTAAGTTGGTTCAAGAAAGGAAGCAGCATAGGGAAAATGTTTGAGGCGACTGCCCGTGGTGCACGTCGT

Amino acid sequence (SEQ ID No.1)

KMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTVLVQIKYEGTDAPCKIPFSTQDERGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVIGAGEKALKLSWFKKGSSIGKMFEATARGARR

The nucleotide of D2E and amino acid sequence (372 nucleotide, 124 amino acid of encoding)

Nucleotide sequence (SEQ ID No.4)

CGTATGGACAAACTACAGCTCAAAGGAATGTCATACTCTATGTGCACAGGAAAGTTTAAAGTTGTGAAGGAAATAGCAGAAACACAACATGGAACAATAGTTATCAGAGTACAATATGAAGGGGACGGTTCTCCATGTAAGATCCCTTTTGAGATAATGGATTTGGAAAAAAGACATGTTTTAGGTCGCCTGATTACAGTCAACCCAATCGTAACAGAAAAAGATAGCCCAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATCATCATAGGAGTAGAGCCGGGACAATTGAAGCTCAACTGGTTTAAGAAAGGAAGTTCTATCGGCCAAATGATTGAGACAACAATGCGTGGTGCGAAGCGT

Amino acid sequence (SEQ ID No.3)

RMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKR

The nucleotide of D3E and amino acid sequence (372 nucleotide, 124 amino acid of encoding)

Nucleotide sequence (SEQ ID No.6)

AAGATGGACAAATTGAAACTCAAGGGGATGAGCTATGCAATGTGCTTGAATACCTTTGTGTTGAAGAAAGAAGTCTCCGAAACGCAGCATGGGACAATACTCATTAAGGTTGAGTACAAAGGGGAAGATGCACCCTGCAAGATTCCTTTCTCCACGGAGGATGGACAAGGGAAAGCTCACAATGGCAGACTGATCACAGCCAATCCAGTGGTGACCAAGAAGGAGGAGCCTGTCAACATTGAGGCTGAACCTCCTTTTGGGGAAAGTAATATAGTAATTGGAATTGGAGACAAAGCCCTGAAAATCAACTGGTACAGGAAGGGAAGCTCGATTGGGAAGATGTTCGAGGCCACTGCCCGTGGTGCACGTCGT

Amino acid sequence (SEQ ID No.5)

KMDKLKLKGMSYAMCLNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDKALKINWYRKGSSIGKMFEATARGARR

The nucleotide of D4E and amino acid sequence (369 nucleotide, 123 amino acid of encoding)

Nucleotide sequence (SEQ ID No.8)

CGTATGGAGAAATTGCGTATTAAGGGAATGTCATACACGATGTGCTCAGGAAAGTTCTCAATTGACCAAAGAGATGGCAGAAACACAGCATGGGACAACAGTGGTAAAAGTCAAGTATGAGGGTGCTGGAGCTCCATGTAAAGTTCCCATAGAGATAAGAGATGTGAACAAGGAAAAAGTGGTAGGGCGTATCATCTCACCTACCCCTTTTGCTGAGAATACCAACAGTGTAACCAACATAGAATTAGAACGCCCTTTGGACAGCTACATAGTAATAGGTGTTGGAGACAGCGCATTAACACTCCATTGGTTCAGGAAAGGGAGTTCCATTGGCAAGATGTTTGAGTCCACATACCGTGGCGCAAAGCGT

Amino acid sequence (SEQ ID No.7)

RMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISPTPFAENTNSVTNIELERPLDSYIVIGVGDSALTLHWFRKGSSIGKMFESTYRGAKR

5. the structure of expression plasmid

Select correct plasmid of D1E, D2E, D3E and D4E sequencing result and carrier pET22b (+) plasmid to carry out enzyme and cut, reclaim enzyme and cut product, purified endonuclease bamhi is connected 2 hours with carrier in 16 ℃.The structure synoptic diagram of expression plasmid is seen Fig. 1.

6. transformed competence colibacillus cell

Rapidly 20 μ l are connected product and add in the competent cell ice bath 30min; 42 ℃ of 2min add 1ml LB nutrient culture media then; 37 ℃ of water-bath 1hr also shake frequently; Then 600 μ l nutrient culture media are coated on respectively with glass bar and contain on the 120 μ g/ml ampicillin LB flat boards; Dull and stereotyped dry back 37 ℃ of inversions are spent the night.

7.DNA extract

Contain 120 μ g/ml ampicillin LB nutrient culture media shaking table incubated overnight from picking recon on the flat board to 2ml; Extract DNA with QIAprep Spin miniprep Kit, whether the size of cutting the evaluation fragment with PCR and enzyme is correct.

8. inoculation and cultivation

With dengue virus 1-4 type recombinant plasmid transformed e. coli bl21 Star ^TM(DE3), the single colony inoculation of each picking contains to 20ml in the LB nutrient solution of 120 μ g/ml ampicillins, and 37 ℃ of joltings are to the next morning 8:30.Respectively get the 8ml seed liquor; Add 200ml and contain 120 μ g/ml ampicillin LB nutrient solutions; Change for 37 ℃ 180 shake soon to OD600 be about 0.6, add IPTG to 100 μ g/ml (IPTG concentration can be optimized), replenish once new ampicillin to 120 μ g/ml (twice is 240 μ g/ml altogether) simultaneously again; Continue to jolt and cultivated 3-5 hour, collect thalline.

During bulk fermentation,, bacterium liquid is mixed with said method equivalent fermentation 1-4 type bacterium liquid, the centrifugal collection supernatant in ultrasonic back, mixed protein is dialysed the rapid purifying of multisteps such as cation exchange and gel filtration through affinity chromatography.

9. polyacrylamide gel electrophoresis (SDS-PAGE)

With the centrifugal supernatant in the ultrasonic back of 1-4 type expression product, the albumen that affinity chromatography, cation exchange and molecular sieve purification are collected is walked the SDS-PAGE protein electrophoresis.Electrophoresis pattern is seen Fig. 2.Among Fig. 2,1: protein standard P7708V; 2: the centrifugal supernatant in ultrasonic back; 3: the albumen of affinitive layer purification; 4: the albumen of cation exchange purification; 5: the albumen of molecular sieve purification.Through the multistep purifying, do not seen that at last assorted band is arranged.Finally obtain the destination protein size and be about 16KDa.

10, the Preliminary Identification of antigen active

Western Blot (WB) experiment: with recombinant protein dilution carrying out SDS-PAGE electrophoresis, gel changes the NC film, and film is cut into four; Get three and the reaction of dengue virus positive serum, recombinant protein and positive serum are specific reaction as a result, the visible WB reaction zone at about 16 * 103 places; Consistent with SDS-PAGE recombinant protein band size, healthy subjects serum is reaction not, and the WB experimental patterns is seen Fig. 3; Among Fig. 3, M: protein marker P7708V; The 1-3:WB reaction zone; 4: negative control.

Embodiment two: recombinant antigen is to the mensuration of dengue virus IgM antibody reactivity

With indirect ELISA method purifying is extracted albumen and carry out the serology detection, to confirm its reactivity dengue virus antibody positive serum.

The antigen of embodiment one gained is encapsulated elisa plate after by 1:500,1:1000,1:2000 dilution, and step on the reaction of leather positive serum, confirm envelope antigen concentration.The antigen active of stepping on leather antigen reaches 1:1000.

Recombinant antigen encapsulates by selected concentration, detects dengue virus with indirect method ELISA and separates positive and dengue fever epidemic situation serum IgM antibody, measures the reactivity of recombinant antigen to dengue virus IgM antibody.

1. recombinant antigen separates the reactivity of positive serum to virus

Separate the positive (13 parts on I type, 3 parts on II type) seroreaction with 16 parts of dengue virus with recombinant antigen, the OD value is more than or equal to 0.29 as a result, and some is up to more than 3.0, and the cases morbidity just can detect dengue virus IgM antibody after the same day or 1 day individually, and susceptibility is very high.Explain that recombinant antigen separates positive serum IgM antibody to dengue virus good antigen reactivity is arranged, the result sees table 1.

Table 1 recombinant antigen separates the reactivity of positive serum to virus

Sample number	Date of the onset	Draw materials the date	The morbidity fate	Former assay	IgM OD value
						D010044	2001-8-23	2001-8-24	1	Be separated to I type dengue virus	0.455
D010046	2001-8-19	2001-8-24	5	Be separated to I type dengue virus	0.653
						D010047	2001-8-22	2001-8-24	2	Be separated to I type dengue virus	0.438
D010050	2001-8-24	2001-8-25	1	Be separated to I type dengue virus	0.541
						D010052	2001-8-22	2001-8-25	3	Be separated to I type dengue virus	0.602

D010053	2001-8-20	2001-8-25	5	Be separated to I type dengue virus	1.117
						D010054	/	2001-8-25	/	Be separated to I type dengue virus	1.978
D010073	2001-9-5	2001-9-6	1	Detect II type dengue virus	2.622
						D010074	2001-9-2	2001-9-6	4	Detect II type dengue virus	0.31
D010093	2001-9-7	2001-9-7	0	Detect II type dengue virus	3.319
						D020031	2002-7-12	2002-7-16	4	Detect I type dengue virus	0.29
D020134	2002-9-13	2002-9-17	4	Detect I type dengue virus	0.568
						A040147	2004-9-28	2004-10-3	5	Detect I type dengue virus	0.346
A040150	2004-9-23	2004-9-28	5	Detect I type dengue virus	2.626
						A040151	2004-9-24	2004-10-2	8	Detect I type dengue virus	1.729
A040152	2004-9-28	2004-10-3	5	Detect I type dengue virus	1.299

2. recombinant antigen is to the reactivity of Zhongshan city's dengue fever epidemic situation serum in 2004

Detect Zhongshan city in 2004 65 parts of dengue fever epidemic situations serum (sample that 36 routine patient's different onset times gathered) with recombinant antigen; 36 portions of acute phase serums (gathering in disease time 3-13 days) dengue virus IgM antibody total positives; The sample OD value of some disease time weak point is on the low side, and 13 portions of convalescent serums (gathering in disease time 37-60 days) OD value is all very high, and 15 portions of convalescent serums (gathering in disease time 71-86 days) OD value descends; But the result is still the positive; The OD value more from high to low, meets generation and the Changing Pattern of IgM behind the dengue virus infection with disease time from low to high.Reagent with the preparation of this recombinant antigen can accurately reflect dengue virus IgM antibody horizontal and situation of change in the patient body, and the recall rate of epidemic situation sample is reached 100%, does not have a omission.Explain that recombinant antigen susceptibility is high, the result sees table 2.

Table 2 recombinant antigen is to the reactivity of mountain dengue fever epidemic situation serum in 2004

Patient number	Sample number	Date of the onset	Date collected	The morbidity fate	IgM?OD
						1	1	2004-10-7	2004-10-12	5	2.56
1	62	2004-10-7	2004-11-13	37	2.386
						2	2	2004-10-6	2004-10-12	6	0.381
2	60	2004-10-6	2004-11-13	38	1.006
						3	3	2004-9-30	2004-10-8	8	1.5
4	4	2004-10-1			1.64
						5	5	2004-10-2	2004-10-7	5	0.606
5	55	2004-10-2	2004-11-13	42	1.565
						6	6	2004-9-20	2004-9-30	10	2.373
6	54	2004-9-20	2004-11-13	54	2.111
						7	7	2004-9-29	2004-10-4	5	2.222
7	71	2004-9-29	2004-12-9	71	0.886
						8	8	2004-10-1	2004-10-4	3	0.309
9	9	2004-9-30	2004-10-3	3	2.512
						10	10	2004-9-30	2004-10-3	3	2.797

11	11	2004-9-27	2004-10-3	6	0.53
						11	72	2004-9-27	2004-12-9	73	0.328
12	12	2004-9-28	2004-10-3	5	1.029
						12	69	2004-9-28	2004-12-9	72	0.346
13	13	2004-9-28	2004-10-3	5	1.299
						13	65	2004-9-28	2004-11-13	46	2.047
14	14	2004-9-28	2004-10-3	5	0.548
						15	15	2004-9-27	2004-10-3	6	1.877
15	53	2004-9-27	2004-11-13	47	1.949
						15	78	2004-9-27	2004-12-9	73	0.445
16	16	2004-9-26	2004-10-2	6	1.041
						17	17	2004-9-24	2004-10-2	8	1.729
18	18	2004-9-24	2004-10-2	8	2.536
						19	19	2004-9-20	2004-9-26	6	2.287
19	61	2004-9-20	2004-11-13	54	1.82
						19	75	2004-9-20	2004-12-9	80	0.411
20	20	2004-9-17	2004-9-27	10	1.606
						21	21	2004-9-21	2004-9-27	6	2.884
21	80	2004-9-21	2004-12-9	79	1.236
						22	22	2004-9-18	2004-9-25	7	0.392
22	76	2004-9-18	2004-12-9	82	0.244
						23	23	2004-9-20	2004-9-25	5	2.568
23	66	2004-9-20	2004-12-9	80	0.542
						24	24	2004-9-10	2004-9-23	13	2.547
25	25	2004-9-14	2004-9-23	9	2.546
						25	57	2004-9-14	2004-11-13	60	1.151
25	68	2004-9-14	2004-12-9	86	0.316
						26	26	2004-9-22	2004-9-27	5	0.292
27	27	2004-9-23	2004-9-28	5	2.626
						28	28	2004-9-22	2004-9-27	5	0.999
28	73	2004-9-22	2004-12-9	78	0.247
						29	29	2004-9-17	2004-9-25	8	2.791
29	59	2004-9-17	2004-11-13	53	2.476
						30	30	2004-9-16	2004-9-26	10	1.244
31	31	2004-9-18	2004-9-25	7	2.746
						32	32	2004-9-17	2004-9-25	8	2.186
32	79	2004-9-17	2004-12-9	74	0.209
						33	33	2004-9-17	2004-9-23	6	0.23
33	63	2004-9-17	2004-11-13	57	1.839
						33	70	2004-9-17	2004-12-9	83	0.513
34	34	2004-9-15	2004-9-23	8	0.307
						34	64	2004-9-15	2004-11-13	59	0.955
34	74	2004-9-15	2004-12-9	85	0.542
						35	35	2004-9-19	2004-9-24	5	1.85
35	58	2004-9-19	2004-11-13	55	1.747
						35	77	2004-9-19	2004-12-9	81	0.372
36	36	2004-10-10	2004-10-21	11	2.141
						36	56	2004-10-10	2004-11-13	33	2.506
36	67	2004-10-10	2004-12-9	59	1.231

Embodiment three: recombinant antigen detects the susceptibility and the specificity of dengue virus IgM antibody

The recombinant antigen of embodiment one gained is prepared into dengue virus IgM antibody test reagent (to call in the following text from grinding reagent), has detected great amount of samples, the susceptibility and the specificity of recombinant antigen are assessed with this reagent.

1 with the comparing result analysis of import reagent (Australian PanBio)

Virus is separated positive sample D010044, D010046, D010047, D010050, D010052, D010073, D010074 and dengue fever epidemic situation sample A040124 in 2004, A040134, A040135, A040137 to grind the reagent testing result with oneself all positive in the table 1; The testing result of stepping on leather reagent (Dengue IgM Capture ELISA) with Australian PanBio is all negative; Detect for the early stage patients serum of dengue fever morbidity, the reagent that grinds certainly than Australian PanBio to step on leather reagent more responsive.

With from grind with Australian PanBio step on leather IgM antibody test reagent, detects the dengue virus IgM antibody in 116 parts of dengue fever epidemic situations and the clinical dengue fever patient suspected serum, the result removes from office reagent and detects 65 parts of positives from grinding to step on, positive rate is 56%; Australia PanBio steps on leather reagent and only detects 52 parts of positives, and positive rate is 44.8%.From grinding reagent the recall rate of the dengue virus IgM antibody in dengue fever epidemic situation and the clinical dengue fever patient suspected serum is stepped on leather reagent apparently higher than Australian PanBio, the result sees table 3.

Table 3 is stepped on leather reagent comparison result from grinding with Australian PanBio

Reagent	Number positive	Negative number	Detect number	Positive rate (%)
					From grinding reagent	65	51	116	56
Contrast agents (PanBio)	52	64	116	44.8

2. to the mensuration result of entomophila laboratory, Guangdong Province Serum Bank

Guangdong Province every year almost all occurred stepping on the leather epidemic situation since the eighties.We are with having detected the dengue fever epidemic situation sent in the city-level disease prevention and control centers, 617 parts of various places (hereinafter to be referred as CDC) and the dengue virus IgM antibody in the clinical dengue fever patient suspected serum from grinding reagent; The result (wherein steps on 410 parts of 1 type serum of leather, 15 parts of 2 type serum, 5 parts of 3 type serum, 6 parts of 4 type serum for positive 436 parts; It is thus clear that these article all have reactivity to stepping on leather 1-4 type serum; The situation that a certain type omission do not occur), negative 181 parts, positive rate is 70%; The recall rate of clinical affirmation dengue fever patient's sample is 90% (2002 hospital for infectious diseases provide sample), and the result sees table 4.

Entomophila laboratory, table 4 Guangdong Province Serum Bank dengue virus IgM TPPA result

Time (year)	Positive	Negative	Total sample number	Positive rate (%)
					1987	4	3	7	57.14

1990	4	8	12	33.33
					1991	3	3	6	50.00
1999	8	1	9	88.88
					2000	24	13	37	64.86
2001	43	45	88	48.86
					2002 (Guangdong CDC)	73	59	132	55.30
2002 (Guangzhou CDC)	90	21	111	81.00
					2002 (hospitals for infectious diseases)	111	12	123	90.24
2003	6	13	19	31.58
					2004	70	3	73	95.89
Add up to	436	181	617	70.66

3. fever patient and healthy population serum are stepped on leather IgM antibody test result

With grinding 245 parts of fever patients and 120 parts of healthy population serum that kit has detected Zhongshan city and the CDC of Zhanjiang City censorship certainly; Detect positive 24 parts altogether; Wherein there are 12 parts to be dengue fever epidemic situation patients serum in 2004 in 14 of the Zhongshan city parts of positive samples; This reagent can detect the dengue fever patient in the fever patient, detects 3 parts of positives in the healthy population serum altogether, and false positive rate is 2.5%.Have good sensitivity and specificity from grinding reagent.The result sees table 5:

Table 52004 year fever patient and healthy population serum are stepped on leather IgM antibody test result

The sample source	Total sample number	Positive	Negative	Positive rate (%)
					Middle mountain fever patient	100	14	86	14
Zhanjiang fever patient	145	7	138	4.8
					Healthy population	120	3	117	2.5
Add up to	365	24	341

4 detections to other arboviruse positive serum

With detecting 25 parts of encephalitis and 16 parts of Hemorrhagic fever (EHF) IgM antibody positive serum from grinding kit, to have only 1 part of OD value be 0.327 to encephalitis as a result, and all the other 24 parts all negative, and 16 parts of EHF serum testing results are all negative.Explain that there is not cross reaction in the very low and EHF serum of recombinant antigen and encephalitis antibody cross reaction property.The specificity that this reagent tool is good.

5. Interference Detection

With detecting the plasma sample that high blood ester, high cholerythrin, high haemoglobin, jaundice and anti-coagulants are handled (EDTA, sodium citrate) from grinding kit, testing result is all negative.Explain that these serum do not have obvious interference to the detection of Dengue IgM.HCV positive serum to belonging to flaviviridae together does not have obvious cross reaction, and specificity is good.

6.. the stability to reagent detects

The antigen plate is put into 37 ℃ of incubators, examined 6 days, detect it the activity under the thermal environment.Detection comprises positive and negative serum, critical quality controlled serum in the factory, the result that the antigen plate of preserving under its result and the 4 ℃ of conditions is detected relatively, antigen active descends and surpasses the requirement of working out rules.

The above results shows that the antigen of expression is good to the reaction of stepping on leather IgM serum.

Embodiment four: the production technology of dengue virus IgM antibody ELISA diagnostic kit of the present invention

(1) material

1. step on leather antigen: embodiment one is seen in preparation.Said antigen 1: 1000 when encapsulating, and the positive coincidence rate of reference article in the factory is reached 98%, and specificity reaches 97%.

2. goat-anti people IgM-HRP:SIGMA product.

3.TMB: Berlin, Germany lattice product.

4. positive control serum: in the PBS of the pH7.4 of the 10mM that contains 20% lowlenthal serum and 0.02% Sodium azide, add the positive serum of confirming concentration, the A value of positive control should be greater than 1.5.

5. negative control sera: in the PBS of the pH7.4 of the 10mM that contains 20% lowlenthal serum and 0.02% Sodium azide, the ratio in 5% adds the negative serum that mixes.The A value of negative control should be less than 0.1.

6. ELISA Plate: 12 orifice plate bars, CV (%)≤10%.Can adopt the product of Shenzhen bright magnificent company limited of gold.

7. reference article in the factory: screening and preparation in the Serum Bank of the little inspection in Guangdong Prov. Disease Prevention-control Center institute insect-borne diseases research department.

8. lowlenthal serum: available from Zhejiang three sharp biological factories, protein content is 3.3 ± 0.5%, and sterility test is negative, to the unrestraint of kit reactive system.

9. other reagent: be homemade AR.

(2) preparation process

1. antigen preparation and calibrating (seeing embodiment one to three)

2. encapsulate confirming of technology

(1) screening of solid phase carrier

CV pH-value determination pH: get 1 part of Dengue-IgM weak positive serum (CV serum) after encapsulating and measure 10 hole CV values with indirect method;

Adsorbability is measured: take from system Dengue gene engineering antigen and begin the two-fold dilution from 1:250 and encapsulate, get 1 part of Dengue-IgM positive serum and measure with indirect method, observe OD>0.5 Kong Xu.

(2) selection of sealing condition

Select existing multiple confining liquid prescription to make an experiment, selected at last with the 10mM PBS (PH7.4) that contains 20% lowlenthal serum and 0.02% Sodium azide, the sealing of 150ul/ hole, 4 ℃ of back buttons that spend the night are done.

(3) encapsulate the plate preparation

The dengue virus envelope protein specific antigen that described genetic engineering is recombinant expressed is after carbonate buffer solution dilutes by the concentration of confirming with the CB of the pH9.6 of 0.05M; Encapsulate in the microwell plate by the 0.1ml/ hole; 2 ℃～8 ℃ absorption 24～26 hours after; With 10mM PBST is that the tween phosphate buffer is washed plate by the 0.25ml/ hole, presses the 0.15ml/ hole at 2 ℃～8 ℃ sealings 18～20 hours, the confining liquid that reject is unnecessary with confining liquid again; After vacuum drying treatment,, put 2～8 ℃ of preservations with the aluminum foil bag sealing.

3, the selection of enzyme labelled antibody dilution and the titration of tiring

Adopt the enzyme mark dilution (the 10mM PBS of pH7.4 contains 10% lowlenthal serum and 0.2% Tween-20,0.2%Bronidox) through screening, press working concentration preparation goat-anti people IgM-HRP working fluid, its stability meets the quality inspection requirement.Through titration, tiring of goat-anti people IgM-HRP is 1:12000.

4, the cut-off value confirms

The serum specimen of the normal health check-up that fetch Pok Oi Hospital, institute of traditional Chinese medicine, the People's Hospital from the Zhongshan city, outpatient's serum (non-dengue fever patient) detect for 463 parts; And carry out statistical study; Drawing average is 0.043; SD=0.0519, X+3SD=0.199 confirms that the cut-off value is 0.2+ negative control average (this value less than 0.050 in 0.050).

5, system inspection rules confirms

According to above experiment and process,, work out the manufacturing and the vertification regulation of this kit with reference to the form of " Chinese biological goods rules II portion ".

SEQUENCE LISTING (sequence table)

< 110>Guangdong Prov. Disease Prevention-control Center

< 120>dengue virus IgM antibody ELISA diagnostic kit

<130>

<141>2007-05-15

<160>16

<170>PatentIn?version3.4

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< 213>dengue virus 1 type E protein B district

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Claims

1. a dengue virus IgM antibody ELISA diagnostic kit comprises enzyme labeling plate, negative control sera, positive control serum, enzyme labeling thing, sample diluting liquid, concentrated cleaning solution, substrate colour developing liquid, stop buffer, shrouding glue, it is characterized in that:

Said enzyme labeling thing comprises goat-anti people IgM – HRP, i.e. goat-anti people IgM – horseradish peroxidase;

Envelope antigen on the said enzyme labeling plate is the recombinant expressed dengue virus envelope protein specific antigen of genetic engineering, and said antigen comprises: amino acid sequence is the reorganization 1 type dengue virus envelope protein specific antigen of SEQ ID No.1; Amino acid sequence is the reorganization 2 type dengue virus envelope protein specific antigens of SEQ ID No.3; Amino acid sequence is the reorganization 3 type dengue virus envelope protein specific antigens of SEQ ID No.5; And amino acid sequence is the reorganization 4 type dengue virus envelope protein specific antigens of SEQ ID No.7;

Described positive control serum is in the PBS of the pH7.4 of the 10mM that contains 20% lowlenthal serum and 0.02% Sodium azide, adds the positive serum of confirming concentration and processes, and the OD value of positive control should be greater than 1.5;

Negative control sera is in the PBS of the pH7.4 of the 10mM that contains 20% lowlenthal serum and 0.02% Sodium azide, and the ratio in 5% adds the negative serum that mixes to be processed, and the OD value of negative control should be less than 0.1.

2 .Dengue virus antibody ELISA diagnostic kit according to claim 1 is characterized in that:

Said reorganization 1 type dengue virus envelope protein specific antigen is coded by the dna sequence dna of SEQ ID No.2;

Said reorganization 2 type dengue virus envelope protein specific antigens are coded by the dna sequence dna of SEQ ID No.4;

Said reorganization 3 type dengue virus envelope protein specific antigens are coded by the dna sequence dna of SEQ ID No.6;

Said reorganization 4 type dengue virus envelope protein specific antigens are coded by the dna sequence dna of SEQ ID No.8.

3 .Dengue virus antibody ELISA diagnostic kit according to claim 1; It is characterized in that: the preparation method of said enzyme labeling plate is: the dengue virus envelope protein specific antigen that described genetic engineering is recombinant expressed is after carbonate buffer solution dilutes by the concentration of confirming with the CB of the pH9.6 of 0.05M; Encapsulating in the microwell plate by the 0.1ml/ hole, after 24～26 hours, is that tween phosphate buffer by 0.25ml/ hole wash plate with 10mM PBST 2 ℃～8 ℃ absorption; Press the 0.15ml/ hole 2 ℃～8 ℃ sealings 18～20 hours with confining liquid again; The confining liquid that reject is unnecessary with the aluminum foil bag sealing, is put 2～8 ℃ of preservations after vacuum drying treatment.

4 .Dengue virus antibody ELISA diagnostic kit according to claim 3 is characterized in that: the consisting of of described confining liquid: the PBS of pH7.4 that contains the 10mM of 20% lowlenthal serum and 0.02% Sodium azide is a phosphate buffer.

5 .Dengue virus antibody ELISA diagnostic kit according to claim 1; It is characterized in that: described said enzyme labeling thing is with the goat-anti people IgM – HRP working fluid of enzyme mark dilution by the working concentration preparation; Consisting of of described enzyme mark dilution: the 10mM phosphate buffer of pH7.4 is PBS, wherein contains 10% lowlenthal serum, 0.2% Tween-20 and 0.02% Sodium azide.