CN101643743A - Test paper for detecting HIV 1/2 antibody of exudates in oral mucosa and preparation method thereof - Google Patents
Test paper for detecting HIV 1/2 antibody of exudates in oral mucosa and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a test paper for detecting HIV 1/2 antibody of exudates in oral mucosa and preparation method thereof and provides a method for obtaining antibody sample from the oral mucosa,comprising contacting a tool to the oral mucosa and contacting the tool with the oral mucosa to a solution to form a liquid sample. The invention provides a method for synthesizing representative of pathogenic microorganism, comprising selecting antigens from the pathogenic microorganism, obtaining a gene of the antigens to form a gene fusion, recombining and expressing to obtain a substance representing the pathogenic microorganism. The invention provides a detecting method, comprising preparing a test paper strip or a device by adopting the substance representing the pathogenic microorganismand analyzing the antibody sample obtained from the oral mucosa by the test paper strip or the device. The invention also provides a kit comprising a test paper strip and a sampling device for sampling from the oral mucosa. The invention also provides a kit, comprising the test paper strip, a sampling device for sampling from the oral mucosa, and an instruction for use.
Description
Technical field
The invention belongs to field of medical examination, the apparatus and method of antibody materials (antibody that comprises known various infectious agents) in a kind of rapid detection oral mucosa transudate are provided.More particularly, the present invention relates to a kind of reagent, prepare this compositions and methods and use this reagent to be used for human oral mucous membrane transudate HIV1/2 detection of antibodies.
The invention provides and a kind ofly obtain to contain the method for antibody sample, comprise with utensil contacting oral mucosa that the utensil that will have oral mucosa contacts with solution, the formation liquid sample from oral mucosa.The invention provides the synthetic method of representing the representative of pathogenic micro-organism, comprise from pathogenic micro-organism and select antigen, obtain these antigenic genes, form the gene fusion thing, the material of pathogenic micro-organism is represented in recombinant expressed acquisition.The invention provides a kind of detection method, comprise adopting and represent the material of pathogenic micro-organism to prepare test strip or device, and will gather the antibody sample that contains that obtains from oral mucosa and analyze with described test strip or device.The present invention also provides test kit, comprises test strip and the sampling unit of taking a sample from oral mucosa.The present invention also provides test kit, comprises test strip and the sampling unit of taking a sample from oral mucosa, and working instructions.
Background technology
(Acquired Immunodeficiency Syndrome AIDS), claims acquired immune deficiency syndrome (AIDS) again to acquired immune deficiency syndrome (AIDS), is the viral communicable disease of interior serious harm human health of a class world wide and life.In less than 20 years, 3,000 ten thousand people are infected, 1,000 ten thousand people lose one's life.At present, have ten thousand every day in the world surplus the new aids infection virus of people.According to statistics, at the bottom of calendar year 2001, the number of the infected of China's acquired immune deficiency syndrome (AIDS) is about 1,000,000, now enters the quick rise period, and according to the data of the Ministry of Health, the patients infected hiv quantity of report in first half of the year calendar year 2001 was compared with last one year and risen 67.4%.The result of study in the whole world show HIV1/2 be by contaminated blood, blood products, blood transfusion or closely between individuality contact propagate infectious, in special crowd even the sign of cross infection occurs.Not only medical circle is in the research diagnosis prophylactic treatment acquired immune deficiency syndrome (AIDS) of trying one's best, and national governments, all orders of society all put into the motion of resisting the AIDS one after another.But up to the present, the mankind also do not find a kind of this sick method for the treatment of.Thereby early diagnosis in time finds infected patient, controls it and further propagates and remain one of important means of AIDS preventing and controlling.
In recent years, main both at home and abroad methods detection HIV1/2 antibody or the viruses such as enzyme-linked immunosorbent assay (ELISA), PCR, cell cultures of adopting.These methods are in various degree some shortcomings of existence all.
Complicated as ELISA method schedule of operation, easily cause false positive and false negative, and easily cause the operator to be injured and environmental pollution, test period is longer, needs just can draw detected result more than two hours.Must possess simultaneously microplate reader and wash the plate machine, this basic unit laboratory and small-sized outpatient service difficulty reach, and test is subjected to condition restriction such as temperature, makes troubles to detection.
Molecular testing (being called for short the PCR method) is long its detection time, and testing sequence is loaded down with trivial details, needs to use special instrument and equipment simultaneously, and reagent needs refrigeration, and the testing cost costliness has limited the popularization and application of reagent simultaneously.
IFA (immunofluorescence assay) must possess fluorescence immunoassay microscope and dark room conditions, must cooperate suitable spectral filter for obtaining good Fluirescence observation effect, experimental period needs two hours, the result detects and carries out under fluorescent microscope, must judge detected result by experienced professional and technical personnel.Whether negative antibody test result can not get rid of and infect, and need be after surpassing 21 days still negatively could determine not to be subjected to the HIV1/2 infection.
Cell cultures is a kind of aseptic technique, requirement possesses very high cut-and-try work environment and condition (between aseptic technique, Bechtop, operation room etc.), need a large amount of plant and instrument (incubator, whizzer, microscope etc.), need a large amount of Specialty Experiment equipment, the experimental technique complexity, need empirical technician's operation, and experimental period is very long, is not suitable for the rapid detection of disease.
Agglutination test comprises that gelatin particle (PA) and latex particle (LAT) belong to semiquantitative determination, and repeatedly doubling dilution is measured and taken reagent, and poor repeatability as a result, and sensitivity is low.
And immune-gold labeled law technology (Immunochromatography Assay) is to grow up middle nineteen nineties in last century, but because of it is quick, easy and simple to handle, the accumulating of stable reagent room temperature, be difficult for pollution characteristics and be widely used.It is the combination of immune affine technology, engram technology, immunolabelling technique and chromatographic technique.With coated antibody, colloid gold label antibody immobilization is combined with sample sorbing material etc., is prepared as immunochromatography diagnosis test paper/plate, only need be during use inserting sample solution under the test strip, and several minutes just can judged result.With the immunity percolation method relatively, good stability operate easylier, quick, and owing to for doing test strip/plate form, need not cryopreservation, accumulating conveniently.
At present, immune colloidal gold technique is confined to serum sample in the application that detects HIV1/2 antibody, and detection must be in the laboratory of obtaining the authorization and gone to finish by the doctor or the technician of qualified certificate.Also have this year the reagent that detects whole blood sample to occur, must take a sample with sharp device, the operating safety that causes thus is also worrying.
Owing to these and other reasons, need a kind of improved reagent, be used for detecting the antibody materials that exists at the body fluid trace reliably, need such reagent to pack in some way simultaneously, thereby the individual can easily use in oneself's detection at home.
Summary of the invention
Main technical schemes provided by the invention is enumerated as follows.
A kind of method for preparing the representative of causal organism, described method comprises: select antigen protein from causal organism; Obtain the gene of these antigen proteins; Make up the recombinant vectors of these genes of amalgamation and expression; Give expression to fused protein in the host, it is the representative of causal organism.
A kind of sampling method, described method comprises: wipe away from oral mucosa and get or scrape district's material.Described sampling method further comprises: with described from oral mucosa wipe away get or scrape the district substance transfer to solution.
A kind of detection kit, it comprises: test strip is used for measuring; Swab is used for taking a sample from mucous membrane; Solution is used for shifting sample from swab.
A kind of test kit that detects the HIV1/2 antibody in the human oral mucous membrane transudate, it comprises: oral mucosa transudate HIV1/2 antibody test test paper; Be used to collect the receptor of oral mucosa transudate; Divider, it is used for the sample liquid absorption portion that shifts out the sample liquid of predetermined amount and be released into proofing unit from described receptor.The test kit of the HIV1/2 antibody in the described detection human oral mucous membrane transudate, it is characterized in that: described oral mucosa transudate HIV1/2 antibody test test paper is included in pastes sample pad (2), Radioactive colloidal gold pad (3), coated film (4) and absorbent pad (5) in turn mutually overlap joint on the base plate (1), coated film (4) has detection zone (6) and control region (7), detection zone (6) bag is by HIV 1/2 chimeric antigen, and wrap by the goat anti-human igg control region (7).The test kit of the HIV1/2 antibody in the described detection human oral mucous membrane transudate is characterized in that: described receptor is buccal swab or cotton swab class mucus sample collecting device.The test kit of the HIV1/2 antibody in the described detection human oral mucous membrane transudate, it is characterized in that: the acquisition mode of described receptor is for swabbing the upper and lower gum of subjects with the collection swab, then swab is put in the band cap test tube that fills about 1000 μ l sample diluents, in vitro mix, after liquid in the swab extracted, discard swab; Described sample diluting liquid is to contain 1% to 5%BSA 0.01M Tris solution, and PH is 7.0 to 9.0, and contains 0.1% to 0.5%PEG and 0.01% to the 0.05%TWEEN-20 tensio-active agent.The test kit of the HIV1/2 antibody in the described detection human oral mucous membrane transudate is characterized in that: described divider is pipettor or Siphonata liquid-transfering device, and presetting and moving liquid measure is 100 μ l.The test kit of the HIV1/2 antibody in the described detection human oral mucous membrane transudate, it is characterized in that: it is characterized in that described envelope antigen is the escherichia coli expression genetically engineered Recombinant HIV antigen through preferred cultivation, it includes the major antigen epi-position in HIV-1gp120, gp41 and the HIV-2gp36 section, molecular weight is 35KD, is with 14 little peptides of amino acid whose T7 at the N of recombinant protein end.The test kit of the HIV1/2 antibody in the described detection human oral mucous membrane transudate is characterized in that described method for coating is is 0.5mg/ml with coated film damping fluid dilution antigen to concentration, and spray printing is detection zone on coated film; With the coated film damping fluid dilute commercially available goat anti-human igg to concentration be 0.1mg/ml, be the control region; The machine line, line-to-line is every 5mm.
The object of the present invention is to provide HIV1/2 detection of antibodies method in a kind of rapid detection oral mucosa transudate, the nothing wound that can realize HIV1/2 antibody detects, walk away safety, because directly do not contact blood, the danger coefficient in tester's experimentation is greatly diminished.This detection method may be feared the people of blood examination for those provides another important detection approach, and it has also improved the public health level when improving tester's protection.
Particularly, the test kit of HIV1/2 antibody in the rapid detection human oral mucous membrane transudate of the present invention is characterized in that comprising:
A kind of oral mucosa transudate HIV1/2 antibody test test paper;
A kind of receptor that is used to collect the oral mucosa transudate;
A kind of divider, it is used for the sample liquid absorption portion that shifts out the sample liquid of predetermined amount and be released into proofing unit from described receptor.
Rapid detection HIV1/2 antibody kit of the present invention, it is characterized in that: oral mucosa transudate HIV1/2 antibody test test paper is to paste sample pad (2), Radioactive colloidal gold pad (3), coated film (4) and absorbent pad (5) on base plate (1) in turn mutually overlap joint, coated film (4) has detection zone (6) and control region (7), detection zone (6) bag is by HIV 1/2 chimeric antigen, and wrap by the goat anti-human igg control region (7).
Rapid detection HIV1/2 antibody kit of the present invention, it is characterized in that: described receptor is buccal swab or cotton swab class mucus sample collecting device, acquisition mode is for swabbing the upper and lower gum of subjects with the collection swab, then swab is put in the band cap test tube that fills about 1000 μ l sample diluents, in vitro mix, after liquid in the swab extracted, discard swab.Described sample diluting liquid is to contain 1% to 5%BSA 0.01M Tris solution, and PH is 7.0 to 9.0, and contains 0.1% to 0.5%PEG and 0.01% to the 0.05%TWEEN-20 tensio-active agent.
Rapid detection HIV1/2 antibody kit of the present invention is characterized in that: described divider is as pipettor or Siphonata liquid-transfering device, and presetting and moving liquid measure is 100 μ l.
Rapid detection HIV1/2 antibody kit of the present invention, it is characterized in that described envelope antigen is the escherichia coli expression genetically engineered Recombinant HIV antigen through preferred cultivation, it includes the major antigen epi-position in HIV-1 gp120, gp41 and the HIV-2 gp36 section, molecular weight is 35KD, is with 14 little peptides of amino acid whose T7 at the N of recombinant protein end.Particularly its help has realized this antigenic solubility expression.Thereby susceptibility and the selectivity higher have been obtained than existing method.
Rapid detection HIV1/2 antibody kit of the present invention is characterized in that described method for coating for being 0.5mg/ml with coated film damping fluid dilution antigen to concentration, and spray printing is detection zone on coated film; With the coated film damping fluid dilute commercially available goat anti-human igg to concentration be 0.1mg/ml, be the control region; Machine line, line-to-line should be careful even every 5mm, room temperature airing 20 minutes.Rearmounted 37 ℃ of oven dry were handled 2 hours, and are standby.
Rapid detection HIV1/2 antibody kit of the present invention is characterized in that described sample pad is glass fibre membrane or non-woven fabrics.Soaked 15 minutes in the sample pad treatment soln for preparing with the 3*30cm specification, place drying room, 50 ℃ of temperature, humidity are less than 20%, and be dry more than 18 hours, standby.
Rapid detection HIV1/2 antibody kit of the present invention, the preparation method who it is characterized in that described Radioactive colloidal gold pad is for to prepare the colloidal gold solution that diameter is 30-50nm with hydrochloro-auric acid-trisodium citrate reduction method, get 100ml after preparation is finished and transfer pH to 7.2, press the 100ml Radioactive colloidal gold and add the 0.5mg albumin A with 0.1M salt of wormwood.Stirring at room 30 minutes adds protective material, seals 30 minutes, and centrifugal 50 minutes of 12000rpm abandons supernatant, and the resuspended liquid dissolving with 1/3rd original volumes is coated in by the 500ul/ bar on the non-woven fabrics of 0.5*30cm.Put drying room again, temperature 20--25 ℃, humidity is less than 20%, and is dry 2--4 hour, standby.
Rapid detection HIV1/2 antibody kit of the present invention, the assembly method that it is characterized in that test paper is in kiln, temperature 20--25 ℃, humidity is less than 20%, get the PVC base plate, the NC film that wraps quilt is sticked on the middle part, at NC film detection zone one side overlap joint Radioactive colloidal gold pad, overlap sample pad again at the Radioactive colloidal gold pad, at NC film control region one side overlap joint absorbent pad, paste a marking film topmost, with cutting machine the plate that posts is cut into the test strip of 3mm or 4mm at last, the bar that cuts can be packed in the plastic clip, forms reagent card.
Rapid detection HIV1/2 antibody kit of the present invention, the detection method that it is characterized in that described reagent is for swabbing the upper and lower gum of subjects with the collection swab, then swab is put in the band cap test tube that fills about 1000 μ l sample diluents, in vitro mix, after liquid in the swab extracted, discard swab.Reagent card is kept flat, takes out 100 μ l sample liquid, add the sample application zone of test paper with divider, sample dissolving Radioactive colloidal gold and on the NC film chromatography, situation appears in visual inspection control line and detection line in 30 minutes, and result of determination.
Compare with existing detection reagent, the present invention has the following advantages:
1. be used to detect HIV antigen at present and mostly be the inclusion body expression, detection sensitivity is difficult to reach the level that detects HIV antibody from saliva.We are through preferably cultivating the genetically engineered Recombinant HIV antigen of escherichia coli expression, it includes the major antigen epi-position in HIV-1 gp120, gp41 and the HIV-2 gp36 section, molecular weight is respectively 35KD, we are with 14 little peptides of amino acid whose T7 by embedding one at the N of recombinant protein end, realized antigenic solubility expression, thereby obtained susceptibility and the selectivity higher than existing method.
2. specificity reaches 99.6%, and sensitivity reaches 100%, is enough to and traditional blood test reagent U.S. that combs mutually, and detection time is short, only is 15-30 minute.
3. method is simple, and security is higher.Do not contain virus in the oral mucosa transudate, the collection of sample can not cause skin injury, has eliminated and has collected the danger that blood preparation uses sharp device to cause, makes tester and measured safer.
The present invention is with low cost, easy and simple to handle, when detecting, do not need special plant and instrument, therefore be convenient to HIV INFECTION IN DETECTION and census operations, this test sheet no pain and greatly reduced the measured and the infected risk of medical personnel particularly, all are not suitable for the venous blood collection crowd for children, obese person, vein atrophy person and other provides a kind of more suitably detection mode.
Description of drawings
Fig. 1 is a structural representation of the present invention;
Fig. 2 is a detected result synoptic diagram of the present invention.
Detailed description of the Invention
Sample source in the detection is an important aspect, can affect efficient, accuracy, convenience of detection etc. In current detection, sample source has blood, ight soil, urine, saliva. Disease for the causal organisms such as virus and bacterium cause will detect from the sample of diseased individuals analysis, often help the aspects such as medical diagnosis on disease, medication effect assessment, the expectation of prognosis situation.
Virus and bacterium, and other causal organisms, after infecting body, body can produce antibody. Adopt blood, urine, saliva as sample, there is situation in the antibody that detects wherein, can help the diagnosis of this class disease. But, the personnel that get blood action need specialized training, the personnel that also have higher risk and hazard door to train. Urine sampling neither be very convenient, and can not take a sample at any time. The saliva sampling is subjected to the behavioral implications such as patient takes food, drinking-water easily, and the antibody materials number change is larger in the saliva.
The present inventor finds: the mucous membrane of mouth sampling is a more satisfactory approach. After infecting, Oral Mucosal Cells can secretory antibody and at its adsorption these antibody is arranged, such as upper and lower gingival cell, therefore, can from the mucous membrane of mouth sampling for detection of. And, also can allow the patient position sampling by the mode such as look in the mirror for the position in the oral cavity, therefore, can allow the proofer carry out spot sampling for disease detection, reach the high consistency of sample, increase the uniformity that detects. Before sampling, carry out in the situation of some promptings, such as allowing the patient use the clear water rinse, can further make sampling be subject to less foeign element and disturb. The sample of mucous membrane of mouth sampling does not have peculiar smell yet, and patient oneself can comparatively accurately take a sample, and is easy to oneself operation.
So, the invention that the application provides, in the broadest sense, first large aspect on, be a kind of sampling method, comprise instruments such as adopting brush, scraper plate, obtain to comprise the step of the detection material such as antibody from mucous membrane of mouth. This method can be united use with existing antibody test device, trial-production bar etc., improves their detection sensitivity and the degree of accuracy. The invention is intended to contain all methods from the mucous membrane of mouth sampling, main points are the detection material that obtain to contain antibody from mucous membrane of mouth.
The application also provides with or has been attached with the checkout gear, test strips of the sampling key element of the reagent of realizing above-mentioned sampling process or device etc. Therefore, existing various checkout gears, test strips can be modified, and are modified to from mucous membrane of mouth and take a sample. Mucous membrane of mouth of the present invention sampling can be combined with existing detection method, improve the detection quality of these detection methods or with enforcement simplification, the family oriented of these detection methods.
On the biology of intrusion body the antigen of being identified by body immune system is arranged. The definition of antigen is the material of induction of immunity reaction, comprises protein, carbohydrate and lipid. Antigenic component on the pathogenic organisms such as bacterium, virus, rickettsia is complicated, proteantigen on it, particularly the proteantigen of surface portion can be prepared by engineered method, is easy to be applied in the interactional detection method of utilizing antigen, antibody.
The application notices that a microorganism can have a plurality of proteantigens, comprises glycoprotein, lipoprotein antigen etc. These antigen performance intensity that contain protein portion are compared and study, choose the stronger proteantigen of antigenicity, from this microbial genome, access the gene of these proteantigens, express by gene engineering method, prepare antigenic substance, for the production of products such as test strips. Especially, these proteantigen genes can be carried out single expression, perhaps couple together and express as single fused protein. Preferably, carry out amalgamation and expression. Therefore, the invention that the application provides, just in the broadest sense, second aspect large be how effectively to prepare for detection of antigenic substance, especially adopt the method for amalgamation and expression to express the single fusion of a plurality of proteantigens.
Among the application, for the convenience of narrating, in most cases describe as pathogenic microorganism with HIV virus. But the application's invention never is limited to this virus and is limited to AIDS (AIDS). The application is the invention of a broad sense, is the improvement of above-mentioned two large aspects, also comprises the improvement of any one aspect of above-mentioned two broad aspect, and these all are scopes of the present invention.
So the present invention can be expressed as: the feature of mucous membrane of mouth sampling. The present invention can be expressed as: the antigen preparation method. The present invention can be expressed as: mucous membrane of mouth sampling and antigen preparation method.
In this application, be to utilize the interaction of antigen-antibody to detect for the basis basically. Therefore, in this application, term " antibody " refers to immunoglobulin (Ig), is the material of the antagonism adventive of body generation. Mucous membrane of mouth also has the ability of secretory antibody.
In this application, " material that contains antibody ", " detected material ", " test sample " has identical implication, all refers to derive from the material that wipes or scrape down of mucous membrane of mouth. This sampling method does not form damage to body, is non-intrusion type. The application's sampling is in the situation of not damaging housing construction, the material that obtains to contain antibody from body for detection of. The concrete content of antibody in specimen can be based on the causal organism that detects and is different.
In this application, " antigen " refers to stimulate on the microorganism body to produce the material of immune response. The immunogenicity of antigen has strong and weak other, and in this application, strong antigen is to assert for poor antigen, is a kind of relatively result. Using the quantity of antigen without limits, can be a kind of, two kinds, and three kinds, and more kinds of. The chemical nature of antigen is defined as the antigen that comprises protein portion, and its gene takes the form of nucleotide sequence. Selecting in the situation of two or more antigens, preferably the gene with these antigens carries out amalgamation and expression, in order to carry out high efficiency production. Therefore the power identification of antigen and select understanding situation and the research situation that also depends on about certain concrete pathogenic microorganism also has the situation of using poor antigen and strong antigen combination, and these are also in scope of the present invention.
In this application, after " antigenic substance " refers to the gene of the antigen protein of causal organism obtained, through expressing, obtain can with sample in antibody carry out the material of combination. Antigenic substance is the material with ability of binding antibody molecule, and antigenic substance can be the antigen fragment combination, can be antigenic complex. On function, refer to the material that the antibody of inducing with corresponding causal organism can specific bond. In general antigenic substance, is that surface protein antigen according to causal organism designs and synthesizes. Preferably take the form of fused protein. Generally be re-combined into. Also can modify the antigen gene from causal organism, such as suddenling change, then synthesize the variant fused protein. Antigenic substance can derive from prokaryotic expression, and perhaps eucaryote is expressed. As long as the substance that obtains represents causal organism in conjunction with the ability of its antibody of inducing in body, then these materials are exactly the antigenic substance among the application. Antigenic substance among the application, the expression product of the gene synthetic of a kind of antigen of causal organism or plurality of antigens preferably, expression product preferably a kind of molecular weight approach or consistent protein or the polypeptide of molecular weight.
In this application, sample collecting apparatus is the device that is fit to obtain from mucous membrane of mouth the cell surface secretory substance. Non-limiting example is swab, cytobrush, scraper plate etc. These article are suitable to the mucous membrane surface adsorbent. Proterties, the material of these article do not have special requirement, as long as can obtain mucous membrane secretion from oral mucosal surface.
In this application, sample diluting liquid can use according to concrete condition, and purpose is to obtain antibody molecule from the objects such as swab that sticked mucous membrane of mouth and secretion thereof, forms solution, for detection of. Sample diluting liquid can change according to concrete condition, and concentration and composition all can change.
In this application, " one " and odd number concepts such as " a kind of " in non-situation about offering some clarification on, also represent plural concept. Perhaps in context, can economize and omit the key element that invention understood in these numeral-classifier compound.
The below discusses AIDS and Causative virus thereof.
AIDS is the transliteration of AIDS, and AIDS is the english abbreviation of human acquired immunodeficiency syndrome. The medical science full name of AIDS is " acquired immunodeficiency syndrome ", and English full name is Acquired Immunodeficiency Syndrome.
AIDS is a kind of worldwide, lethal communicable disease. The complete concept of AIDS has been expressed in this name, therefrom we can recognize three of AIDS clearly definition: (1) is acquired: be illustrated in cause of disease aspect and be and obtain the day after tomorrow rather than congenitally have, AIDS is by a kind of retrovirus, that is the infectious disease that causes of AIDS virus. The route infection such as main trafficability characteristic contact, drug abuse, perinatal period. (2) immune deficiency: being illustrated in the pathogenesis aspect, mainly is to cause human immune system's damage and cause that immune safeguard function lowers, forfeiture. (3) syndrome: be illustrated in the clinical symptoms aspect, the complicated syndrome that opportunistic infections, the tumour of each system that causes owing to immune deficiency occurs. Our AIDS of often saying namely.
AIDS is discovery in 1981, and the nineteen eighty-two definite designation is " acquired immunodeficiency syndrome ". The virus that causes " human acquired immunodeficiency syndrome " was found in nineteen eighty-three. Present widely used title " human immunodeficiency virus " is that International Congress of Microbiology in 1996 can reach the unified specific term of naming of virus taxis association.
HIV is the abbreviation of AIDS virus (human immunodeficiency virus). In a kind of blood that can survive in the people and the virus of attacking the human immune system. It engulfs, destroys the T4 lymphocyte to most important T4 lymphocyte among the human immune system in a large number as target of attack, thereby whole human immune system is destroyed, and final human body is lost the resistivity of various diseases is caused death. Scientist is called " human immunodeficiency virus " to this virus.
HIV causes slow gradual infection, has very longly to dive the shape phase without disease, and average latency 7～10 years, annual about 4～10% develop into AIDS, and in the incidence of disease 65%, 15 year 95%, minority is in infection yet morbidity in rear 17 years in 12 years; Course of disease average out to is 1 year half after the morbidity. The HIV primary infection is usually in (2～4 weeks after infecting virus replication incipient stage, between 5 days～March) can cause nonspecific acute HIV Infectious syndrome, performance is monocytosis,mononucleosis seemingly: heating, night sweat, discomfort, myalgia, headache, arthralgia, trunk maculo-papular rash, diarrhoea, lymphadenopathy (are mainly involved armpit, occipitalia and cervical lymph node, also the lymph nodes of body as a whole enlargement can appear, sustainablely there is a several months, even the several years), sustainable 3～14 days. The monocytosis,mononucleosis that it and Epstein-Barr virus cause is mainly differentiated: break out, tonsillotome normal, common canker sore, fash and diarrhoea, aseptic meningitis, peripheral nerve disease, encephalopathic, splenomegaly etc. may occur in addition. Detecting mainly by checking HIV antibody of former acute infection. The infected enters asymptomatic long-term incubation period subsequently, and this moment, virus replication was in low-level state. Virus replication is activated under some factor effect, and since a large amount of propagation of virus, the lymphocyte of damage body, thus progressively strengthening immune deficiency, the infected presents the AIDS related syndromes, changes at last AIDS.
The World Health Organization infected HIV and was divided into the clinical fourth phase nineteen ninety:
One, asymptomatic stage---non-evident sympton or the enlargement of lymph nodes of delay property is arranged.
Two, the light disease phase---lose weight＜10% mild skin mucosal injury such as dermatitis, pruigo, mycotic nail infection, stomatocace, bridou, the upper respiratory tract infection of showing effect repeatedly.Total lymphocyte count＞2000/mm3, cd4 cell 500mm3.
Three, middle disease phase---expendable symptom grouping: lose weight＞10%, pyrexiaof unknown etiology＞January, agnogenio diarrhoea＞January, stomatitis, oral leukoplakia, pulmonary tuberculosis etc.Total lymphocyte count 1000～2000/mm3, cd4 cell 200～500/mm3.
Four, the severe phase---the expendable symptom grouping increases the weight of, various secondary infections, various secondary tumors, agnogenio dyskinesia etc.Total lymphocyte count＜1000/mm3, cd4 cell＜200/mm3 (reduce to about 100, patient was survived 1 year at most again).
AIDS patient and HIV carriers are the AIDS contagium.These human peripheral liquid, tissue juice, seminal fluid, vaginal secretions, milk, cerebrospinal fluid, marrow, central nervous system unify skin all separable to virus.Studies show that at present the route of transmission of AIDS mainly contains following several:
Transmission through sex is the most common circulation way.With the HIV relevant high risk population that spreads through sex intercourse be: men homosexuality person, prostitute, whoring person, property unrest person.U.S.'s investigation finds that homosexual's HIV infection rate is up to 20～30%.HIV the infected of western countries 70% is men homosexuality person.In Africa, some countries of South East Asia, 80% above HIV the infected is because of due to the opposite sex contact, and 81% is the prostitute among the women patient.In the prostitute of economic condition difference, the HIV antibody positive rate is up to 66%.In these countries, different in nature sexuality is the main circulation way of HIV.
This is the important channel of the another kind of HIV of propagation for blood transfusion and blood product.Before the blood donor not being carried out the HIV antibody test, blood transfusion and blood product have the very big danger of infected by HIV.In Africa 30% HIV infected children is due to the blood transfusion.Dangerous maximum that VIII thrombin treatment haemophiliac infects extracted in use from a large amount of blood donor's blood plasma.
It is the important channel that vein drug addiction person propagates AIDS that vein drug addiction person uses syringe, the syringe needle of non-sterile.U.S.'s investigation in 1986 finds that 25% has the intravenous injection drug use history among the AIDS patient.In western countries' shoot up is the second largest factor of infected by HIV.
Mother and baby's vertical transmission can be propagated parent HIV to fetus through blood in the Gestation period; Can make the newborn baby infected by birth canal at term; Also there is report to propagate by lactation.The AIDS case that 2-15% is arranged in Africa is by mother-to-baby transmission.The baby that the pregnant woman gives birth to that HIV infects has 30～70% to be infected by HIV in perinatal period.It is generally acknowledged that the baby is diaplacental infection in the chance of birth canal infected by HIV.Other circulation way is used the medicine equipment without thorough disinfection to undergo surgery, have tooth pulled out, puncture all can to draw HIV and is infected.But can not confirm still that at present HIV may cross air, food tableware, drinks water, shakes hands, daily apparatus is propagated.
An importance of the diagnosis of acquired immune deficiency syndrome (AIDS) and treatment is early diagnosis, comprises that also the human individual's knows situation in early days.Because the special pathogenesis of acquired immune deficiency syndrome (AIDS), also promptly with the height cognation of bad behavior, the human individual public situation of being unwilling itself detects.The detection means that exploitation is fit to individual's use is crucial.The application develops a kind of suitable individual detection means of using by setting up from the oral mucosa sampling.Simultaneously, the application has proposed a kind of antigenic substance manufacture method that is easy to produce, and detects with other virus detections with acquired immune deficiency syndrome (AIDS) to adapt.The application also can show as the array configuration of above-mentioned two aspects.
So, from big aspect, the invention provides the method that obtains the antibody sample from oral mucosa, contain the sample of the antibody of anti-HIV such as acquisition.From another big aspect, the invention provides the method for the representative of determining causal organism, just determine to screen these surface antigens by its surface antigen, obtain these surface antigen genes, and the representative that obtains to represent this causal organism with gene engineering method.The 3rd big aspect is the combination of above-mentioned two broad aspect.
The main aspect that the present invention includes has:
The invention provides and a kind ofly obtain to contain the method for antibody sample, comprise with utensil contacting oral mucosa that the utensil that will have oral mucosa contacts with solution, the formation liquid sample from oral mucosa.The invention provides the synthetic method of representing the representative of pathogenic micro-organism, comprise from pathogenic micro-organism and select antigen, obtain these antigenic genes, form the gene fusion thing, the material of pathogenic micro-organism is represented in recombinant expressed acquisition.The invention provides a kind of detection method, comprise adopting and represent the material of pathogenic micro-organism to prepare test strip or device, and will gather the antibody sample that contains that obtains from oral mucosa and analyze with described test strip or device.The present invention also provides test kit, comprises test strip and the sampling unit of taking a sample from oral mucosa.The present invention also provides test kit, comprises test strip and the sampling unit of taking a sample from oral mucosa, and working instructions.
Setting forth from concrete aspect below, is that example describes with the HIV detection.
First concrete aspect, the invention provides the test kit of HIV1/2 antibody in a kind of rapid detection human oral mucous membrane transudate, it comprises
A kind of oral mucosa transudate HIV1/2 antibody test test paper;
A kind of receptor that is used to collect the oral mucosa transudate;
A kind of divider, it is used for the sample liquid absorption portion that shifts out the sample liquid of predetermined amount and be released into proofing unit from described receptor.
According to test kit recited above, it is characterized in that: oral mucosa transudate HIV1/2 antibody test test paper is to paste sample pad (2), Radioactive colloidal gold pad (3), coated film (4) and absorbent pad (5) on base plate (1) in turn mutually overlap joint, coated film (4) has detection zone (6) and control region (7), detection zone (6) bag is by HIV 1/2 chimeric antigen, and wrap by the goat anti-human igg control region (7).
According to test kit recited above, it is characterized in that: described receptor is buccal swab or cotton swab class mucus sample collecting device, acquisition mode is for swabbing the upper and lower gum of subjects with the collection swab, then swab is put in the band cap test tube that fills about 1000 μ l sample diluents, in vitro mix, after liquid in the swab extracted, discard swab.Described sample diluting liquid is the 0.01MTris solution that contains 1% 1 5%BSA, and PH is 7.0---9.0, and contains 0.1%--0.5%PEG and 0.01%--0.05%TWEEN-20 tensio-active agent.
According to test kit recited above, it is characterized in that: described divider is as pipettor or Siphonata liquid-transfering device, and presetting and moving liquid measure is 100 μ l.
According to test kit recited above, it is characterized in that described envelope antigen is the escherichia coli expression genetically engineered Recombinant HIV antigen through preferred cultivation, it includes the major antigen epi-position in HIV-1gp120, gp41 and the HIV-2gp36 section, molecular weight is 35KD, is with 14 little peptides of amino acid whose T7 at the N of recombinant protein end.Particularly its help has realized this antigenic solubility expression.Thereby susceptibility and the selectivity higher have been obtained than existing method.
According to test kit recited above, it is characterized in that described method for coating for being 0.5mg/ml with coated film damping fluid dilution antigen to concentration, spray printing is detection zone on coated film; With the coated film damping fluid dilute commercially available goat anti-human igg to concentration be 0.1mg/ml, be the control region; Machine line, line-to-line should be careful even every 5mm, room temperature airing 20 minutes.Rearmounted 37 ℃ of oven dry were handled 2 hours, and are standby.
According to test kit recited above, it is characterized in that described sample pad is glass fibre membrane or non-woven fabrics.Soaked 15 minutes in the sample pad treatment soln for preparing with the 3*30cm specification, place drying room, 50 ℃ of temperature, humidity are less than 20%, and be dry more than 18 hours, standby.
According to test kit recited above, the preparation method who it is characterized in that described Radioactive colloidal gold pad is for to prepare the colloidal gold solution that diameter is 30-50nm with hydrochloro-auric acid-trisodium citrate reduction method, get 100ml after preparation is finished and transfer PH to 7.2, press the 100ml Radioactive colloidal gold and add the 0.5mg albumin A with 0.1M salt of wormwood.Stirring at room 30 minutes adds protective material, seals 30 minutes, and centrifugal 50 minutes of 12000rpm abandons supernatant, and the resuspended liquid dissolving with 1/3rd original volumes is coated in by the 500ul/ bar on the non-woven fabrics of 0.5*30cm.Put drying room again, temperature 20--25 ℃, humidity is less than 20%, and is dry 2--4 hour, standby.
According to test kit recited above, the assembly method that it is characterized in that test paper is in kiln, temperature 20--25 ℃, humidity is less than 20%, get the PVC base plate, the NC film that wraps quilt is sticked on the middle part, at NC film detection zone one side overlap joint Radioactive colloidal gold pad, overlap sample pad again at the Radioactive colloidal gold pad, at NC film control region one side overlap joint absorbent pad, paste a marking film topmost, with cutting machine the plate that posts is cut into the test strip of 3mm or 4mm at last, the bar that cuts can be packed in the plastic clip, forms reagent card.
According to test kit recited above, the detection method that it is characterized in that described reagent is for swabbing the upper and lower gum of subjects with the collection swab, then swab is put in the band cap test tube that fills about 1000 μ l sample diluents, in vitro mix, after liquid in the swab extracted, discard swab.Reagent card is kept flat, takes out 100 μ l sample liquid, add the sample application zone of test paper with divider, sample dissolving Radioactive colloidal gold and on the NC film chromatography, situation appears in visual inspection control line and detection line in 30 minutes, and result of determination.
So, the present invention includes the work in the test sample sampling; Comprise the work on the test strip raw material sources; Comprise the combination of test strip and sampling.Above-mentioned any one aspect can be set up separately, and the present invention also is not limited to acquired immune deficiency syndrome (AIDS) and HIV virus.Above-mentioned any one aspect of the present invention can independently constitute the invention of satisfying patent statute, also can be applied to any available reagent box and existing detection based on antigen antibody interaction, the present invention can not only only limit to specific virus, specific bacteria or particular detection mode.Only otherwise deviate from spirit of the present invention and essence, all should be included within the scope of the invention.
Embodiment
The preparation of the anti-position of embodiment one .HIV multilist chimeric antigen:
The anti-position of HIV multilist chimeric antigen is that the genetically engineered recombinant technology is adopted in this laboratory, according to proteinic structural performance and antigenic immunology knowledge, adopt the Goldkey computer aided design (CAD), with the main epitope gene of main epitope gene, HIV-1gp120 of third generation acquired immune deficiency syndrome (AIDS) recombinant antigen HIV-1gp41, " M " group and " O " group and the main epitope gene of HIV-2gp36 flexible structure gene in addition, a full gene DNA connection artificial sequence gene together.This sequence clone to expression vector ectopic expression again, is set up the strain of immunocompetence high expressing cell.Last fermentation culture, inducement efficient are expressed, many epitopes of chromatographic separation purifying hiv virus fused antigen HIVM Ag, because of fused antigen has reduced unnecessary gene order nonspecific reaction is reduced more than 50%, and the synergy of a plurality of epitopes can make the sensitivity that detects increase 2-3 doubly on the same antigen.The usefulness that acquired immune deficiency syndrome (AIDS) recombinant multi-epitope fused antigen can be supplied AIDS diagnosis articles for use manufacturer and medicine clinical research.
Concrete operations: determine the antigenic amino acid position of the different sections of the strong HIV of antigenicity, respectively synthetic primer.Primer series is as follows:
Prime?????Sequence(5’---3’)????????????Genomes
P41a??????GAAGAATTCGCAGTGGGAATAGGAG??????7758----7773bp
P41b??????TTATCTCGAGCAGCCAATTTGTTATGTT???8244----8261bp
P120a?????GACGAATTCACACTCCCATGCAGAATAA???7467-----7485bp
P120b?????AAGCTCGAGAGCTCCTATTCCCACTGC????7758-----7775bp
P36a??????TTAGAATTCGTGTTCGTGCTAGGGTTC????7683-----7700bp
P36b??????AATCTCGAGGACCCAGGAGGTTAAGTCA???8150-----8168bp
Making a definite diagnosis the positive inactivated serum of HIV (You An hospital provides) with Nat'l Pharmaceutical ﹠ Biological Products Control Institute's HIV PCR somatotype is template, carry out pcr amplification, obtain HIV/gp41, HIV/gp36, HIV/O, the HIV/gp120 gene fragment, again with EcoRI, behind the BamHI double digestion, make up the pGEM-T carrier, and antigen is connected into the multi-epitope chimeric antigen be inserted into respectively in the pGEM-T carrier, obtain expression plasmid pGEM-T HIV/gp41, pGEM-T HIV/GP gp36, pGEM-T HIV/gp120 utilizes pGEM-T a plurality of goal gene can be made things convenient for connection characteristics, pGEM-T one HIV/gp41, pGEM-T one HIV/gp36pGEM-T one HIV/gp120 connects, and is built into pGEM-T one HIVgp41 one gp120 one gp36 expression plasmid.Be template with pGEM-T one HIVM Ag plasmid then, carry out pcr amplification, obtain gene fragment, be inserted in the carrier after enzyme is cut, obtain expression plasmid.The expression plasmid of above acquisition is transformed in the intestinal bacteria recipient bacterium, efficiently expresses, because be with 14 little peptides of amino acid whose T7, finally obtain the HIV antigen of solubility expression especially in the embedding of the N of recombinant protein end.
Carry out purifying with mistake Q one Sepharose one FF negatively charged ion handing-over post, His-Ni Seprose gel, SDS--PAGE identifies that the application of sample amount is not less than 5 micrograms, and the result should not have any foreign protein band, and purity is greater than 95%, and antigenic molecular weight is 35KD.Frost drying is preserved down at 25 ℃.
Embodiment two. the preparation of oral mucosa transudate HIV1/2 antibody test test paper
The preparation of 1 coated film
1.1 bag is cushioned the preparation of liquid: preferred 0.05M pH 9.6 carbonate buffer solutions for bag by solution, behind the 0.22um membrane filtration, put 2 ℃～8 ℃ standby, one week of validity period.
1.2 coated film preparation: debugging Membrane jetter (Bio-Dot), preferred spray film liquid measure is 20ul/30cm, be cushioned liquid dilution envelope antigen with bag, concentration is 0.5mg/ml, diluting commercially available SIGMA goat anti-human igg with the coated film damping fluid is 0.1mg/ml to concentration, the machine line, and line-to-line is every 5mm, should be careful even, room temperature airing 20 minutes.Rearmounted 37 ℃ of oven dry were handled 2 hours, and envelope is equipped with the test paper plate pasting board and uses.
The preparation of 2 mixture pads
2.1 the preparation of Radioactive colloidal gold: go to select the stirrer that is fit to size for use from place clean glassware (but the silication of having ready conditions) in water 1200ml, middling speed stirs.Be docile and obedient preface and add 1% hydrochloro-auric acid 24ml, trisodium citrate 0.42 gram.Boiled 10 minutes.Carry out spectral scan then, be advisable maximum absorption band to occur between 500 1 600nm.Colloid gold particle is that 30-50nm is advisable.
2.2A protein labeling: adjusting Radioactive colloidal gold OD value with deionized water is 1.5, and transferring pH with 0.1M salt of wormwood is 7.2.The A albumen (SIGMA product) that in the 100ml colloidal gold solution, adds 0.5mg, mixing, mark 30 minutes.The BSA of adding 10% is 1% to final concentration, and mixing was placed 30 minutes.Centrifugal 50 minutes of 12000r/min abandons supernatant, and precipitation is dissolved with the resuspended liquid of 1/3rd initial Radioactive colloidal golds.Resuspended liquid formula is: 2% bovine serum albumin BsA, 2% sucrose, 0.15%tweeen-20,0.01M pH7.0PBS solution, the 0.22p membrane filtration, put 2 ℃～8 ℃ standby, two weeks of validity period.
2.3 the preparation of mixture pad: the Radioactive colloidal gold that mark is good is layered on the non-woven fabrics equably, is coated in by the 500ul/ bar on the glass fibre membrane of 0.5*30cm, puts drying room again, and temperature 20--25 ℃, humidity is less than 20%, and is dry 2--4 hour, standby.
The preparation of 3 sample pad
3.1 in the sample pad treatment soln for preparing, soaked 15 minutes with the 3*30cm specification, place drying room, 50 ℃ of temperature, humidity are less than 20%, and be dry more than 18 hours, standby.
3.2 the prescription of sample pad treatment solution is: the 0.01MTris solution of 2%BSA, PH are 8.0, and contain 0.2%PEG, 1% hydrolysis network albumen and 0.02%TWEEN-20 tensio-active agent.
The preparation of 4 test cards:
In kiln, temperature 20--25 ℃, humidity is got the PVC base plate less than 20%, the NC film that wraps quilt is sticked on the middle part, at NC film detection zone one side overlap joint Radioactive colloidal gold pad, overlap sample pad again at the Radioactive colloidal gold pad, at NC film control region one side overlap joint absorbent pad, paste a marking film topmost, with cutting machine the plate that posts is cut into the test strip of 3mm or 4mm at last, the bar that cuts can be packed in the plastic clip, forms reagent card.
Test strip is fixed on the base plate of test kit, the upper cover plate that closes makes the sample pad of the corresponding test strip of application of sample window of loam cake, as a result corresponding detection zone of display window and control region.
The preparation of 5 sample diluting liquids:
5.1 through preferred, sample diluting liquid is defined as containing the 0.01M Tris solution of 2%BSA, PH is 8.0, and contains 0.2%PEG and 0.02%TWEEN-20 tensio-active agent.
5.2 prop up by 1ml/ and to be divided in sealing in vitro.
6. detect principle and method
Its detect principle be at the detection zone bag that detects the test paper nitrocellulose filter by HIVgp120, gp41 and gp36 gene engineering antigen, at check plot bag quilt the goat anti-human igg.During detection, swab the upper and lower gum of subjects, then swab is put in the test tube that fills about 1000 μ l sample diluents, in vitro mix, after the liquid in the swab is extracted, throw away swab with the collection swab.The sample application zone of the sample mixed solution of 100 μ l being transferred to test paper detects.Blush albumin A-colloid gold label thing on the test paper is dissolved, same albumin A/the colloidal gold particle of IgG in the sample combines, form " IgG-albumin A-Jin " mixture, " IgG-albumin A-Jin " mixture is moved upward along test paper, at first arrives bag by HIV detection of antigens district.If contain the antibody of HIV in the sample, " IgG-albumin A-Jin " mixture is the synantigen combination, and under being detained on the detection line, forms an erythroid line, and this represents positive findings.The depth of line color is with the disproportional relation of antibody quantity in the sample.Do not represent not contain in the sample anti-HIV antibody if there are colored lines in the detection zone.Free " IgG-albumin A-Jin " mixture continues to move to the test paper top, arrives the check plot of test paper.The check plot bag is fixed on the control line of test paper by the goat anti-human igg." IgG-albumin A-Jin " mixture will combine and will be enriched on the control line with the goat anti-human igg, form an erythroid line.The appearance proof test strip measuring ability of control line is normal.No matter whether contain HIV-1/HIV-2 antibody in the sample, in efficiency test, the check plot of test paper all should occur little red-the purple control line.Sample continues to move, and crosses the check plot and finally enters the uptake zone.The effect of uptake zone is absorption remaining " IgG-albumin A-Jin " mixture, and it is moved in test paper, and eliminates background colour.
Embodiment three. the performance analysis of oral mucosa transudate HIV1/2 antibody test reagent
1. main raw
1.1 oral mucosa transudate HIV1/2 antibody test reagent
HIV1/2 antibody diagnosing reagent kit (colloidal gold method): Shanghai Kehua Bio-engineering Co., Ltd, Beijing Tso Biological Pharmaceutical Co
1.2HIV antibody test reagent country reference material (colloidal gold method): Nat'l Pharmaceutical ﹠ Biological Products Control Institute
1.3HIV the inner quality control product (saliva) of antibody test reagent: Nat'l Pharmaceutical ﹠ Biological Products Control Institute
1.4 clinical serum: ditan hospital collects, 324 parts.
1.5 oral mucosa transudate sample: ditan hospital collects, 324 parts.Corresponding one by one with serum sample.
2. method
2.1 clinical sample detection method: reference is specification sheets separately, result of determination in 30 minutes.
2.2HIV the inner quality control product of antibody test reagent (saliva) detects: take out back balance to room temperature, detect result of determination in 30 minutes with this reagent.
2.3 the clinical sample experiment is collected clinical serum and corresponding oral mucosa transudate sample by ditan hospital, carries out control test with a kind of colloid gold reagent and this reagent, if meet the inconsistent sample of result, checks with another colloid gold reagent again.Two kinds of all positive result of determination of reagent are positive, and two kinds of equal feminine genders are judged to be feminine gender.
3. result
3.1 the detection to HIV antibody test reagent country's reference material (saliva) meets national standard fully.
Concrete outcome sees Table one.
3.2 the clinical sample experiment has detected 324 parts in ditan hospital's outpatient service sample altogether, two kinds of colloid gold reagents are confirmed positive 56 parts, and negative 268 parts, this reagent detects positive 57 parts, negative 267 parts.The sensitivity of this reagent is 100%, specificity 99.6%.
Table one. inner quality control product (saliva) detected result of this reagent HIV antibody test reagent
| Interventions Requested | Quantity | Standard code | The result |
| ??HIV(+) | ??20 | ??20/20 | 20/20 is up to specification |
| ??HIV(-) | ??20 | ??≥18/20 | 20/20 is up to specification |
| The accuracy sample | ??5 | ??5/5 | 5/5 is up to specification |
| ??S1 | ??- | -up to specification | |
| The sensitivity sample | ??S2 | ??+/- | + up to specification |
| ??S3 | ??+ | + up to specification | |
| ??S4 | ??+ | + up to specification | |
| ??S5 | ??+ | + up to specification |
Claims (11)
1. method for preparing the representative of causal organism, described method comprises:
Select antigen protein from causal organism;
Obtain the gene of these antigen proteins;
Make up the recombinant vectors of these genes of amalgamation and expression;
Give expression to fused protein in the host, it is the representative of causal organism.
2. sampling method, described method comprises:
Wipe away to get or scrape from oral mucosa and get material.
3. sampling method as claimed in claim 2, described method also comprises:
The substance transfer of getting is got or scraped to described wiping away from oral mucosa to solution.
4. detection kit, it comprises:
Test strip,
Swab,
Solution.
5. test kit that detects the HIV1/2 antibody in the human oral mucous membrane transudate, it comprises:
Oral mucosa transudate HIV1/2 antibody test test paper;
Be used to collect the receptor of oral mucosa transudate;
Divider, it is used for the sample liquid absorption portion that shifts out the sample liquid of predetermined amount and be released into proofing unit from described receptor.
6. according to the test kit of claim 5, it is characterized in that: described oral mucosa transudate HIV1/2 antibody test test paper is included in pastes sample pad (2), Radioactive colloidal gold pad (3), coated film (4) and absorbent pad (5) in turn mutually overlap joint on the base plate (1), coated film (4) has detection zone (6) and control region (7), detection zone (6) bag is by HIV 1/2 chimeric antigen, and wrap by the goat anti-human igg control region (7).
7. according to the test kit of claim 5, it is characterized in that: described receptor is buccal swab or cotton swab class mucus sample collecting device.
8. according to the test kit of claim 7, it is characterized in that: the acquisition mode of described receptor is for swabbing the upper and lower gum of subjects with the collection swab, then swab is put in the band cap test tube that fills about 1000 μ l sample diluents, in vitro mix, after liquid in the swab extracted, discard swab;
Described sample diluting liquid is to contain 1% to 5%BSA 0.01M Tris solution, and PH is 7.0 to 9.0, and contains 0.1% to 0.5%PEG and 0.01% to the 0.05%TWEEN-20 tensio-active agent.
9. according to the test kit of claim 5, it is characterized in that: described divider is pipettor or Siphonata liquid-transfering device, and presetting and moving liquid measure is 100 μ l.
10. according to the test kit of claim 6, it is characterized in that: it is characterized in that described envelope antigen is the escherichia coli expression genetically engineered Recombinant HIV antigen through preferred cultivation, it includes the major antigen epi-position in HIV-1gp120, gp41 and the HIV-2gp36 section, molecular weight is 35KD, is with 14 little peptides of amino acid whose T7 at the N of recombinant protein end.
11. according to the test kit of claim 9, it is characterized in that described method for coating is is 0.5mg/ml with coated film damping fluid dilution antigen to concentration, spray printing is detection zone on coated film; With the coated film damping fluid dilute commercially available goat anti-human igg to concentration be 0.1mg/ml, be the control region; The machine line, line-to-line is every 5mm.
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