CN101566631A - Magnetic immunochromatographic test strip for combined detection of HIV-1+2 antibody and P24 antigen and preparation method thereof - Google Patents

Abstract

The invention relates to a magnetic immunochromatographic test strip for combined detection of HIV-1+2 antibody and P24 antigen and a preparation method thereof. The test strip is assembled by pasting a coating film, magnetic particles combined with the HIV-1+2 antibody and the P24 antigen, a sample pad and a water-absorbing pad which are mutually staggered by 2 millimeters in turn on a bottom plate and then covering an upper layer with a transparent plastic sealing film, wherein the coated film is precoated with an HIV-1+2 antibody detection line, an HIV P24 antigen detection line and a quality control line. As the test strip introduces a magnetic immunochromatography technique and a biotin-avidin system into the combined detection of the HIV-1+2 antibody and the P24 antigen, the test strip has the advantages of greatly increasing detection sensitivity result accuracy, shortening detection window period and reducing the labor intensity of operators.

Description

Magnetic immuno-chromatographic test paper strip of a kind of joint-detection HIV-1+2 antibody and P24 antigen and preparation method thereof

Technical field

The invention belongs to field of medical examination, particularly relate to magnetic immuno-chromatographic test paper strip of a kind of joint-detection HIV-1+2 antibody and P24 antigen and preparation method thereof.

Background technology

Acquired immune deficiency syndrome (AIDS), be that (Acquired Immure Deficiency Syndrome AIDS), is by human immunodeficiency virus (human immunodeficiency virus to aids, HIV) cause the chronic progressive disease that still can not cure at present, case fatality rate is high.The UNAIDS and the World Health Organization (WHO) unite the issue report and point out that global AIDS virus carrier's number was 3,950 ten thousand in 2006; Newly-increased HIV-positive 4,300,000 in 2006; The whole world had 2,900,000 people to die from acquired immune deficiency syndrome (AIDS) in 2006.According to ministry of Health of China " China's AIDS is prevented and treated joint assessment report (2007) ", to the end of the year 2007, about 700,000 (550,000～850,000) people of existing patients infected hiv of China and patient, total man group's infection rate is 0.05% (0.04%-0.07%).Acquired immune deficiency syndrome (AIDS) patient 8.5 ten thousand (80,000～90,000) people wherein; New Development patients infected hiv 50,000 (40,000～60,000) people in 2007 is because of dead 20,000 (1.5 ten thousand～2.5 ten thousand) people of acquired immune deficiency syndrome (AIDS).Above data show that acquired immune deficiency syndrome (AIDS) is just threatening the development of human peacefulness, society with unprecedented degree and stablizing, and China has entered the quick infection period of acquired immune deficiency syndrome (AIDS) at present, if catastrophic consequence will appear in untimely control.But up to the present, medical circle does not still have a kind of method of effective treatment acquired immune deficiency syndrome (AIDS), and therefore aspect the control of acquired immune deficiency syndrome (AIDS), early diagnosis is in time found the infected and controlled the most important thing that its further propagation just becomes AIDS preventing and controlling work.

According to the difference of acquired immune deficiency syndrome (AIDS) clinical manifestation, it can be divided into four periods of acute infection period, latent period, pre-AIDS, typical AIDS stadium.The AIDS of acute infection period only shows as light symptoms such as heating, fash, enlargement of lymph nodes, weak, perspiration, nausea,vomiting,diarrhea, pharyngitis usually, most patients are after HIV1/2 infected for 2～6 weeks, HIV antibody can occur in the serum, can be used as the important indicator of early diagnosis and disorder in screening.Acute stage symptom continued after 3～14 days, and the asymptomatic latent period of (average 2～10 years) of a varying length appears in AIDS, and HIV virus continues breeding, has strong destruction, and the patient does not but have any clinical symptoms.Carry out examination widely, particularly hold blood transfusion and close, can reduce the probability that AIDS propagates greatly.After latent period, disease continues development and promptly enters pre-AIDS and typical AIDS stadium, and the infected causes various infection owing to immunodeficiency, the later stage of course of disease development, various opportunistic infections and malignant tumour appear in the infected, and serious consequence and high mortality ratio take place.

Present AIDS etiological diagnosis technology mainly comprises special viral antibody enzyme linked immune assay (ELISA), chemiluminescence (CLIA), immunochromatographic method (collaurum or latex particle method), Western blot (WB), cell culture method, PCR method, and these methods all have characteristics separately and use object.

The ELISA method is generally to use detection technique in the present clinical blood examination, third generation reagent is used to measure antiviral antibody, occur the end of the nineties the 4th generation reagent, on the basis of detecting antibody, detect P24 antigen simultaneously, but ELISA test operation program complexity, false positive or false negative result appear easily, and sensitivity is low, CLIA and ELISA method are similar, just sensitivity has improved some, the problem that the not basic ELISA of solution exists, and all there is complex operation in the two, the problem that the reaction time is long, and all need microplate reader or light-emitting appearance and wash complex apparatus such as plate machine and incubator, and can not single part detect, further limited them at some basic hospitals, the application of clinic.Occurred both at home and abroad in recent years with collaurum or latex particle is the quick detection test paper bar of representative, but because the result is the naked eyes visualizations, be subjected to the influence of observer's subjective judgement easily, sensitivity is low, result precision is not high yet, but also can not use the P24 detection of antigens, the detection window phase is longer.

Western blot (WB) is the confirmation method that present clinical AIDS is detected, the result accurately and reliably, but its technical difficulty is big, at present the whole world also has only several company to produce, high use cost makes it only be used for the affirmation of suspicious sample at present, and seldom is used for screening.

Cell culture method and PCR method, all need very high experiment work environment and condition (between sterile working, superclean bench) and a large amount of instrument and equipment (incubators, hydro-extractor, PCR instrument etc.), and operation is extremely complicated, need be subjected to the personnel operation of strict professional training,, and the test period is very long, is not suitable for clinical detection and uses.

Magnetic immuno-chromatographic (Mgnetic ImmunoChromatographic Test, MICT) be occur in recent years the single part fast quantification detection technique of a kind of a new generation.Be to replace traditional label (collaurum, latex particle etc.) to carry out immunochromatography, be combined in biochemical substances on the super-paramagnetism nano particulate by detection detection by quantitative data to biological sample are provided with supperparamagnetic particles (superPMPs).Come the amount of presentation markup by detecting the measurement magnetic field intensity, adopt the typical curve of the immune complex-magnetic field intensity of mark, thereby reach quantitative purpose in sample area.This technology is compared with conventional art has following advantages: 1) sensitivity for analysis: than the sensitive 10-100 of all kinds of range estimation quick diagnosis methods doubly; 2) analysis speed: can in 15 seconds, measure the nearly data of 6 site of analysis; 3) linear range: at 1-10 ⁴Concentration range in be linear; 4) the used magnetic detecting instrument adopts the solid phase element, and miniaturized design is had a style of one's own, independent operating, and volume is little, and is easy and simple to handle; 5) the super-paramagnetism nano particulate can not decayed in time by polymer coating; Independently quick diagnosis chromatogram card (MAR Cassette) can directly insert the MAR detector, for the integration of many quick reagents for clinical diagnosis at present provides development space widely; But also personnel or the contingent cross pollution of instrument in the operating process, the security that has improved analysis and process have been avoided.This technology has been inherited traditional immunochromatographic method (collaurum, latex particle etc.) easy to be quick, the advantage of single part of operation, it is low to have remedied traditional immunochromatography technique sensitivity again, can only be qualitative, shortcoming that can not be quantitative, represented current real-time test (Point of Care Test, POCT) direction of technical development and trend are once appearance, development has become the first-selection that substitutes traditional immunochromatography technique at present rapidly.

Mark magnetic particle commonly used at present is a super paramagnetic particle (superPMPs), do not have any magnetic in the absence of externally-applied magnetic field, only just can show magnetic adding under the action of a magnetic field, the super paramagnetic particle of commercialization all passes through finishing, greatly facilitate labeling process, mark is easy, good reproducibility.

Biotin-avidin system (BAS) is a kind of technology of widespread use, with the Avidin bag by solid phase, with biotin labeling antigen or antibody, utilize affinity high between the biotin Avidin and Avidin characteristic, can improve the sensitivity of reaction greatly, through the development of decades in conjunction with four biotins, a large amount of derivants have all appearred in Avidin and biotin at present, can select for use according to different needs, labeling method is also very easy, has increased their ease for use greatly.Biotin Avidin system is introduced in the magnetic immuno-chromatographic; in the sensitivity that has greatly improved detection method, a kind of fabulous current techique platform is provided again, in large-scale production, reduced markers step; increase the versatility of technology, reduced variable factor.

Summary of the invention

Purpose of the present invention promptly is with in magnetic immuno-chromatographic technology and the system combined HIV of the being applied in immunoassay of biotin Avidin.With the streptavidin covalent coupling on super paramagnetic particle, biotinylation HIV1+2 antigen and biotinylation P24 antibody are mixed with it afterwards as detecting moving phase, HIV1+2 antigen and P24 antibody wrapped respectively on nitrocellulose filter, made antibody detection line and Detection of antigen line as catching solid phase, carry out the detection of sample according to routine immunization chromatography ratio juris, detect in conjunction with simple and easy to do magnetism detector, when realizing high-sensitivity detection, can shorten the window phase of detection again greatly, can avoid the deficiency of aforementioned several detection methods, combine the advantage of aforementioned several method again: can single part detect, also can batch detection, and can provide quantitative result immediately, surveying instrument is simple and reliable, easy and simple to handle, convenient and practical.

For reaching above-mentioned purpose, technical scheme of the present invention is as follows: the magnetic immuno-chromatographic test paper strip of detection HIV-1+2 antibody of the present invention and P24 antigen is with coated film, the magnetic mat of particles that combines HIV-1+2 antigen and P24 antibody, sample pad, adsorptive pads, sticks on the base plate successively interlaced 2mm, cover the transparent plastic diaphragm seal then on the upper strata and make, be coated with the HIVP24 Detection of antigen line of HIV1+2 HIV antigens-1+2 antibody detection line and P24 antibody on the wherein said coated film in advance, and the nature controlling line of biotinylation bovine serum albumin(BSA).

The base plate of selecting for use is the transparent plastic base plate, and coated film is the nitrocellulose filter of 35mm width, and the adsorptive pads of selecting for use is a cellulose membrane, and the magnetic mat of particles is a fiberglass packing, and sample pad is the pretreated cellulose membrane of process sample pad treating fluid.Described sample pad treating fluid is the polyvinyl alcohol (PVA) (PVP) that contains 1%-5% casein (casein) and 0.1%-1%, and the 0.02M of 0.01-0.2% Tween-20 (Tween-20), the phosphate buffered solution of pH7.0-7.6 (PBS).

Detect the preparation method of the magnetic immuno-chromatographic test paper strip of HIV-1+2 antibody and P24 antigen, may further comprise the steps:

The processing of A, antigen: select for use commercialization HIV-1+2 recombinant antigen (the HIV fusion antigengp120-41-36 of CTK company, cat:A3641); To 20mM, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6;

The processing of B, antibody: select for use the commercialization height tire P24 pairing monoclonal antibody (day strong company, cat:210-1011,210-1012); 20mM, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6;

The preparation of C, magnetic particle: selecting diameter for use is the super paramagnetic particle of 100-300nm, the mode of using carbodiimide (EDC) and succinimide (NHS) covalent coupling with the streptavidin mark to the magnetic particle, select for use pre-activation biotin to carry out the mark of HIV-1+2 antigen/P24 antibody, biotinylated antigen/antibody that mark is good is with 1: 2-1: 10 ratio (volume ratio) is mixed with streptavidin magnetic particle, guarantees that streptavidin magnetic particle is excessive;

D, use quantitative liquid-jet device to be sprayed on the magnetic mat of particles magnetic particle for preparing with the amount of 25 μ l/cm-50 μ l/cm;

The preparation of E, coated film: use bag to be cushioned the concentration that liquid is diluted to HIV-1+2 envelope antigen and P24 coated antibody and biotinylation BSA 0.5-2mg/m respectively, use quantitative liquid-jet device respectively with three's being interval on the nitrocellulose filter with 0.5-1.0cm, dry the back and in confining liquid, soak after 10 minutes, add drying agent and seal up for safekeeping standby in 25-35 ℃ of oven dry 8 hours;

The processing of F, sample pad: sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour;

The assembling of G, test strips: interlaced successively 2mm sticks coated film, magnetic mat of particles, sample pad, adsorptive pads on the transparent plastic base plate, covers the transparent plastic diaphragm seal then on the upper strata, obtains test paper plate, and the width cutting promptly obtains test strips as requested.

Described step C comprises following three steps:

1) preparation of streptavidin magnetic particle: use the 50mM that contains 0.1%Tween-20, the sodium-acetate buffer washing magnetic particle of pH4.5-5.0, adding EDC and NHS makes the two final concentration be 20mmol, room temperature reaction 1 hour, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer of pH4.5-5.0 fully washs and adds streptavidin behind the magnetic particle to make the molecule ratio of streptavidin and magnetic particle be 5: 1 (mol ratio), room temperature reaction 3 hours, add the 0.02M that contains 0.5%BSA, the PBS room temperature sealing of pH7.0-7.6 30 minutes, washing magnetic particle, use contains i%PVP, 1%Casein, 0.5%Tween-20, the boric acid of the 50mM pH8.2-9.0 of 5% sucrose is preserved damping fluid redissolution magnetic particle, and 4 ℃ of preservations are standby;

2) preparation of biotinylation HIV-1+2 antigen and P24 antibody: with HIV-1+2 antigen and P24 antibody to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6, adjustment concentration is 2mg/ml, to activate biotin in advance and use dimethyl sulfoxide (DMSO) (DMSO) dissolving, final concentration is 50mM, adds the biotin solution of aequum, room temperature reaction 1 hour with 20: 1 molecule ratios in antigen or antibody-solutions, to 4 ℃ of dialysed overnight of 0.02M PBS ,-20 ℃ of preservations are standby behind the adding equal-volume glycerine;

3) biotinylated antigen/antibody and streptavidin magnetic particle mixes, amount with 0.5 μ l/mg magnetic particle adds biotinylation HIV-1+2 antigen in streptavidin magnetic particle solution, amount with 0.25 μ l/mg magnetic particle adds biotinylation P24 antibody, and fully use behind the mixing and preserve damping fluid with 1: 5-1: 20 ratio (volume ratio) is used mixture diluted fiberglass packing to be sprayed.

Among the described step D, the spraying method of magnetic particle is: use quantitative spray film device evenly to be sprayed on the glass fibre with the amount of 50 μ l/cm the magnetic particle for preparing, add drying agent after the freeze drying and seal up for safekeeping standby.

In the described step e, the preparation method of coated film is: be cushioned liquid (0.02M PB with bag, pH7.0-7.6) be 0.5mg/ml with HIV-1+2 antigen and P24 antibody dilution, biotinylation BSA dilution is 1mg/ml, use quantitative spray film device with the amount of 1 μ l/cm with the three with the interval spray printing of 0.6cm on nitrocellulose filter, room temperature is dried after 30 minutes and to be soaked in confining liquid after 10 minutes in 25-35 ℃ of oven dry 8 hours, adds drying agent and seals up for safekeeping standby.

Compare with existing quick detection test paper bar, the present invention has the following advantages:

1) by improvement to test strips, with in HIV 1+2 antibody and the antibody combined introducing test strips of P24, realize the antigen-antibody joint inspection first, shortened the window phase that HIV detects, and can roughly judge the infected's infective stage, for further diagnosis and treatment provide foundation.

2) by on the magnetic particle, introducing biotin-avidin system, make the preparation process of test strips simplify greatly, be fit to large-scale production.

3) the utilization magnetism detector carries out result's interpretation, carries out yin and yang attribute according to the magnetic detection value ratio of detection line and nature controlling line and judges, has reduced subjectivity, and the result accurately, reliably.

The present invention is easy and simple to handle, be fit to large-scale production, detect required portable set and also go on the market, therefore can be widely used in short run or single part of unit use of using such as applying unit and some blood sampling scenes, rural area and basic unit clinic in enormous quantities such as hospital, blood station, epidemic prevention station, health check-up.Primary dcreening operation etiologic diagnosis for HIV has positive meaning.

Description of drawings

Fig. 1 is the magnetic immuno-chromatographic test paper strip structural representation of joint-detection HIV-1+2 antibody of the present invention and P24 antigen.

For magnetic immuno-chromatographic test paper strip of further specifying joint-detection HIV-1+2 antibody of the present invention and P24 antigen and preparation method thereof, describe especially exemplified by following embodiment, this embodiment is in order to explain rather than limit by any way the present invention.

Embodiment

The magnetic immuno-chromatographic test paper strip of HIV-1+2 antibody and P24 antigen in the joint-detection blood of the present invention, as shown in Figure 1, this test strips is at base plate 1) on interlaced successively 2mm ground paste to go up coated film 2), combine the magnetic mat of particles 3 of HIV-1+2 antigen and P24 antibody), sample pad 4), adsorptive pads 5), and cover transparent plastic diaphragm seal 6 on the upper strata) test strips that assembles, coated film 2) on be coated with HIV-1+2 antibody detection line T1 and HIV P24 Detection of antigen line T2 and nature controlling line C in advance.

In specific embodiment, the HIV1+2 antigen that adopts to for commercialization antigen, HIV P24 antibody is to being the commercialization monoclonal antibody.The principle of utilizing double antigens sandwich to detect HIV antibody and double-antibody sandwich detection P24 antigen detects sample, when containing HIV P24 antigen in the sample to be measured, the antibodies of combination on antigen meeting elder generation and the magnetic particle, carrying out along with the chromatography effect, bond moves forward and arrives P24 antibody sandwich line T2 place, antigen can accumulate in the T2 place with coated antibody in conjunction with forming the double-antibody sandwich compound once more, in like manner, when containing HIV 1+2 antibody in the sample, also can form the double antigens sandwich compound at the antigen coated line T1 of HIV1+2 place and assemble.In addition, can not continue to move ahead when arriving nature controlling line C, thereby biotinylation BSA can combine at C line place with streptavidin mark magnetic particle and occurs the magnetic particle aggregation equally in conjunction with the streptavidin mark magnetic particle of biotinylated antibody or antigen.Entire reaction was carried out in 30 minutes fully, general reaction can be used magnetic immuno-chromatographic instrument Card Reader after 15 minutes, the T1 line, and T2 line and C line all can produce corresponding magnetic signal value, calculate the ratio of T1/C and/or T2/C, get final product the yin and yang attribute of result of determination according to default boundary ratio.Whole Card Reader, calculating, with the process sequencing fully of preset bounds value comparison, magnetism detector can directly provide the yin and yang attribute result.

The preparation method of the magnetic immuno-chromatographic test paper strip of HIV-1+2 antibody and P24 antigen sees following example in the joint-detection blood of the present invention:

Embodiment 1

The magnetic immuno-chromatographic test paper strip of HIV-1+2 antibody and P24 antigen and the preparation method of paper box in the joint-detection blood

The test strips of present embodiment and the preparation method of paper box may further comprise the steps:

The preparation of A, antigen and antibody: select commercial HIV1+2 recombinant antigen and P24 monoclonal antibody for use, to 20mM, the PBS of pH7.2 (pH7.0-7.6 all is suitable for), 4 ℃ of dialysed overnight are standby.

The preparation of B, coated film:

Bag is cushioned the preparation of liquid: the phosphate buffer of 0.02M pH 7.2 (PBS) is cushioned liquid for bag, and the rearmounted 4 ℃ of preservations of 0.22 μ m filtering with microporous membrane degerming are standby, two weeks of the term of validity.

The preparation of confining liquid: contain the phosphate buffer (PBS) of the 0.02M pH7.2 (pH7.0-7.6 all is suitable for) of 0.5%BSA, it is standby that 0.22 μ m filtering with microporous membrane degerming is placed on 4 ℃ of preservations, one week of the term of validity.

The preparation of coated film: being cushioned liquid (PB of 0.02M pH7.2 (pH7.0-7.6 all is suitable for)) with bag is 0.5mg/ml with HIV-1+2 antigen and P24 antibody dilution, biotinylation BSA dilution is 1mg/ml, use quantitative spray film device with the amount of 1 μ l/cm with the three with the even spray printing in the interval of 0.6cm on 3.5cm width nitrocellulose filter, room temperature is dried after 30 minutes in the confining liquid (PBS that contains the 0.02M pH7.2 (pH7.0-7.6 all is suitable for) of 0.5%BSA,) in 25-35 ℃ of oven dry 8 hours, the adding drying agent was sealed up for safekeeping standby after 10 minutes in middle immersion.

The preparation of C, magnetic particle:

The preparation of sodium-acetate buffer: with distilled water and sodium acetate and glacial acetic acid secure ph is 4.7 (pH4.5-5.0 all is suitable for), concentration is the acetate buffer solution of 50mM, adding Tween-20 is that 4 ℃ of preservations are standby after 0.1%, the 0.22 μ m filtering with microporous membrane degerming to final concentration, two weeks of the term of validity.

Boric acid is preserved the preparation of damping fluid: use distilled water, boric acid and borax preparation pH are 8.5 (pH8.2-9.0 all is suitable for), and final concentration is the borate buffer of 50mM, add PVP, Casine, Tween-20, sucrose, final concentration is respectively 1%, 1%, 0.5%, 5%, 0.22 4 ℃ of preservations are standby after the degerming of μ m filtering with microporous membrane, one week of the term of validity.

The preparation of streptavidin magnetic particle: use 50mM pH4.7 (pH4.5-5.0 all is suitable for) the sodium-acetate buffer washing magnetic particle that contains 0.1%Tween-20, adding EDC and NHS makes the two final concentration be 20mmol, room temperature reaction 1 hour, fully adding streptavidin behind the washing magnetic particle, to make the molecule ratio of streptavidin and magnetic particle be 5: 1 (mol ratio), room temperature reaction 3 hours, the PBS that adds the 0.02M pH7.2 (pH7.0-7.6 all is suitable for) that contains 0.5%BSA, room temperature sealing 30 minutes, washing magnetic particle, use contains 1%PVP, 1%Casein, 0.5%Tween-20, the boric acid of the 50mmolpH8.5 of 5% sucrose (pH8.2-9.0 all is suitable for) is preserved damping fluid redissolution magnetic particle, and 4 ℃ of preservations are standby.

The preparation of biotinylation HIV-1+2 antigen and P24 antibody: with HIV-1+2 antigen and P24 antibody 4 ℃ of dialysed overnight of PBS to 0.02MpH7.2 (pH7.0-7.6 all is suitable for), adjustment concentration is 2mg/ml, to activate biotin in advance and use the DMSO dissolving, final concentration is 50mM, the biotin solution that in antigen or antibody-solutions, adds aequum with 20: 1 molecule ratios, room temperature reaction 1 hour, to 4 ℃ of dialysed overnight of PBS of 0.02M pH7.2 (pH7.0-7.6 all is suitable for) ,-20 ℃ of preservations are standby behind the adding equal-volume glycerine.

Biotinylated antigen/antibody mixes with streptavidin magnetic particle: the amount with 0.5 μ l/mg adds biotinylation HIV-1+2 antigen in streptavidin magnetic particle solution, add biotinylation P24 antibody with 0.25 μ l/mg magnetic proportion of particles, fully use the preservation damping fluid mixture diluted fiberglass packing to be sprayed to be used behind the mixing with 1: 5 ratio.

The spraying of D, magnetic particle and freeze-drying

That uses BioDot spray film instrument nozzle specially usedly evenly is sprayed at the magnetic particle handled well the amount with 50 μ l/cm on the 0.8cm width fiberglass packing, the frozen overnight drying, add drying agent seal up for safekeeping standby,

The processing of E, sample pad

1.8cm width sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour.

The sample pad treating fluid is the PBS solution that contains the PVA of 1%-5%Casein and 0.1%-1% and the 0.02M pH7.2 of 0.01-0.2%Tween-20 (pH7.0-7.6 all is suitable for).

The assembling of F, test strips and cutting

Following all operations all must carry out in temperature 20-25 ℃ the room in humidity less than 20%.

The assembling of test paper plate: use as requested that 3.5cm is the wide coated film of BioDot LM5000 type assembling instrument, 2.5cm wide thieving paper, the magnetic mat of particles that 0.8cm is wide, the wide sample pad of 1.8cm are assembled on the 9.8cm width transparent plastic base plate, stick upper strata transparent plastic cover plate, be assembled into test paper plate.

Cutting of test strips: use BioDot CM4000 type cutting cutter that the test paper plate that assembles is cut into the wide finished product test strips of 0.5cm.

The assembling of G, test card

The single part test strips of well cutting of the present invention is placed in the draw-in groove on the plastic bottom card, covers loam cake, use card press machine up and down two plastic clips compress, guarantee that whole test strips is in tensioned state.Adding the drying agent room temperature seals up for safekeeping standby.

H, determine the 2 D code information of this batch

The name of an article: HIV 1+2﹠amp; P24 magnetic detection card

Batch: on the assembling date of test card, form is: Year/Month/Day, XXXX/XX/XX

Determining of yin and yang attribute interpretation standard: get 100 parts and confirm HIV1+2 antibody positive sample (power all has), 50 parts of affirmation HIV P24 antigen positive samples (power all has), 500 parts at random sample use this batch test card to detect, use the magnetism detector testing result, calculate the T1/C value of each test card, the T2/C value, use statistical method computation of mean values and standard deviation, determine: T1/C＜0.1 and T2/C＜0.05 is negative.T1/C＞0.2, or T2/C＞0.1 is positive, is gray area between the two.

The printing of I, two-dimension code is pasted

With in the above-mentioned 2 D code information input two-dimension code printer and print, two-dimension code is pasted on the ad-hoc location of test card, use two-dimension code paste position detecting device to inspect 2% at random by random samples and guarantee that two-dimension code pastes errorless.

J, finished product packing

The single part test card and that the posts two-dimension code drying prescription of being responsible for a task until it is completed is sealed in the aluminium foil bag, 100 person-portions are that a packing places in the packing box, and a instructions of a box and 1 bottle of 10ml dress chromatography damping fluid are promptly made paper box, this paper box keeps in Dark Place in room temperature, and the shelf-life is 18 months.The chromatography buffer formulation is: 1%Tween-20, and 0.5%Triton X-100,1%NP-40,0.05%NaN3, the PBS of 20mmol pH7.2 (pH7.0-7.6 all is suitable for).

Embodiment 2

Except; In the step of the preparation of streptavidin magnetic particle: it is 1: 2 that streptavidin makes streptavidin and magnetic proportion of particles.Other step is with embodiment 1,

Embodiment 3

In the step except the preparation of streptavidin magnetic particle: it is 1: 10 that streptavidin makes streptavidin and magnetic proportion of particles.Other step is with embodiment 1.

Embodiment 4

In the blend step except biotinylated antigen/antibody and streptavidin magnetic particle: fully use the preservation damping fluid with 1: 10 ratio mixture diluted fiberglass packing to be sprayed to be used behind the mixing, other step is with embodiment 1.

Embodiment 5

In the blend step except biotinylated antigen/antibody and streptavidin magnetic particle: fully use the preservation damping fluid with 1: 20 ratio mixture diluted fiberglass packing to be sprayed to be used behind the mixing, other step is with embodiment 1.

Embodiment 6

The using method of test card of the present invention

1, application of sample

From packing box, take out single part test card, tear the aluminium foil strip packing, test card is placed on the smooth desktop, get 50 μ l sample serum with micropipettor and add in the well on the card, add 50 μ l chromatography damping fluids again, wait question response to carry out 15 minutes.

2, measurement and result output

The MICT detector is started shooting in advance, test card is inserted the card inserting mouth of detector, the operation instrument, instrument can read the 2 D code information on the card automatically and measure, and prints measurement result immediately, and the yin and yang attribute result can show in print result.

Embodiment 7

Test card of the present invention detects clinical samples and detects and testing result

Table 1: normal person's detection result of specimen

The sample numbering	The T1/C value	The T2/C value	This method result of determination	The collaurum result of determination	The sample numbering	The T1/C value	The T2/C value	This method result of determination	The collaurum result of determination
										1	0.018	0.049	N	N	427	0.086	0.011	N	N
2	0.004	0.028	N	N	428	0.078	0.024	N	N
										3	0.048	0.017	N	N	429	0.040	0.027	N	N
4	0.023	0.031	N	N	430	0.088	0.036	N	N
										5	0.028	0.027	N	N	431	0.020	0.028	N	N
6	0.025	0.009	N	N	432	0.063	0.047	N	N
										7	0.029	0.025	N	N	433	0.084	0.004	N	N
8	0.058	0.047	N	N	434	0.039	0.039	N	N
										9	0.057	0.040	N	N	435	0.067	0.020	N	N
10	0.062	0.029	N	N	436	0.072	0.009	N	N
										11	0.089	0.044	N	N	437	0.008	0.039	N	N
12	0.005	0.004	N	N	438	0.070	0.036	N	N
										13	0.018	0.047	N	N	439	0.055	0.015	N	N
14	0.022	0.043	N	N	440	0.043	0.015	N	N
										15	0.024	0.039	N	N	441	0.005	0.042	N	N
16	0.009	0.000	N	N	442	0.052	0.026	N	N
										17	0.044	0.025	N	N	443	0.082	0.009	N	N
18	0.012	0.035	N	N	444	0.012	0.048	N	N
										19	0.064	0.048	N	N	445	0.018	0.001	N	N
20	0.055	0.039	N	N	446	0.023	0.040	N	N
										21	0.005	0.017	N	N	447	0.056	0.039	N	N
22	0.003	0.000	N	N	448	0.048	0.048	N	N
										23	0.049	0.025	N	N	449	0.040	0.033	N	N
24	0.019	0.024	N	N	450	0.035	0.022	N	N
										25	0.041	0.025	N	N	451	0.053	0.026	N	N
26	0.021	0.032	N	N	452	0.003	0.017	N	N
										27	0.049	0.023	N	N	453	0.019	0.020	N	N
28	0.071	0.023	N	N	454	0.068	0.012	N	N
										29	0.073	0.024	N	N	455	0.034	0.018	N	N
30	0.070	0.036	N	N	456	0.006	0.017	N	N
										31	0.067	0.026	N	N	457	0.046	0.039	N	N
32	0.017	0.032	N	N	458	0.085	0.044	N	N
										33	0.089	0.009	N	N	459	0.052	0.018	N	N
34	0.084	0.048	N	N	460	0.046	0.045	N	N
										35	0.021	0.022	N	N	461	0.024	0.030	N	N

36	0.000	0.003	N	N	462	0.008	0.024	N	N
										37	0.030	0.035	N	N	463	0.054	0.025	N	N
38	0.068	0.020	N	N	464	0.002	0.018	N	N
										39	0.053	0.005	N	N	465	0.050	0.046	N	N
40	0.063	0.020	N	N	466	0.087	0.049	N	P
										41	0.086	0.043	N	N	467	0.007	0.039	N	N
42	0.072	0.045	N	N	468	0.040	0.003	N	N
										43	0.039	0.010	N	N	469	0.076	0.028	N	N
44	0.041	0.036	N	N	470	0.088	0.010	N	N
										45	0.010	0.032	N	N	471	0.015	0.024	N	N
46	0.001	0.008	N	N	472	0.015	0.008	N	N
										47	0.085	0.009	N	N	473	0.062	0.045	N	N
48	0.052	0.046	N	N	474	0.083	0.012	N	N
										49	0.045	0.013	N	N	475	0.088	0.036	N	N
50	0.086	0.047	N	N	476	0.073	0.004	N	N
										51	0.030	0.002	N	N	477	0.053	0.010	N	N
52	0.019	0.041	N	N	478	0.042	0.004	N	N
										53	0.004	0.039	N	N	479	0.088	0.008	N	N
54	0.020	0.042	N	N	480	0.034	0.021	N	N
										55	0.031	0.007	N	N	481	0.053	0.032	N	N
56	0.068	0.012	N	N	482	0.013	0.019	N	N
										57	0.085	0.026	N	N	483	0.060	0.029	N	N
58	0.074	0.014	N	N	484	0.009	0.004	N	N
										59	0.027	0.010	N	N	485	0.033	0.022	N	N
60	0.090	0.037	N	N	486	0.059	0.011	N	N
										61	0.015	0.024	N	N	487	0.066	0.043	N	N
62	0.026	0.012	N	N	488	0.007	0.039	N	N
										63	0.033	0.036	N	N	489	0.023	0.015	N	N
64	0.037	0.031	N	N	490	0.083	0.044	N	N
										65	0.033	0.039	N	N	491	0.076	0.035	N	N
66	0.052	0.032	N	N	492	0.059	0.036	N	N
										67	0.027	0.028	N	N	493	0.035	0.023	N	N
68	0.002	0.037	N	N	494	0.088	0.009	N	N
										69	0.062	0.016	N	N	495	0.046	0.045	N	N
70	0.055	0.000	N	N	496	0.078	0.013	N	N
										71	0.060	0.034	N	N	497	0.073	0.020	N	N
72	0.006	0.011	N	N	498	0.074	0.047	N	N
										73	0.021	0.022	N	N	499	0.020	0.022	N	N
74	0.056	0.046	N	N	500	0.015	0.009	N	N
										75	0.019	0.019	N	N	501	0.076	0.043	N	N
76	0.052	0.024	N	N	502	0.021	0.019	N	N
										77	0.024	0.007	N	N	503	0.070	0.017	N	N
78	0.042	0.028	N	N	504	0.027	0.044	N	N
										79	0.067	0.018	N	N	505	0.025	0.046	N	N

80	0.053	0.047	N	N	506	0.023	0.002	N	N
										81	0.071	0.013	N	N	507	0.084	0.047	N	P
82	0.065	0.011	N	N	508	0.014	0.041	N	N
										83	0.012	0.030	N	N	509	0.026	0.047	N	N
84	0.011	0.004	N	N	510	0.022	0.007	N	N
										85	0.010	0.025	N	N	511	0.049	0.002	N	N
86	0.056	0.038	N	N	512	0.079	0.018	N	N
										87	0.008	0.048	N	N	513	0.018	0.000	N	N
88	0.033	0.029	N	N	514	0.017	0.033	N	N
										89	0.052	0.033	N	N	515	0.083	0.019	N	N
90	0.038	0.029	N	N	516	0.046	0.036	N	N
										91	0.013	0.036	N	N	517	0.004	0.016	N	N
92	0.021	0.013	N	N	518	0.071	0.048	N	N
										93	0.074	0.041	N	N	519	0.002	0.038	N	N
94	0.009	0.025	N	N	520	0.023	0.021	N	N
										95	0.040	0.033	N	N	521	0.048	0.004	N	N
96	0.037	0.006	N	N	522	0.019	0.035	N	N
										97	0.020	0.004	N	N	523	0.014	0.022	N	N
98	0.022	0.008	N	N	524	0.085	0.018	N	N
										99	0.067	0.001	N	N	525	0.015	0.033	N	N
100	0.067	0.033	N	N	526	0.032	0.002	N	N
										101	0.029	0.046	N	N	527	0.063	0.009	N	N
102	0.007	0.044	N	N	528	0.073	0.025	N	N
										103	0.066	0.046	N	N	529	0.070	0.004	N	N
104	0.018	0.039	N	N	530	0.070	0.003	N	N
										105	0.005	0.033	N	N	531	0.055	0.038	N	N
106	0.068	0.043	N	N	532	0.033	0.043	N	N
										107	0.086	0.007	N	N	533	0.011	0.013	N	N
108	0.008	0.009	N	N	534	0.036	0.000	N	N
										109	0.067	0.034	N	N	535	0.020	0.042	N	N
110	0.044	0.048	N	N	536	0.029	0.031	N	N
										111	0.026	0.047	N	N	537	0.030	0.005	N	N
112	0.046	0.034	N	N	538	0.021	0.016	N	N
										113	0.032	0.038	N	N	539	0.059	0.043	N	N
114	0.076	0.046	N	N	540	0.006	0.046	N	N
										115	0.056	0.045	N	N	541	0.008	0.029	N	N
116	0.037	0.033	N	N	542	0.074	0.043	N	N
										117	0.027	0.033	N	N	543	0.086	0.002	N	N
118	0.056	0.041	N	N	544	0.042	0.005	N	N
										119	0.014	0.028	N	N	545	0.058	0.018	N	N
120	0.012	0.010	N	N	546	0.067	0.009	N	N
										121	0.072	0.022	N	N	547	0.087	0.018	N	N
122	0.004	0.040	N	N	548	0.062	0.043	N	N
										123	0.086	0.029	N	N	549	0.003	0.036	N	N

124	0.028	0.048	N	N	550	0.023	0.007	N	N
										125	0.002	0.044	N	N	551	0.055	0.016	N	N
126	0.010	0.027	N	N	552	0.060	0.010	N	N
										127	0.010	0.025	N	N	553	0.022	0.000	N	N
128	0.000	0.006	N	N	554	0.026	0.014	N	N
										129	0.055	0.002	N	N	555	0.043	0.038	N	N
130	0.012	0.021	N	N	556	0.080	0.042	N	N
										131	0.005	0.048	N	N	557	0.078	0.045	N	N
132	0.050	0.020	N	N	558	0.067	0.036	N	N
										133	0.008	0.003	N	N	559	0.058	0.045	N	N
134	0.058	0.035	N	N	560	0.039	0.012	N	N
										135	0.088	0.021	N	N	561	0.034	0.044	N	N
136	0.026	0.027	N	N	562	0.024	0.024	N	N
										137	0.014	0.011	N	N	563	0.079	0.023	N	N
138	0.079	0.039	N	N	564	0.013	0.021	N	N
										139	0.047	0.014	N	N	565	0.084	0.033	N	N
140	0.045	0.018	N	N	566	0.059	0.040	N	N
										141	0.042	0.008	N	N	567	0.045	0.047	N	N
142	0.003	0.025	N	N	568	0.023	0.039	N	N
										143	0.015	0.031	N	N	569	0.003	0.025	N	N
144	0.056	0.044	N	N	570	0.010	0.035	N	N
										145	0.057	0.009	N	N	571	0.000	0.020	N	N
146	0.029	0.015	N	N	572	0.063	0.012	N	N
										147	0.030	0.011	N	N	573	0.056	0.036	N	N
148	0.071	0.007	N	N	574	0.021	0.006	N	N
										149	0.030	0.025	N	N	575	0.045	0.019	N	N
150	0.016	0.023	N	N	576	0.066	0.002	N	N
										151	0.050	0.022	N	N	577	0.037	0.019	N	N
152	0.080	0.047	N	N	578	0.028	0.029	N	N
										153	0.060	0.018	N	N	579	0.013	0.029	N	N
154	0.025	0.001	N	N	580	0.082	0.006	N	N
										155	0.059	0.038	N	N	581	0.075	0.019	N	N
156	0.075	0.018	N	N	582	0.073	0.006	N	N
										157	0.086	0.043	N	N	583	0.017	0.027	N	N
158	0.081	0.034	N	N	584	0.008	0.020	N	N
										159	0.084	0.019	N	N	585	0.021	0.033	N	N
160	0.065	0.012	N	N	586	0.023	0.023	N	N
										161	0.046	0.029	N	N	587	0.060	0.026	N	N
162	0.024	0.044	N	N	588	0.087	0.036	N	N
										163	0.054	0.006	N	N	589	0.020	0.013	N	N
164	0.068	0.023	N	N	590	0.017	0.025	N	N
										165	0.009	0.000	N	N	591	0.016	0.027	N	N
166	0.011	0.047	N	N	592	0.045	0.000	N	N
										167	0.078	0.046	N	N	593	0.070	0.038	N	N

168	0.046	0.020	N	N	594	0.062	0.026	N	N
										169	0.036	0.028	N	N	595	0.041	0.029	N	N
170	0.027	0.016	N	N	596	0.033	0.041	N	N
										171	0.070	0.001	N	N	597	0.004	0.022	N	N
172	0.070	0.018	N	N	598	0.009	0.035	N	N
										173	0.053	0.041	N	N	599	0.048	0.008	N	N
174	0.030	0.046	N	N	600	0.029	0.045	N	N
										175	0.041	0.007	N	N	601	0.030	0.033	N	N
176	0.021	0.025	N	N	602	0.084	0.020	N	N
										177	0.062	0.007	N	N	603	0.039	0.034	N	N
178	0.025	0.043	N	N	604	0.051	0.045	N	N
										179	0.014	0.003	N	N	605	0.077	0.043	N	N
180	0.014	0.035	N	N	606	0.061	0.030	N	N
										181	0.031	0.047	N	N	607	0.079	0.044	N	N
182	0.049	0.049	N	N	608	0.065	0.025	N	N
										183	0.005	0.023	N	N	609	0.027	0.006	N	N
184	0.010	0.049	N	N	610	0.039	0.040	N	N
										185	0.075	0.028	N	N	611	0.034	0.004	N	N
186	0.028	0.003	N	N	612	0.000	0.010	N	N
										187	0.079	0.048	N	N	613	0.041	0.034	N	N
188	0.024	0.030	N	N	614	0.078	0.035	N	N
										189	0.025	0.038	N	N	615	0.086	0.024	N	N
190	0.063	0.038	N	N	616	0.049	0.030	N	N
										191	0.007	0.040	N	N	617	0.015	0.030	N	N
192	0.014	0.011	N	N	618	0.071	0.047	N	N
										193	0.073	0.020	N	N	619	0.078	0.018	N	N
194	0.050	0.031	N	N	620	0.076	0.046	N	N
										195	0.061	0.030	N	N	621	0.020	0.044	N	N
196	0.067	0.043	N	N	622	0.049	0.001	N	N
										197	0.023	0.009	N	N	623	0.027	0.028	N	N
198	0.074	0.014	N	N	624	0.012	0.043	N	N
										199	0.002	0.031	N	N	625	0.033	0.043	N	N
200	0.006	0.020	N	N	626	0.025	0.014	N	N
										201	0.006	0.007	N	N	627	0.051	0.041	N	N
202	0.090	0.034	N	N	628	0.015	0.002	N	N
										203	0.032	0.029	N	N	629	0.089	0.035	N	N
204	0.036	0.034	N	N	630	0.018	0.048	N	N
										205	0.012	0.034	N	N	631	0.045	0.017	N	N
206	0.030	0.006	N	N	632	0.048	0.038	N	N
										207	0.036	0.020	N	N	633	0.049	0.037	N	N
208	0.082	0.019	N	N	634	0.050	0.021	N	N
										209	0.054	0.009	N	N	635	0.083	0.038	N	N
210	0.042	0.014	N	N	636	0.083	0.017	N	N
										211	0.036	0.016	N	N	637	0.015	0.043	N	N

212	0.006	0.034	N	N	638	0.037	0.048	N	N
										213	0.057	0.048	N	N	639	0.072	0.027	N	N
214	0.069	0.025	N	N	640	0.054	0.012	N	N
										215	0.089	0.004	N	N	641	0.002	0.011	N	N
216	0.077	0.004	N	N	642	0.065	0.025	N	N
										217	0.030	0.047	N	N	643	0.064	0.027	N	N
218	0.038	0.018	N	N	644	0.024	0.004	N	N
										219	0.005	0.011	N	N	645	0.073	0.048	N	N
220	0.032	0.025	N	N	646	0.077	0.014	N	N
										221	0.061	0.027	N	N	647	0.001	0.005	N	N
222	0.039	0.033	N	N	648	0.035	0.032	N	N
										223	0.036	0.001	N	N	649	0.086	0.023	N	N
224	0.029	0.042	N	N	650	0.075	0.039	N	N
										225	0.080	0.008	N	N	651	0.047	0.029	N	N
226	0.032	0.008	N	N	652	0.020	0.002	N	N
										227	0.071	0.028	N	N	653	0.009	0.017	N	N
228	0.035	0.035	N	N	654	0.008	0.013	N	N
										229	0.017	0.025	N	N	655	0.079	0.006	N	N
230	0.015	0.028	N	N	656	0.062	0.007	N	N
										231	0.019	0.043	N	N	657	0.072	0.022	N	N
232	0.044	0.000	N	N	658	0.044	0.034	N	N
										233	0.077	0.040	N	N	659	0.004	0.048	N	N
234	0.000	0.037	N	N	660	0.052	0.017	N	N
										235	0.002	0.016	N	N	661	0.019	0.034	N	N
236	0.057	0.047	N	N	662	0.038	0.037	N	N
										237	0.090	0.024	N	N	663	0.078	0.006	N	N
238	0.068	0.017	N	N	664	0.004	0.043	N	N
										239	0.087	0.033	N	N	665	0.016	0.000	N	N
240	0.004	0.000	N	N	666	0.038	0.009	N	N
										241	0.076	0.007	N	N	667	0.050	0.043	N	N
242	0.040	0.001	N	N	668	0.083	0.039	N	N
										243	0.035	0.002	N	N	669	0.057	0.022	N	N
244	0.037	0.047	N	N	670	0.002	0.012	N	N
										245	0.048	0.046	N	N	671	0.027	0.007	N	N
246	0.086	0.005	N	N	672	0.041	0.014	N	N
										247	0.006	0.048	N	N	673	0.088	0.012	N	N
248	0.039	0.004	N	N	674	0.032	0.005	N	N
										249	0.024	0.041	N	N	675	0.051	0.030	N	N
250	0.030	0.010	N	N	676	0.029	0.010	N	N
										251	0.023	0.006	N	N	677	0.013	0.026	N	N
252	0.024	0.022	N	N	678	0.079	0.012	N	N
										253	0.060	0.011	N	N	679	0.030	0.006	N	N
254	0.073	0.020	N	N	680	0.054	0.042	N	N
										255	0.025	0.033	N	N	681	0.056	0.030	N	N

256	0.056	0.011	N	N	682	0.061	0.013	N	N
										257	0.007	0.031	N	N	683	0.061	0.044	N	N
258	0.085	0.030	N	N	684	0.067	0.008	N	N
										259	0.008	0.011	N	N	685	0.072	0.015	N	N
260	0.026	0.005	N	N	686	0.048	0.006	N	N
										261	0.037	0.017	N	N	687	0.077	0.011	N	N
262	0.065	0.038	N	N	688	0.042	0.030	N	N
										263	0.039	0.014	N	N	689	0.019	0.037	N	N
264	0.019	0.041	N	N	690	0.049	0.007	N	N
										265	0.064	0.035	N	N	691	0.031	0.043	N	N
266	0.062	0.023	N	N	692	0.070	0.018	N	N
										267	0.064	0.006	N	N	693	0.043	0.011	N	N
268	0.002	0.004	N	N	694	0.046	0.010	N	N
										269	0.004	0.014	N	N	695	0.069	0.008	N	N
270	0.065	0.047	N	N	696	0.039	0.008	N	N
										271	0.074	0.031	N	N	697	0.039	0.011	N	N
272	0.057	0.033	N	N	698	0.038	0.047	N	N
										273	0.084	0.041	N	N	699	0.038	0.016	N	N
274	0.024	0.000	N	N	700	0.046	0.046	N	N
										275	0.060	0.040	N	N	701	0.040	0.019	N	N
276	0.006	0.028	N	N	702	0.004	0.026	N	N
										277	0.020	0.044	N	N	703	0.019	0.005	N	N
278	0.043	0.031	N	N	704	0.031	0.034	N	N
										279	0.066	0.035	N	N	705	0.070	0.048	N	N
280	0.024	0.025	N	N	706	0.077	0.025	N	N
										281	0.036	0.006	N	N	707	0.007	0.003	N	N
282	0.089	0.016	N	N	708	0.038	0.019	N	N
										283	0.031	0.040	N	N	709	0.030	0.018	N	N
284	0.072	0.005	N	N	710	0.002	0.009	N	N
										285	0.085	0.046	N	N	711	0.070	0.009	N	N
286	0.080	0.047	N	N	712	0.073	0.011	N	N
										287	0.009	0.024	N	N	713	0.020	0.025	N	N
288	0.030	0.019	N	N	714	0.034	0.013	N	N
										289	0.045	0.023	N	N	715	0.076	0.047	N	N
290	0.065	0.019	N	N	716	0.036	0.030	N	N
										291	0.078	0.026	N	N	717	0.012	0.025	N	N
292	0.017	0.028	N	N	718	0.073	0.038	N	N
										293	0.069	0.001	N	N	719	0.057	0.020	N	N
294	0.030	0.049	N	N	720	0.013	0.009	N	N
										295	0.004	0.044	N	N	721	0.053	0.038	N	N
296	0.032	0.031	N	N	722	0.066	0.044	N	N
										297	0.004	0.042	N	N	723	0.002	0.012	N	N
298	0.026	0.025	N	N	724	0.057	0.046	N	N
										299	0.074	0.039	N	N	725	0.013	0.036	N	N

300	0.057	0.027	N	N	726	0.019	0.035	N	N
										301	0.035	0.032	N	N	727	0.003	0.035	N	N
302	0.056	0.001	N	N	728	0.006	0.036	N	N
										303	0.004	0.040	N	N	729	0.069	0.039	N	N
304	0.073	0.005	N	N	730	0.086	0.044	N	N
										305	0.077	0.024	N	N	731	0.030	0.034	N	N
306	0.080	0.014	N	N	732	0.004	0.035	N	N
										307	0.078	0.024	N	N	733	0.082	0.006	N	N
308	0.004	0.012	N	N	734	0.026	0.027	N	N
										309	0.014	0.040	N	N	735	0.084	0.048	N	N
310	0.013	0.047	N	N	736	0.058	0.027	N	N
										311	0.041	0.006	N	N	737	0.063	0.008	N	N
312	0.037	0.023	N	N	738	0.064	0.020	N	N
										313	0.086	0.022	N	N	739	0.056	0.028	N	N
314	0.053	0.010	N	N	740	0.065	0.040	N	N
										315	0.038	0.015	N	N	741	0.062	0.002	N	N
316	0.021	0.005	N	N	742	0.005	0.033	N	N
										317	0.051	0.013	N	N	743	0.023	0.017	N	N
318	0.041	0.001	N	N	744	0.016	0.013	N	N
										319	0.071	0.035	N	N	745	0.054	0.025	N	N
320	0.031	0.006	N	N	746	0.082	0.000	N	N
										321	0.035	0.049	N	N	747	0.073	0.008	N	N
322	0.008	0.025	N	N	748	0.067	0.020	N	N
										323	0.040	0.004	N	N	749	0.084	0.021	N	N
324	0.079	0.027	N	N	750	0.018	0.038	N	N
										325	0.022	0.030	N	N	751	0.050	0.032	N	N
326	0.028	0.041	N	N	752	0.062	0.016	N	N
										327	0.083	0.039	N	N	753	0.081	0.029	N	N
328	0.023	0.033	N	N	754	0.058	0.040	N	N
										329	0.075	0.040	N	N	755	0.031	0.009	N	N
330	0.045	0.031	N	N	756	0.048	0.001	N	N
										331	0.085	0.037	N	N	757	0.021	0.014	N	N
332	0.044	0.029	N	N	758	0.048	0.043	N	N
										333	0.020	0.017	N	N	759	0.055	0.006	N	N
334	0.060	0.007	N	N	760	0.082	0.039	N	N
										335	0.045	0.039	N	N	761	0.043	0.047	N	N
336	0.018	0.006	N	N	762	0.088	0.001	N	N
										337	0.050	0.005	N	N	763	0.080	0.030	N	N
338	0.057	0.017	N	N	764	0.064	0.038	N	N
										339	0.003	0.042	N	N	765	0.020	0.046	N	N
340	0.054	0.017	N	N	766	0.025	0.021	N	N
										341	0.038	0.003	N	N	767	0.044	0.013	N	N
342	0.044	0.027	N	N	768	0.057	0.031	N	N
										343	0.088	0.001	N	N	769	0.011	0.044	N	N

344	0.059	0.045	N	N	770	0.007	0.046	N	N
										345	0.056	0.049	N	N	771	0.055	0.008	N	N
346	0.031	0.028	N	N	772	0.084	0.000	N	N
										347	0.047	0.044	N	N	773	0.050	0.036	N	N
348	0.044	0.018	N	N	774	0.017	0.032	N	N
										349	0.043	0.025	N	N	775	0.087	0.036	N	N
350	0.039	0.021	N	N	776	0.090	0.029	N	N
										351	0.074	0.039	N	N	777	0.087	0.023	N	N
352	0.055	0.040	N	N	778	0.057	0.018	N	N
										353	0.047	0.026	N	N	779	0.048	0.035	N	N
354	0.033	0.035	N	N	780	0.051	0.028	N	N
										355	0.064	0.021	N	N	781	0.030	0.027	N	N
356	0.062	0.016	N	N	782	0.070	0.015	N	N
										357	0.045	0.044	N	N	783	0.033	0.016	N	N
358	0.056	0.024	N	N	784	0.057	0.009	N	N
										359	0.073	0.044	N	N	785	0.030	0.032	N	N
360	0.076	0.030	N	N	786	0.011	0.003	N	N
										361	0.089	0.020	N	N	787	0.053	0.033	N	N
362	0.027	0.005	N	N	788	0.056	0.049	N	N
										363	0.034	0.010	N	N	789	0.049	0.006	N	N
364	0.032	0.038	N	N	790	0.066	0.032	N	N
										365	0.058	0.018	N	N	791	0.085	0.012	N	N
366	0.042	0.038	N	N	792	0.009	0.045	N	N
										367	0.063	0.016	N	N	793	0.064	0.001	N	N
368	0.068	0.048	N	N	794	0.016	0.004	N	N
										369	0.023	0.014	N	N	795	0.055	0.015	N	N
370	0.067	0.016	N	N	796	0.019	0.027	N	N
										371	0.162	0.017	Gray area	P	797	0.034	0.039	N	N
372	0.063	0.035	N	N	798	0.013	0.032	N	N
										373	0.047	0.017	N	N	799	0.089	0.037	N	N
374	0.010	0.031	N	N	800	0.079	0.039	N	N
										375	0.020	0.000	N	N	801	0.078	0.036	N	N
376	0.005	0.031	N	N	802	0.062	0.007	N	N
										377	0.054	0.018	N	N	803	0.007	0.004	N	N
378	0.035	0.028	N	N	804	0.008	0.048	N	N
										379	0.004	0.018	N	N	805	0.001	0.020	N	N
380	0.036	0.030	N	N	806	0.058	0.044	N	N
										381	0.051	0.026	N	N	807	0.028	0.046	N	N
382	0.023	0.023	N	N	808	0.009	0.001	N	N
										383	0.082	0.037	N	N	809	0.013	0.020	N	N
384	0.009	0.003	N	N	810	0.053	0.047	N	N
										385	0.004	0.034	N	N	811	0.085	0.010	N	N
386	0.042	0.023	N	N	812	0.080	0.020	N	N
										387	0.035	0.043	N	N	813	0.082	0.038	N	N

388	0.077	0.042	N	N	814	0.008	0.038	N	N
										389	0.053	0.048	N	N	815	0.034	0.033	N	N
390	0.029	0.007	N	N	816	0.049	0.036	N	N
										391	0.000	0.008	N	N	817	0.042	0.001	N	N
392	0.002	0.024	N	N	818	0.031	0.004	N	N
										393	0.014	0.031	N	N	819	0.080	0.035	N	N
394	0.067	0.016	N	N	820	0.055	0.011	N	N
										395	0.086	0.024	N	N	821	0.060	0.031	N	N
396	0.061	0.027	N	N	822	0.015	0.028	N	N
										397	0.045	0.045	N	N	823	0.072	0.035	N	N
398	0.028	0.028	N	N	824	0.124	0.044	Gray area	P
										399	0.058	0.024	N	N	825	0.023	0.041	N	N
400	0.034	0.001	N	N	826	0.040	0.020	N	N
										401	0.001	0.018	N	N	827	0.090	0.039	N	N
402	0.075	0.002	N	N	828	0.043	0.001	N	N
										403	0.033	0.048	N	N	829	0.054	0.033	N	N
404	0.029	0.040	N	N	830	0.084	0.033	N	N
										405	0.090	0.015	N	N	831	0.068	0.011	N	N
406	0.084	0.043	N	N	832	0.073	0.047	N	N
										407	0.004	0.030	N	N	833	0.046	0.021	N	N
408	0.065	0.033	N	N	834	0.058	0.041	N	N
										409	0.036	0.046	N	N	835	0.072	0.031	N	N
410	0.035	0.026	N	N	836	0.078	0.012	N	N
										411	0.037	0.027	N	N	837	0.027	0.021	N	N
412	0.074	0.035	N	N	838	0.041	0.025	N	N
										413	0.077	0.009	N	N	839	0.042	0.017	N	N
414	0.046	0.013	N	N	840	0.062	0.047	N	N
										415	0.089	0.021	N	N	841	0.081	0.027	N	N
416	0.080	0.032	N	N	842	0.067	0.004	N	N
										417	0.016	0.010	N	N	843	0.007	0.017	N	N
418	0.082	0.019	N	N	844	0.013	0.017	N	N
										419	0.072	0.032	N	N	845	0.033	0.008	N	N
420	0.024	0.044	N	N	846	0.062	0.032	N	N
										421	0.045	0.044	N	N	847	0.002	0.047	N	N
422	0.070	0.038	N	N	848	0.034	0.039	N	N
										423	0.081	0.003	N	N	849	0.027	0.007	N	N
424	0.073	0.002	N	N	850	0.047	0.047	N	N
										425	0.015	0.005	N	N	851	0.085	0.025	N	P
426	0.071	0.048	N	N	852	0.013	0.027	N	N
															853	0.033	0.008	N	N

N: feminine gender

Table 2:HIV-1+2 antibody positive detection result of specimen

The sample numbering	The T1/C value	The T2/C value	This method result of determination	The sample numbering	The T1/C value	The T2/C value	This method result of determination
								1	1.894	0.021	P	157	0.818	0.057	P
2	0.948	0.040	P	158	1.402	0.033	P
								3	0.666	0.034	P	159	0.031	0.036	P
4	0.054	0.059	P	160	0.844	0.085	P
								5	1.339	0.049	P	161	0.367	0.042	P
6	1.125	0.006	P	162	0.947	0.000	P
								7	1.839	0.018	P	163	1.137	0.054	P
8	0.112	0.049	P	164	0.663	0.010	P
								9	1.201	0.040	P	165	0.423	0.020	P
10	0.888	0.054	P	166	0.430	0.006	P
								11	0.701	0.073	P	167	1.721	0.044	P
12	1.523	0.011	P	168	0.073	0.016	P
								13	1.787	0.076	P	169	1.998	0.055	P
14	1.884	0.044	P	170	1.082	0.065	P
								15	1.899	0.061	P	171	1.721	0.060	P
16	1.229	0.099	P	172	0.546	0.039	P
								17	1.095	0.001	P	173	0.952	0.042	P
18	1.699	0.089	P	174	0.975	0.095	P
								19	0.675	0.082	P	175	1.076	0.037	P
20	0.543	0.043	P	176	0.604	0.098	P
								21	0.742	0.055	P	177	0.930	0.080	P
22	0.978	0.029	P	178	0.375	0.056	P
								23	0.874	0.024	P	179	1.697	0.038	P
24	0.290	0.031	P	180	0.011	0.061	P
								25	0.700	0.036	P	181	0.075	0.009	P
26	1.628	0.024	P	182	1.521	0.096	P
								27	0.493	0.026	P	183	0.530	0.002	P
28	0.118	0.078	P	184	0.725	0.077	P
								29	0.779	0.019	P	185	0.212	0.059	P
30	0.310	0.057	P	186	0.733	0.081	P
								31	1.468	0.073	P	187	0.426	0.018	P
32	0.221	0.000	P	188	1.470	0.093	P
								33	1.385	0.083	P	189	0.400	0.062	P
34	1.692	0.038	P	190	1.628	0.054	P
								35	0.913	0.096	P	191	1.408	0.045	P

36	1.487	0.055	P	192	1.020	0.086	P
								37	1.775	0.029	P	193	1.907	0.065	P
38	1.475	0.006	P	194	0.239	0.066	P
								39	0.385	0.009	P	195	1.219	0.052	P
40	1.634	0.022	P	196	1.693	0.064	P
								41	1.376	0.027	P	197	0.581	0.029	P
42	1.113	0.098	P	198	0.178	0.039	P
								43	1.240	0.015	P	199	0.329	0.008	P
44	1.250	0.035	P	200	1.149	0.076	P
								45	1.809	0.065	P	201	1.732	0.021	P
46	1.547	0.035	P	202	1.557	0.030	P
								47	0.310	0.015	P	203	1.455	0.063	P
48	1.599	0.042	P	204	0.400	0.020	P
								49	0.237	0.023	P	205	1.810	0.098	P
50	0.476	0.052	P	206	0.135	0.040	P
								51	1.561	0.054	P	207	0.432	0.071	P
52	1.549	0.080	P	208	0.877	0.033	P
								53	1.554	0.088	P	209	0.609	0.049	P
54	1.845	0.051	P	210	0.897	0.030	P
								55	1.799	0.058	P	211	0.604	0.041	P
56	0.364	0.082	P	212	1.090	0.015	P
								57	0.419	0.012	P	213	1.405	0.034	P
58	1.708	0.012	P	214	1.820	0.068	P
								59	1.221	0.015	P	215	1.932	0.067	P
60	0.656	0.009	P	216	0.575	0.083	P
								61	0.866	0.050	P	217	0.310	0.032	P
62	0.842	0.059	P	218	1.483	0.075	P
								63	0.268	0.094	P	219	1.947	0.035	P
64	1.697	0.041	P	220	1.806	0.035	P
								65	0.461	0.053	P	221	1.330	0.019	P
66	0.537	0.065	P	222	1.148	0.095	P
								67	1.556	0.085	P	223	0.169	0.034	P
68	0.423	0.048	P	224	1.519	0.079	P
								69	0.487	0.078	P	225	1.488	0.003	P
70	1.998	0.054	P	226	0.656	0.094	P
								71	1.411	0.035	P	227	1.949	0.068	P
72	0.210	0.008	P	228	0.061	0.001	P
								73	0.949	0.082	P	229	0.767	0.008	P
74	1.627	0.048	P	230	1.214	0.081	P
								75	0.959	0.053	P	231	0.260	0.040	P
76	1.277	0.062	P	232	0.399	0.003	P
								77	0.116	0.074	P	233	0.422	0.018	P
78	0.964	0.027	P	234	1.558	0.000	P
								79	0.478	0.052	P	235	1.991	0.077	P

80	0.426	0.022	P	236	0.916	0.062	P
								81	1.664	0.042	P	237	1.496	0.090	P
82	0.347	0.046	P	238	1.806	0.078	P
								83	1.206	0.074	P	239	1.446	0.068	P
84	0.539	0.002	P	240	1.465	0.039	P
								85	0.762	0.033	P	241	1.967	0.069	P
86	0.973	0.015	P	242	0.154	0.009	P
								87	0.246	0.031	P	243	0.994	0.084	P
88	0.804	0.064	P	244	0.169	0.017	P
								89	0.392	0.004	P	245	1.078	0.096	P
90	0.450	0.077	P	246	0.672	0.063	P
								91	1.533	0.045	P	247	1.348	0.089	P
92	1.223	0.061	P	248	1.301	0.052	P
								93	1.274	0.054	P	249	1.547	0.094	P
94	1.421	0.075	P	250	0.604	0.058	P
								95	0.945	0.051	P	251	1.490	0.062	P
96	0.344	0.096	P	252	0.368	0.091	P
								97	0.830	0.029	P	253	1.460	0.073	P
98	1.377	0.080	P	254	0.270	0.014	P
								99	1.876	0.069	P	255	0.849	0.018	P
100	0.362	0.020	P	256	1.729	0.083	P
								101	0.955	0.098	P	257	0.259	0.092	P
102	1.646	0.062	P	258	1.276	0.048	P
								103	1.139	0.001	P	259	1.291	0.045	P
104	0.553	0.093	P	260	0.931	0.003	P
								105	1.940	0.049	P	261	1.543	0.095	P
106	1.685	0.021	P	262	1.066	0.087	P
								107	0.330	0.085	P	263	1.859	0.093	P
108	0.924	0.019	P	264	1.230	0.067	P
								109	0.437	0.007	P	265	0.314	0.002	P
110	0.949	0.003	P	266	0.266	0.024	P
								111	0.371	0.056	P	267	1.392	0.025	P
112	1.278	0.048	P	268	0.589	0.002	P
								113	0.411	0.095	P	269	1.950	0.055	P
114	1.957	0.005	P	270	1.835	0.071	P
								115	0.484	0.006	P	271	0.386	0.076	P
116	0.555	0.024	P	272	0.275	0.048	P
								117	1.894	0.016	P	273	0.991	0.063	P
118	0.738	0.010	P	274	1.546	0.090	P
								119	0.218	0.032	P	275	1.894	0.064	P
120	0.628	0.036	P	276	0.925	0.086	P
								121	0.526	0.050	P	277	0.360	0.074	P
122	1.258	0.082	P	278	1.684	0.022	P
								123	1.829	0.097	P	279	0.352	0.050	P

124	1.703	0.055	P	280	0.402	0.096	P
								125	1.459	0.086	P	281	1.754	0.005	P
126	1.814	0.054	P	282	1.312	0.044	P
								127	0.287	0.066	P	283	1.404	0.009	P
128	1.525	0.060	P	284	1.888	0.038	P
								129	0.998	0.036	P	285	1.564	0.072	P
130	0.572	0.051	P	286	1.073	0.083	P
								131	1.424	0.013	P	287	1.871	0.055	P
132	0.990	0.041	P	288	0.968	0.032	P
								133	0.537	0.005	P	289	0.620	0.020	P
134	1.062	0.052	P	290	1.705	0.067	P
								135	0.650	0.075	P	291	1.179	0.099	P
136	1.415	0.036	P	292	1.390	0.012	P
								137	0.712	0.091	P	293	1.688	0.038	P
138	1.737	0.073	P	294	0.608	0.099	P
								139	1.271	0.070	P	295	1.356	0.031	P
140	0.333	0.099	P	296	1.961	0.078	P
								141	1.251	0.093	P	297	0.711	0.098	P
142	0.763	0.090	P	298	0.408	0.074	P
								143	1.774	0.015	P	299	1.339	0.000	P
144	1.777	0.019	P	300	0.805	0.039	P
								145	0.897	0.062	P	301	0.323	0.027	P
146	1.318	0.079	P	302	1.856	0.011	P
								147	1.712	0.048	P	303	1.470	0.004	P
148	0.691	0.076	P	304	1.241	0.019	P
								149	1.008	0.097	P	305	0.650	0.063	P
150	1.067	0.058	P	306	1.199	0.006	P
								151	1.941	0.010	P	307	1.078	0.061	P
152	0.501	0.038	P	308	1.129	0.029	P
								153	1.626	0.074	P	309	0.263	0.046	P
154	1.146	0.047	P	310	1.093	0.044	P
								155	0.470	0.091	P	311	0.822	0.054	P
156	0.287	0.015	P	312	1.620	0.024	P

P: the positive

Table 3:P24 positive sample testing result

The sample numbering	T1/C	T2/C	This method result of determination	The collaurum result of determination	The sample numbering	T1/C	T2/C	This method result of determination	The collaurum result of determination
										1	0.04	0.17	P	N	12	0.00	0.57	P	N
2	0.17	0.60	P	N	13	0.07	0.34	P	N

3	0.07	0.17	P	N	14	0.17	0.51	P	N
										4	0.04	0.57	P	N	15	0.18	0.61	P	N
5	0.05	0.18	P	N	16	0.06	0.61	P	N
										6	0.19	0.40	P	N	17	0.02	0.60	P	N
7	0.15	0.41	P	N	18	0.12	0.15	P	N
										8	0.03	0.35	P	N	19	0.05	0.69	P	N
9	0.01	0.11	P	N	20	0.13	0.49	P	N
										10	0.04	0.02	P	N	21	0.04	0.16	P	N
11	0.18	0.25	P	N	22	0.18	0.24	P	N
															23	0.09	0.52	P	N

P: the positive; N: feminine gender

Table 4:HIV-1+2 antibody and the equal positive sample of P24 antigen

The sample numbering	T1/C	T2/C	This method result of determination	The collaurum result of determination	The sample numbering	T1/C	T2/C	This method result of determination	The collaurum result of determination
										1	1.108	0.109	P	P	29	0.284	0.102	P	P
2	1.021	0.111	P	P	30	1.432	0.341	P	P
										3	0.825	0.420	P	P	31	0.572	0.064	P	P
4	1.189	0.352	P	P	32	0.311	0.129	P	N
										5	0.196	0.491	P	P	33	1.445	0.429	P	P
6	0.898	0.289	P	P	34	0.838	0.097	P	P
										7	0.564	0.277	P	P	35	1.293	0.287	P	P
8	0.336	0.317	P	P	36	0.471	0.431	P	P
										9	1.317	0.168	P	P	37	0.978	0.457	P	P
10	0.122	0.372	P	P	38	0.956	0.348	P	P
										11	0.737	0.136	P	P	39	0.117	0.144	P	P
12	0.338	0.215	P	P	40	0.830	0.267	P	P
										13	0.715	0.049	P	P	41	0.335	0.129	P	P
14	0.584	0.378	P	P	42	1.422	0.232	P	P
										15	1.449	0.398	P	P	43	0.660	0.182	P	P
16	0.918	0.044	P	P	44	1.071	0.122	P	P
										17	0.557	0.296	P	P	45	1.313	0.349	P	P
18	1.101	0.039	P	P	46	0.428	0.360	P	P
										19	0.852	0.182	P	P	47	0.827	0.341	P	P
20	0.229	0.406	P	P	48	1.421	0.214	P	P
										21	0.016	0.299	P	P	49	0.628	0.125	P	P
22	1.033	0.404	P	P	50	0.652	0.191	P	P
										23	0.383	0.200	P	P	51	0.773	0.155	P	P
24	0.998	0.111	P	P	52	0.246	0.444	P	P
										25	0.677	0.124	P	P	53	0.184	0.267	P	N

26	0.281	0.435	P	P	54	0.845	0.468	P	P
										27	1.124	0.417	P	P	55	0.820	0.115	P	P
28	1.464	0.479	P	P	56	0.799	0.311	P	P
															57	0.206	0.422	P	P

P: the positive; N: feminine gender

Table 5: clinical detection interpretation of result

The clinical trial project	MICT result	Interpretation of result	The collaurum result
				853 parts in normal human serum sample	851 parts of feminine genders	Specificity is 99.76%	848 parts of feminine genders
312 parts of HIV antibody positive blood serum samples	312 parts of positives	Sensitivity is 100.00%	311 parts of positives
				23 parts of P24 antigen positive blood serum samples	23 parts of positives	Sensitivity is 100.00%	0 part
HIV antibody and P24 antigen are 57 parts in positive serum sample	57 parts of positives	Sensitivity is 100.00%	55 parts

Test card of the present invention and import HIV 1+2 colloid gold reagent have carried out contrast test, the result all is being better than colloid gold reagent far away aspect specificity and the sensitivity, especially for p24 antigen positive sample, test card of the present invention has shown the advantage that colloid gold test paper is incomparable, improved the rate of wiping out and shortened and detected window phase, for the detection of early infection HIV provides good method.

Claims (6)

1, the magnetic immuno-chromatographic test paper strip of a kind of joint-detection HIV-1+2 antibody and P24 antigen, it is characterized in that: this test strips is that coated film, the magnetic mat of particles that combines HIV-1+2 antigen and P24 antibody, sample pad, adsorptive pads are sticked on the base plate successively interlaced 2mm, cover the transparent plastic diaphragm seal then on the upper strata and assemble, be coated with HIV-1+2 antibody detection line and HIV P24 Detection of antigen line and nature controlling line on the wherein said coated film in advance.

2, the magnetic immuno-chromatographic test paper strip of joint-detection HIV-1+2 antibody according to claim 1 and P24 antigen, it is characterized in that: described sample pad is through the pretreated cellulose membrane of sample pad treating fluid, described sample pad treating fluid is the polyvinyl alcohol (PVA) that contains 1%-5% casein and 0.1%-1%, and the 0.02M of 0.01-0.2% Tween-20, the phosphate buffered solution of pH7.0-7.6.

3, the preparation method of the magnetic immuno-chromatographic test paper strip of joint-detection HIV-1+2 antibody according to claim 1 and P24 antigen is characterized in that: may further comprise the steps:

The preparation of A, antigen: select the commercialization HIV-1+2 reorganization pairing antigen of technique for gene engineering preparation for use, to 20mM, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6;

The preparation of B, antibody: select the commercial height P24 pairing monoclonal antibody of tiring for use, to 4 ℃ of dialysed overnight of PBS of 20mM pH7.0-7.6;

The preparation of C, magnetic particle: selecting diameter for use is the super paramagnetic particle of 100-300nm, the mode of using carbodiimide and succinimide covalent coupling with the streptavidin mark to the magnetic particle, select for use pre-activation biotin to carry out the mark of HIV-1+2 antigen/P24 antibody, biotinylated antigen/antibody that mark is good is with 1: 2-1: 10 ratio (volume ratio) is mixed with streptavidin magnetic particle, guarantees that streptavidin magnetic particle is excessive;

D, use quantitative liquid-jet device to be sprayed on the magnetic mat of particles magnetic particle for preparing with the amount of 25 μ l/cm-50 μ l/cm;

The preparation of E, coated film: use 0.02mM, the PBS of pH7.0-7.6, respectively HIV-1+2 envelope antigen and P24 coated antibody and biotinylation bovine serum albumin(BSA) are diluted to the concentration of 0.5-2mg/ml, use quantitative liquid-jet device respectively with the two with the interval spray printing of 0.5-1.0cm on nitrocellulose filter, dry the back and in confining liquid, soak after 10 minutes, add drying agent and seal up for safekeeping standby in 25-35 ℃ of oven dry 8 hours;

The processing of F, sample pad: sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour;

The assembling of G, test strips: interlaced successively 2mm sticks coated film, magnetic mat of particles, sample pad, adsorptive pads on the transparent plastic base plate, covers the transparent plastic diaphragm seal then on the upper strata and obtains test paper plate, and width cutting as requested promptly obtains test strips.

4, the preparation method of the magnetic immuno-chromatographic test paper strip of joint-detection HIV-1+2 antibody according to claim 3 and P24 antigen is characterized in that described step C comprises following three steps:

1) preparation of streptavidin magnetic particle: use the 50mM that contains 0.1%Tween-20, the sodium-acetate buffer washing magnetic particle of pH4.5-5.0, adding carbodiimide and succinimide makes the two final concentration be 20mmol/L, room temperature reaction 1 hour, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer of pH4.5-5.0 fully washs and adds streptavidin behind the magnetic particle to make the molecule ratio of streptavidin and magnetic particle be 5: 1 (mol ratio), room temperature reaction 3 hours, add the 0.02M that contains 0.5%BSA, the PBS room temperature sealing of pH7.0-7.6 30 minutes, use contains the 50mM of 0.1%Tween-20, the sodium-acetate buffer washing magnetic particle of pH4.5-5.0, use then and contain 1%PVP, 1%casein, 0.5%Tween-20, the 50mM of 5% sucrose, the boric acid of pH8.2-9.0 is preserved damping fluid redissolution magnetic particle, and 4 ℃ of preservations are standby;

2) preparation of biotinylation HIV-1+2 antigen and P24 antibody: with HIV-1+2 antigen and P24 antibody to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6, adjustment concentration is 2mg/ml, to activate biotin in advance and use dmso solution, final concentration was 50mM, added the biotin solution of aequum in antigen or antibody-solutions with 20: 1 molecule ratios, room temperature reaction 1 hour, to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6 ,-20 ℃ of preservations are standby behind the adding equal-volume glycerine;

3) biotinylated antigen/antibody and streptavidin magnetic particle mixes, amount with 0.5 μ l/mg magnetic particle adds biotinylation HIV-1+2 antigen in streptavidin magnetic particle solution, amount with 0.25 μ l/mg magnetic particle adds biotinylation P24 antibody, and fully use behind the mixing and preserve damping fluid with 1: 5-1: 20 ratio (volume ratio) is used mixture diluted fiberglass packing to be sprayed.

5, preparation method according to claim 3, it is characterized in that: among the described step D, the spraying method of magnetic particle is: use quantitative spray film device evenly to be sprayed on the glass fibre with the amount of 50 μ l/cm the magnetic particle for preparing, add drying agent after the freeze drying and seal up for safekeeping standby.

6, preparation method according to claim 3, it is characterized in that: in the described step e, the preparation method of coated film is: being cushioned liquid with bag is 0.5mg/ml with HIV-1+2 antigen and P24 antibody dilution, biotinylation BSA dilution is 1mg/ml, use quantitative spray film device with the amount of 1ul/cm with the three with the interval spray printing of 0.6cm on nitrocellulose filter, room temperature is dried after 30 minutes and to be soaked in confining liquid after 10 minutes in 25-35 ℃ of oven dry 8 hours, adds drying agent and seals up for safekeeping standby.

Images