CN101685097B - ELISA kits for detecting antibody of Schistosoma japonicum, detection method and uses thereof - Google Patents
ELISA kits for detecting antibody of Schistosoma japonicum, detection method and uses thereof Download PDFInfo
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Abstract
The invention discloses an ELISA kits for detecting antibody of Schistosoma japonicum, and corresponding detection method and uses thereof. Antigen of the ELISA kits according to the invention adopts recombinant antigen SjE16 or antigen SjP50 of Schistosoma japonicum. The detection method of the present invention has high sensitivity and specificity, which can detect schistosomiasis japonica quickly, and the ELISA kits according to the invention has wide application prospect in prevention and control and diagnosis of schistosomiasis japonica.
Description
Technical field
The present invention relates to field of biological detection, relate in particular to the ELISA kit that detects antibody of Schistosoma japonicum, and corresponding detection method and application.
Background technology
Japanese schistosomiasis is a kind of being widely current in the infecting both domestic animals and human parasitic disease of the countries such as China in Asia, Philippine, only has a kind of effective medicine-praziquantel for Japanese schistosomiasis at present.Because the long-term extensive this medicine that uses might make blood fluke develop immunity to drugs, the diagnosis of snail fever remains the important means of prevention and cure of schistosomiasis.Bilharzial diagnostic techniques is to seek and determine the direct or indirect evidence that the indication blood fluke exists at body, for the clinical diagnosis of infection by Schistosoma provides foundation.Can also understand the transmission of disease situation by diagnostic result, might in time find at the initial stage of infecting, take appropriate measures immediately and effectively control the wide-scale distribution of disease.Although the excrement detecting method of KatoKatz remains the goldstandard of Japanese schistosomiasis diagnosis, the method is also unsatisfactory in the susceptibility in moderate or low Endemic Areas of Schistosomiasis Japonica territory, and loss is higher.In addition, when assessment schistosomiasis endemic situation was collected the excrement sample of same individuality especially continuously, some resident was for repeatedly checking to seem not to be extremely to cooperate.In order to address this problem, exceed so far the research that the half a century researchers are devoted to amynologic diagnostic method always, develop the panimmunity diagnostic method and done relevant optimization.According to estimates, the quantity of Chinese snail fever amynologic diagnostic method be the whole world.Immunology diagnosis has become the Common Diagnostic Methods of current snail fever because of advantages such as its good susceptibility, specificity and practicality.
The immune diagnostic method that is usually used at present Japanese schistosomiasis mainly comprises: indirect hemagglutination test (IHA), immune enzyme-linked sorbent assay (ELISA), colloidal gold-labeled method etc.
Immune enzyme-linked sorbent assay (enzyme-linked immunosorbent assay, ELISA) be detection method take the antibody of enzyme labeling or antigen as main agents, belong to a kind of immuno-labelling technique, in various diagnostic methods, ELISA be people study the most detailed a kind of.
Engvall in 1971 and Perlmann have carried out quantitative mensuration with ELISA to IgG.Tight self-service and Lv Zaiying at first detects the specific antibody of Rabbits Infected With Schistosoma Japonicum with ELISA, obtain preferably effect (sternly self-service, Lv Zaiying. the Primary Study of immunoenzymatic technique and application. Chinese Medical Journal, 1978,88:470-473).Because so circulating antibody still can long-term existence can not be distinguished existing disease infection and previous infection after the patient accepts effectively treatment, should not be used as efficacy assessment, and the normal prompting of circulating antigen has worm alive to exist, and therefore it is generally acknowledged and can judge existing disease patient and examination curative effect by detecting circulating antigen.
Along with the introducing of hybridoma technology, the monoclonal antibody that multiplex specificity is higher detects circulating antigen.Be used to study the circulating antigen that detects in the Patients with Schistosomiasis Japonica body in the multiple monoclonal antibody of China, but to the person under inspection of low infectiosity, the sensitivity that detects circulating antigen is still to await the difficult problem that solves.Once report had: McAb IIID10 (Lou Wenxian, Qian Zongli, Xue Chunliang, Deng. the preparation of anti schistosoma monoclonal antibody and the application in diagnosis thereof. Chinese parasitology and parasitic disease magazine, 1991,9 (4): 254-257), 1B10A is (sternly self-service, Lv Zaiying, Wang Wen, Deng. monoclonal antibody Dot-ELISA detects the research II. China prevention and cure of snail fever magazine of Circulating Antigen In Schistosomiasis, 1990,2 (2): 39-41), 3D8A is (sternly self-service, Wang Wen, Lv Zaiying, Deng. monoclonal antibody Dot-ELISA detects research I. China's parasitology and the parasitic disease magazine of Japanese schistosomiasis circulating antigen, 1990,8 (3): 161-163), 8SE4 (Qiu Lishu, Xue Hai raises, Lu Jian, Deng. anti-schistosome adult pellicle antigen monoclonal antibody detects the experimental study of circulating antigen and immune complex. Chinese parasitology and parasitic disease magazine, 1990,8 (3): 177-179), NP30 (pipe Xiao Hong, Chou Zhenning, Ma Lei builds strain and Preliminary Identification etc. Schistosoma japonicum Monoclonal Anti-idiotypic Antibodies NP30. Chinese parasitic disease and parasite blood magazine, 1991,9 (4): 261-264) etc., but these monoclonal antibodies still have much room for improvement for the susceptibility of Patients With Schistosomiasis.The monoclonal method of tradition preparation is the hybridoma that obtains only to secrete a strain specific antibodies by hybridoma technology, the method that the humans such as Chen DX make up the phage displaying antibody library prepares monoclonal antibody, in order to obtain antischistosomal a kind of single chain variable fragment antibody (sc-Fv), constructed library is detected with the Schistosoma japonicum adult metabolic antigen with the method for ELISA, the result has shown the potential value of sc-Fv in the blood fluke diagnosis, also provides a kind of new selection for preparation anti schistosoma monoclonal antibody in enormous quantities simultaneously.
Qiu Lishu etc. resist and sandwich ELISA method detection Sera from Schistosomiasis cases circulating antigen with more, in inspected 150 routine Patients with Schistosomiasis Japonicas, susceptibility average out to 84.7%, 40 routine normal persons do not occur among false positive reaction 74 other parasitic infection persons of example, there are 2 routine paragonimus patients positive, all the other cross reaction all do not occur, the result shows that this method is a kind of comparatively desirable circulating antigen detection method (Qiu Lishu, Feng just, Zhang Yonghong, Deng. polyclonal antibody and monoclonal antibody sandwich ELISA method detect Sera from Schistosomiasis cases circulating antigen [J]. Chinese parasitology and parasitic disease magazine, 2000,18 (2): 92-93).Nowadays ELISA has developed into the amynologic diagnostic method for a kind of numerous types.
1. spot-ELISA (Dot-ELISA)
Dot-ELISA as carrier, with the naked eye gets final product judged result after adsorbing trace antigen and being subjected to the reaction of sample product to add two anti-colour developings with nitrocellulose filter, does not need specific apparatus, than the scene of being more suitable for.China prevention of parasitic diseases control institute (1986) at first uses this method diagnosing Japanese schistosomiasis, is that 89%～97%, 65 Healthy Peoples are all negative at inspected 63 positive rates of making a definite diagnosis Schistosomiasis patients.The report Dot-ELISA such as Janitschke have higher specificity and susceptibility.Domestic researcher detects the interior circulating antigen of snail fever human body with the monoclonal antibody of anti-CCA, acute and susceptibility Patients With Schistosomiasis are respectively 90.6% and 83.2%, in detecting, the Pest-or disease-free area personnel do not find false positive reaction, also very low with the intersection reflection of other parasitic diseases, and cure the rear 1 year patient reaction that all is negative with the overwhelming majority, show that this method can be used for the examination of curative effect phase, next the researchist carries out a large amount of research work and experiment, so that Dot-ELISA is widely used in the scene.Shen Dingwen etc. utilize Dot-ELISA to detect acute schistosomiasis japonica patient blood serum special IgG antibody, and compare with conventional ELISA, the positive rate of Dot-ELISA and conventional ELISA is respectively 94.0% and 97.4%, be higher than the positive rate (92.3%) that excrement detects, the coincidence rate of two method testing results is 97.2%, the result shows that two methods are similar aspect the susceptibility that detects and specificity, testing result is without significant difference, and Dot-ELISA is easy and simple to handle, diagnosis fast, have certain practical diagnosis and be worth (Shen Dingwen, the Chen Jia Gui, experimental study Deng .Dot-ELISA fast detecting Japanese schistosomiasis. Xianning medical college journal, 2001,15 (1): 36-38).
2. Q-ELISA (FAST-ELISA)
In 1986, it is fixed that Hancock and Tsang at first are incorporated in dynamics enzyme mapping with U.S. Falcon Assay Screening Test system (being called for short the FAST system), in order to detect anti-Schistosoma mansoni adult particulate antigen among the Manson's schistosomiasis patients serum, obtain satisfactory result.(the Wang Xiuzhen such as Wang Xiuzhen, Li Shitao. the research of Fast ELISA for Diagnosis snail fever and application. Chinese blood fluke control magazine, 1990,2 (4): 31-35) Fast-ELISA has been carried out a series of research, and the development become kit, its susceptibility is 93.88%～96.39%, specificity is 94%～99.22%, the Youden index is 0.93, and its repeatability has also reached more than 90%, and is lower with the cross reaction of other parasitic diseases.In addition, it is comparatively ripe that Shenzhen health hundred gets the Fast-ELISA kit of company's development, generally uses in each large epidemic-stricken area, the whole nation.Compare with traditional ELISA, the whole running time of Fast-ELISA only needs 20-30 minute, need not to carry specialized equipment, but is short of slightly to some extent on the phase at efficacy assessment.
3. biotin-Avidin ELISA (ABC-ELISA)
Biotin-Avidin enzyme linked immunosorbent assay (avidin biotin complex ELISA, ABC-ELISA) be a kind of by the amynologic diagnostic method of extremely strong affinity between Avidin and the biotin, both had immunoreactive specificity, sensitivity than conventional label improves several times again, helps low antibody horizontal patient's diagnosis.The ABC-ELISA that the CCA that the researcher crosses with the monoclonal antibody purifying (CCA) is set up detects the specific antibody in the Sera from Schistosomiasis cases, its specificity is up to 99.0%, can reach respectively 96.2% and 94.1% in the susceptibility of acute patient and chronic patients.Specificity and the susceptibility of its height are that other ELISA method is incomparable, the whole reaction time only needs 1.5h, and this method can repeat, have larger using value in the diagnosis at the scene, domestic relevant report less (realize clear, Ceng Qingren, Zhou Jinchun, Deng. applicating biotin-Avidin system diagnostics Japanese schistosomiasis. parasitology and parasitic disease magazine, 1986,4 (1): 8-11).Owing to the label of ABC-ELISA is expensive, and because it is highly sensitive, when improving positive reaction, also might improves Background Anti and answer, also not have at present widespread use.
The sensitivity of snail fever amynologic diagnostic method and specificity, except from outside the Pass the method for choice experiment different have, main relevant with the characteristic of used diagnostic antigen.Though commonly use at present higher [the Mott KE of susceptibility that rough adult and egg antigen are used for schistosomiasis diagnosis, Dixon N, Carter CE, et al.collaborative study on antigens for immunodiagnosis of schistosomajaponicum infection.Bull WHO, 1987,65 (2): 233-244], but because it often is mixed with host antigen and contains other parasite such as liver fluke, the total antigenic component of lung fluke, easily produce cross reaction, specificity is lower, and [Feng just, Qiu Lishu, Chen Minggang etc. from the present situation of Parallel testing and analysis on monitoring result China schistosomiasis diagnosis reagent. Chinese parasitology and parasitic disease magazine .1998; 16 (5): 321-325].Because traditional antigen mostly is worm source property, and it is less to originate, expensive large in addition, be difficult to adapt to the needs of looking on a large scale disease.Development along with Protocols in Molecular Biology, producing synthetic antigen with gene recombination method is used for diagnosing parasitic disease to become possibility, also studies confirm that both at home and abroad, the use blood fluke is specific, the antigen natural or restructuring of purifying not only can improve susceptibility and the specificity of schistosomiasis diagnosis, and the short distance antibody of anti-some specific antigen of detection has certain efficacy assessment value.Prepare antigen by gene engineering method, the antigen purity height that obtains can be produced in a large number, is easy to quality control.Not only can solve the source of a large amount of diagnostic antigens, and be beneficial to the standardization of detection method.
The SME16 gene of SjE16 and Schistosoma mansoni has higher homology.SME16 is considered to special calbindin of worm's ovum stage, but its function in worm's ovum it be unclear that.Moser in 1992 at in-vitro recombination expression a kind of calbindin Sm16 (SME16) in the Schistosoma mansoni worm's ovum, this albumen can be the identification of immunize rabbit serum, is considered to a kind of diagnostic antigen of tool potentiality.Thereafter the research of Rao finds that Sm16 has anti-infectious effect, is that a kind of immune modulator plays an important role in bilharzial immune evasion, and recombinant expressed Sm16 has strong immunocompetence and can be polyclonal immunize rabbit serum and identify.It should be noted that, calbindin is the protein family that cellular activity is regulated in the important participation of a class, this albuminoid can make latter's occurred conformation change after target protein is combined, thereby excite a series of biologicallies, such as [Silva AC, Reinach FC.Calcium bindinginduces conformational changes in muscle regulatory proteins.TrendsBiochem Sci1991 such as contraction of muscle, neurotransmitter release, enzyme activation and Growth of Cells; 16 (2): 53-7].In view of the high expressed of SME16 in worm's ovum and the important physiological function of calbindin, have reason to think that SME16 must bring into play important and unique effect in regulating worm's ovum cellular activity process.Ironically SME16 and virgin worm, the special calbindin sm20[Avercroft JC of adult, Huggins MC, Dunne DW, et al.Characterisation of Sm20, a 20-kilodalton calcium-binding protein ofSchistosoma manoni.Mol Biochem Parasitol1990; 38 (2): 211-9] and special 8-KDa calbindin [the Am D of cercaria, Grossman Z, Markovics A, et al.Rapidchanges in the expression of a gene encoding a calcium binding protein inSchistosoma mansoni.Mol Biochem Parasitol1989; 34 (2): 167-75] except having similar calcium binding site, do not have each other obvious homology.
Antibody (Immunophilin) is the acceptor of immunodepressant cyclosporin A (CsA), belongs to FKBPL (FKBP) superfamily, with the structural similarity such as heat shock binding immunoassay element p59.It has peptidyl-propyl cis-trans isomerase (PPIase) activity, is chaperone important in the protein folding, and can with multiple intracellular receptor protein combination.The antibody that is present in the blood fluke body can interact with FKBP12 in the human body, plays an important role in infection by Schistosoma.
Because SjP50 and the consistance of Smp50 on the nucleotide level amino acid levels have reached 71%, can think that they probably also have similar function.The P50 that studies show that of Smp50 belongs to FKBPL (antibody) superfamily, with the structural similarity such as heat shock binding immunoassay element P59, has the PPIase activity, can help albumen assembling and protein folding in the blood fluke body, and can interact with heat shock protein 90, consist of together the composition of steroid hormone receptor compound.Known host's hormone is to the bilharzial regulating action of having grown, but up to the present, people not yet positive evidence show have steroid hormone receptor to exist in the blood fluke body.And the existence of SjP50 and Smp50 may provide some supports to the checking steroid receptor.Studies show that in the past, P50 can be identified by Schistosoma mansoni solubility extract and epidermis extract, illustrate that Smp50 may be present in epidermis, and the antiserum of anti-P50 can not be identified Hela cell and colibacillary extract, at general structure many similaritys are arranged although the FKBP that derives from different plant species is described, the difference on entire infrastructure and sequence is enough to produce different antibody responses.
Summary of the invention
The technical problem to be solved in the present invention is, a kind of kit with hypersensitivity and specific quick test Japanese schistosomiasis is provided, and corresponding detection method and application.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of ELISA kit that detects antibody of Schistosoma japonicum is provided, comprise: the solid phase carrier of coated Japan schistosome antigen, the Japan schistosome antigen of enzyme labeling, the substrate of enzyme, negative control product and positive reference substance, coating buffer, cleansing solution and enzyme reaction stop buffer, coated Japan schistosome antigen derives from antigen protein SjE16 or the SjP50 of restructuring on the described solid phase carrier, the antigen protein SjE16 of described restructuring has the amino acid sequence shown in the SEQ ID NO:1, and described antigen protein SjP50 has the amino acid sequence shown in the SEQ ID NO:2.
Described coating buffer adopts the mixed solution of sodium carbonate and NaOH.Preferably, sodium carbonate and the NaOH of 2.9g of described coating buffer by 1.5g mixes when being settled to 1000ml and makes.
Described enzyme-labelled antigen is divided into two kinds, comprising:
No. 1 enzyme conjugates adopts biotin labeled anti-human IgG (H+L) (BiotinylatedAnti-Human IgG (H+L)); The working solution configuration of No. 1 enzyme conjugates: press the 1:10000 dilution proportion with the sample dilution, fully mixing namely is configured to the enzyme conjugates working solution No. 1, needs instant joining herein;
No. 2 enzyme conjugates adopt horseradish peroxidase chain (mould) Avidin (HorseradishPeroxidase Streptavidin); The working solution configuration of No. 2 enzyme conjugates: press the 1:5000 dilution proportion with the sample dilution, fully mixing namely is configured to the enzyme conjugates working solution No. 2, also needs instant joining herein.
Described enzyme reaction stop buffer adopts sulfuric acid solution.
In another aspect of the present invention, a kind of detection antibody of Schistosoma japonicum detection method is provided, comprise following steps:
1, sample dilution: serum to be checked is pressed the 1:100 dilution with the sample dilution;
2, application of sample reaction:
(1), every hole, pattern detection hole adds respectively diluted sample serum 100ul, each sample Parallel testing 2 hole, establish simultaneously each 2 hole of feminine gender, the positive and blank, get feminine gender, each 100ul of positive reference substance adds respectively in the reacting hole, the blank hole only adds 100ul sample dilution;
(2), the reaction of 37 ℃ of lucifuges gets rid of liquid in the hole after 60 minutes, every hole is filled with sample dilution or cleansing solution, gets rid of immediately, fill with again sample dilution or cleansing solution, left standstill 2 minutes, get rid of liquid, repeated washing 3 times all need leave standstill 2 minutes at every turn, got rid of for the last time to pat dry;
3, add enzyme reaction:
(1), every hole adds the working solution 100ul of No. 1 enzyme conjugates, 37 ℃ of lucifuges reactions were got rid of the liquid hole in after 60 minutes, as above washed, and patted dry;
(2), every hole adds the working solution 100ul of No. 2 enzyme conjugates, 37 ℃ of lucifuges reactions were got rid of the liquid hole in after 30 minutes, as above repeated 5 times and washed, and patted dry;
4, chromogenic reaction: every hole adds the substrate nitrite ion 100ul for preparing, and room temperature reaction 1 minute and 30 seconds adds stop buffer 50ul, mixing, and cessation reaction is surveyed with microplate reader immediately and is read;
5, microplate reader is surveyed reading: the ELISA Plate of cessation reaction is surveyed immediately and is read, returns to zero with blank and reads the OD value in 490nm.
In another aspect of the present invention, provide the application of above-mentioned ELISA kit in detecting snail fever.
The ELISA kit of detection antibody of Schistosoma japonicum of the present invention and detection method have important effect in control and treatment Japanese schistosomiasis.
Embodiment
Following examples only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, or the condition of advising according to manufacturer.
The preparation of embodiment 1 Japanese schistosomiasis enzyme connection diagnostic kit
1. the solid phase carrier of envelope antigen
Solid phase carrier can be selected polystyrene or Polyvinylchloride, adopts the form of microtiter plate or small test tube.
Antigen protein SjE16 or SjP50 are coated in polystyrene or the pvc response hole.
1. solution preparation
3.1 the preparation of coated damping fluid
With 1.5gNaCO
3With 2.9g NaHCO
3Adding distil water is settled to 1000ml.
3.2 preparation enzyme conjugates (enzyme-labelled antigen)
Here enzyme-labelled antigen is divided into two kinds, comprising:
No. 1 enzyme conjugates adopts biotin labeled anti-human IgG (H+L) BiotinylatedAnti-Human IgG (H+L).(Vector Laboratories，CAT：BA-3000)
The working solution configuration of No. 1 enzyme conjugates: press the 1:10000 dilution proportion with the sample dilution, fully mixing namely is configured to the enzyme conjugates working solution No. 1, needs instant joining herein.
No. 2 enzyme conjugates adopt horseradish peroxidase chain (mould) Avidin HorseradishPeroxidase Streptavidin.(Vector Laboratories，CAT：SA-5004)
The working solution configuration of No. 2 enzyme conjugates: press the 1:5000 dilution proportion with the sample dilution, fully mixing namely is configured to the enzyme conjugates working solution No. 2, also needs instant joining herein.
3.3 the substrate solution of enzyme (nitrite ion)
The configuration of a.OPD storage liquid: with C6H8O7H
2O (citric acid) 2.1g, OPD (o-phenylenediamine) mixes, and is diluted to the distilled water constant volume of 100ml, and the small component packing, and is frozen under-20 ℃ of environment.
B. with the Na of 0.1M
2HPO412H
2O weight is 7.2g, is diluted to 200ml distilled water constant volume.
In use, the taking-up of a solution is melted, and a, b are mixed in the proportioning of a:b=1:2, add again 3% peroxide water H
2O
2(30ul/10ml) namely be configured to the substrate solution of enzyme.
3.4 sample dilution or cleansing solution configuration
Select following sample and weight:
1)Na
2HPO
4*12H
2O 11.6g
2)NaH
2PO
4*2H
2O 1.2g
3)NaCl 18g
4)Tween-20 2ml
5)Water 2000ml
Be configured as the sample dilution.
3.5 the configuration of stop buffer
Select 100ml distilled water, and with 12.4ml H
2SO
4Add in the distilled water, mixed process need slowly be carried out, and is configured to the H of 2M
2SO
4Stop buffer.
Embodiment 2 detection methods and running program
1. sample dilution
With the sample dilution serum to be checked is pressed the 1:100 dilution, as 500ul dilution, fully mixing will be added in the 5ul serum.The yin, yang reference substance need not dilute.
2. application of sample reaction
2.1 every hole, pattern detection hole adds respectively diluted sample serum 100ul, advises each sample Parallel testing 2 hole.Establish simultaneously each 2 hole of feminine gender, the positive and blank, get feminine gender, each 100ul of positive reference substance adds respectively in the reacting hole, the blank hole only adds 100ul sample dilution.
2.2 37 ℃ of lucifuge reactions were got rid of liquid in the hole after 60 minutes, every hole is filled with sample dilution or cleansing solution, gets rid of immediately, fill with again sample dilution or cleansing solution, left standstill 2 minutes, get rid of liquid, repeated washing 3 times all need leave standstill 2 minutes at every turn, got rid of for the last time to pat dry.
3. add enzyme reaction
3.1 every hole adds working solution (instant joining) 100ul of No. 1 enzyme conjugates, 37 ℃ of lucifuge reactions were got rid of liquid in the hole after 60 minutes, and as above washing pats dry.
3.2 every hole adds working solution (instant joining) 100ul of No. 2 enzyme conjugates, 37 ℃ of lucifuge reactions were got rid of liquid in the hole after 30 minutes, as above repeated 5 washings, patted dry.
4. chromogenic reaction: every hole adds the substrate nitrite ion 100ul for preparing, room temperature reaction 1 minute and 30 seconds.Add stop buffer 50ul, mixing, cessation reaction.Survey with microplate reader immediately and read.
5. microplate reader is surveyed reading: the ELISA Plate of cessation reaction is surveyed immediately and is read, returns to zero with blank and reads the OD value in 490nm.
Here it should be noted that the body series chromogenic reaction is fast, in colour developing, stop and when measuring reading, must every carry out one by one, otherwise very easily cause error.
6. the result judges
Instrument is judged: treat that verify OD value is positive more than or equal to 2.1 times of persons of negative control.When being lower than 0.05, negative control OD value calculates by 0.05.
7. points for attention
(1) when using volley of rifle fire application of sample, must avoid the rifle head to touch elisa plate hole inwall;
(2) kit is 2-8 ℃ of lower the preservation, and wherein No. 1 enzyme conjugates (No. 1 liquid), substrate (No. 3 liquid), negative control product, positive reference substance need be stored in-20 ℃, and elder generation's balance is used to room temperature when taking out at every turn;
When (3) washing dilution or cleansing solution are filled with in the hole, parked after 2 minutes at every turn and get rid of liquid in the hole.Other water sources such as forbidding tap water.
Embodiment 3
Further human serum is tested to kit of the present invention as negative control and positive reference substance, and compare with commonly used kit, with the test effect that draws this kit and application prospect clinically.
1. estimate susceptibility and curative effect that recombinant antigen SjP50 and SjE16 are used for the treatment of front and back epidemic-stricken area crowd ELISA diagnosis
Blood-sampling method: take Jiangxi, the epidemic-stricken area crowd of Sichuan, Anhui, Hubei Si Sheng as blood sampling object Jiangxi, Sichuan, Anhui (being used for the site assessment of efficacy assessment)
(1) selects the excrement inspection positive (A group) of some and the excrement inspection negative patient (B group) of hatching method affirmation; Before treatment, gather for the first time blood sample, treat simultaneously the A group.
(2) 3,4 (Jiangxi, Sichuan), 5 (Anhui) gathered the blood sample second time that the A group is hatched negative patient and B group hatching negative patient with collection ovum hatching method reinspection A group and B group in individual month after the chemotherapy.
Hubei (Japanese schistosomiasis diagnostic kit on-site assessment)
Gather 562 person-portion blood samples in the epidemic-stricken area, this 562 human improvement is added the rattan method carry out egg count (two send three inspections), excrement inspection negative patient is collected the ovum hatching.
All blood samples renumbers, and reaches the requirement of single blind method (Hubei), double-blind study (Jiangxi, Sichuan, Anhui).
2.ELISA detect
2.1 schistosomiasis detection reagent box: adopt Shenzhen health hundred to get the detection kit that reagent company produces
2.2 the laboratory is detected:
Diagnostic antigen: SjP50, SjE162 albumen equal proportion combined packet quilt
ELISA: biotin/avidin
Positive criteria: take Pest-or disease-free area (Beijing) normal person's serum as reference.
3. statistics
Blood sample to four collection in worksite adopts respectively single blind method, double-blind study to carry out the detection of ELISA, the total sample number that detects is 1506 examples, wherein Jiangxi 424 examples, Sichuan 409 examples, Anhui 111 examples, Hubei 562 examples, simultaneously, select after the examination competition, recommended ELISA kit for annual Endemic Areas of Schistosomiasis Japonica epidemic monitoring detects synchronously.
The result shows, recombinant antigen is used for Japanese schistosomiasis diagnostic kit on-site assessment as the serum group ELISA result of purpose (Hubei): the susceptibility of associating antigen reaches 97.3%, the susceptibility that is higher than single antigen near the susceptibility (98.6%) of commercially available reagent box, and the SjP50 specificity reaches 31.1%, is higher than associating antigen (27.4%) and kit (27.2%).Be used for the epidemic-stricken area efficacy assessment as the testing result of the serum (Jiangxi group, Sichuan group, Anhui group) of purpose to estimate recombinant antigen: treat rear 3 months negative conversion rate: Jiangxi group SjE as 20.3%, SjP50 as 15.3%, associating antigen as 10.9%, kit is as 0, Sichuan group SjE be 23.5%, SjP50 is 22.4%, associating antigen is 12%, kit is 1.6%; Treat rear 4 months negative conversion rate: Jiangxi group SjE is 11.4%, SjP50 is 18.1%, associating antigen is 7.6%, kit is 3.8%, and Sichuan group SjE is 22.9%, SjP50 is 50%, associating antigen is 3.4%, kit is 1.5%; Treat rear 5 months negative conversion rate: Anhui group SjE is 3.7%, SjP50 is 29.2%, associating antigen is 39.2%, kit is 9.7%.Concrete statistics is shown in following table 1-4.
Table 1.SjE16 and SjP50ELISA diagnosis are in the blind method assessment result of epidemic-stricken area, Jiangxi efficacy assessment
*Expression is take the epidemic-stricken area Kato negative patient that gathers simultaneously as cloudy ginseng contrast,
*Expression is take the epidemic-stricken area Kato negative patient that gathers simultaneously as cloudy reference
Table 2 SjE and SjP50ELISA diagnosis are in the assessment result of the blind method assessment result of epidemic-stricken area, Sichuan efficacy assessment
Table 3.SjE16 and SjP50ELISA diagnosis are in the blind method assessment result of epidemic-stricken area, Anhui efficacy assessment.
The on-the-spot blind method result of appraisal in table 4.SjE16 and epidemic-stricken area, SjP50 immunologic diagnosis method Hubei
*Expression artificially contrasts so that Pest-or disease-free area is normal,
*Expression is joined as contrast take the moon that commercially available reagent box provides, without exogenous cloudy ginseng contrast,
* *: expression is examined feminine gender as contrast, wherein take the epidemic-stricken area excrement: false positive rate=ELISA false positive number/excrement is examined negative total x100%.
4. interpretation of result
At present the snail fever Site Detection usually adopts Hatching and improvement to add rattan method (be called for short excrement incubate and add the rattan method) and makes a definite diagnosis patient, since the impact of method susceptibility and other disturbing factors, the testing result of these two kinds of methods all can not be strictly according to the facts the number of patients of reflection reality.And patient after healing for a long time, its antibody titer still maintains a high level.The exploitation of the diagnostic kit that therefore, a kind of easy and simple to handle, susceptibility and specificity are higher is particularly important to the control of snail fever.The present invention excavates possible diagnosis target molecule by the research to the Schistosoma japonicum functional gene, utilize Molecular Biology Lab's technology, 2 recombinant proteins with diagnostic value have been obtained, and by the serology detection to the epidemic-stricken area crowd, the result shows, recombinant antigen can be used as diagnostic kit and is used for Site Detection, and its susceptibility is near ripe kit, and specificity is a little more than kit.And detect as the diagnosis of efficacy assessment, 3,4,5 months negative conversion rate all is higher than kit after its treatment, and therefore, these 2 recombinant antigens all have the prospect that further is developed as diagnostic kit.
Sequence table
＜110〉Prevention ﹠ Control Station of Parasitic Disease, China Diseases Prevention ﹠ C
Research Center of Shanghai Human Genome
＜120〉ELISA kit and detection method and the application of detection antibody of Schistosoma japonicum
<130>NP-08-12557
<160>2
<210>1
<211>145
<212>PRT
＜213〉Schistosoma japonicum (Schistosoma japonicum)
<400>1
<210>2
<211>425
<212>PRT
＜213〉Schistosoma japonicum (Schistosoma japonicum)
<400>2
Claims (7)
1. ELISA kit that detects antibody of Schistosoma japonicum, it is characterized in that, comprise: the solid phase carrier of coated Japan schistosome antigen, the antigen of enzyme labeling, substrate, negative control product and positive reference substance, coating buffer, cleansing solution and the enzyme reaction stop buffer of enzyme, coated Japan schistosome antigen derives from antigen protein SjE16 and the SjP50 of restructuring on the described solid phase carrier, the amino acid sequence of the antigen protein SjE16 of described restructuring is shown in SEQ ID NO:1, and the amino acid sequence of described antigen protein SjP50 is shown in SEQ ID NO:2.
2. the ELISA kit of detection antibody of Schistosoma japonicum according to claim 1 is characterized in that, described coating buffer adopts the mixed solution of sodium carbonate and sodium bicarbonate.
3. the ELISA kit of described detection antibody of Schistosoma japonicum according to claim 2 is characterized in that, sodium carbonate and the sodium bicarbonate of 2.9g of described coating buffer by 1.5g mixes when being settled to 1000ml and make.
4. the ELISA kit of detection antibody of Schistosoma japonicum according to claim 1 is characterized in that, described enzyme-labelled antigen is divided into two kinds, comprising:
No. 1 enzyme conjugates adopts biotin labeled anti-human IgG (H+L); The working solution configuration of No. 1 enzyme conjugates: by 1: 10000 dilution proportion, fully mixing namely was configured to the enzyme conjugates working solution No. 1, needs instant joining herein with the sample dilution;
No. 2 enzyme conjugates adopt horseradish peroxidase chain (mould) Avidin; The working solution configuration of No. 2 enzyme conjugates: by 1: 5000 dilution proportion, fully mixing namely was configured to the enzyme conjugates working solution No. 2, also needs instant joining herein with cleansing solution.
5. the ELISA kit of detection antibody of Schistosoma japonicum according to claim 1 is characterized in that, described enzyme reaction stop buffer adopts sulfuric acid solution.
6. the detection method of an antibody of Schistosoma japonicum, described method does not comprise methods for the diagnosis of diseases, comprises following steps:
Step 1, sample dilution: serum to be checked was diluted by 1: 100 with the sample dilution;
Step 2, application of sample reaction:
(1) every hole, pattern detection hole adds respectively diluted sample serum 100ul, each sample Parallel testing 2 hole, establish simultaneously each 2 hole of feminine gender, the positive and blank, get feminine gender, each 100ul of positive reference substance adds respectively in the reacting hole, the blank hole only adds 100ul sample dilution;
(2) 37 ℃ of lucifuge reactions were got rid of liquid in the hole after 60 minutes, and every hole is filled with sample dilution or cleansing solution, gets rid of immediately, fill with again sample dilution or cleansing solution, left standstill 2 minutes, get rid of liquid, repeated washing 3 times all need leave standstill 2 minutes at every turn, got rid of for the last time to pat dry;
Step 3, add enzyme reaction:
(1) every hole adds the working solution 100ul of No. 1 enzyme conjugates, and 37 ℃ of lucifuge reactions were got rid of liquid in the hole after 60 minutes, and as above washing pats dry;
(2) every hole adds the working solution 100ul of No. 2 enzyme conjugates, and 37 ℃ of lucifuge reactions were got rid of liquid in the hole after 30 minutes, as above repeated 5 washings, patted dry;
Step 4, chromogenic reaction: every hole adds the substrate nitrite ion 100ul for preparing, and room temperature reaction 1 minute and 30 seconds adds stop buffer 50ul, mixing, and cessation reaction is surveyed with microplate reader immediately and is read;
Step 5, microplate reader are surveyed reading: the ELISA Plate of cessation reaction is surveyed immediately and is read, returns to zero with blank and reads the OD value in 490nm.
7. the application of ELISA kit as claimed in claim 1 in detecting antibody of Schistosoma japonicum, this application is not included in the application of medical diagnosis on disease aspect.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101968485A (en) * | 2010-04-09 | 2011-02-09 | 中国疾病预防控制中心寄生虫病预防控制所 | Method for detecting schistosome circulating antigen and enzyme-linked immune kit thereof |
| CN102279259A (en) * | 2010-06-10 | 2011-12-14 | 中国疾病预防控制中心寄生虫病预防控制所 | Diagnostic reagent kit for paragonimiasis detection and preparation method thereof |
| CN103575883A (en) * | 2012-07-19 | 2014-02-12 | 普莱柯生物工程股份有限公司 | ELISA detection kit for detecting PCV2 antibody |
| CN103197054A (en) * | 2013-04-07 | 2013-07-10 | 中国科学院上海应用物理研究所 | Antigens jointly assembled sensing interface |
| CN103197059B (en) * | 2013-04-07 | 2015-05-20 | 中国科学院上海应用物理研究所 | Schistosomiasis electrochemistry sensing quick determination kit, detection method and preparation method of kit |
Citations (1)
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| CN1952140A (en) * | 2005-10-20 | 2007-04-25 | 中国疾病预防控制中心寄生虫病预防控制所 | Cloning of Japanese blood fluke vaccine antigen gene and its expression and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952140A (en) * | 2005-10-20 | 2007-04-25 | 中国疾病预防控制中心寄生虫病预防控制所 | Cloning of Japanese blood fluke vaccine antigen gene and its expression and application |
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| 徐宜为.免疫检测技术（第二版）.《免疫检测技术（第二版）》.1991, * |
| 王兆军等.日本血吸虫SjE16基因的原核表达及其免疫诊断应用潜能.《中国寄生虫学与寄生虫病杂志》.2003,第21卷(第2期), * |
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