CN202975018U - Human immunodeficiency virus antibody confirmation reagent - Google Patents

Abstract

Provided is a human immunodeficiency virus antibody confirmation reagent. The reagent comprises human immunodeficiency virus (HIV)antibody test paper, washing liquid, a chromogenic system, a positive control body, a negative control body, and an immunity chromatography result identification instrument. Ten protein bands respectively including an HIV1 band, a gp160 band, a gp120 band, a P66 band, a p51band, a gp41 band, a p24 band, a p17 band, an HIV 2 band and a gp36 band, and two human IgG bands different in concentration are coated on the HIV antibody test paper. The chromogenic system is composed of anti-human IgG or SPA labeled by colloidal gold or anti-human IgG or SPA labeled by biotin, and avidin labeled by colloidal gold. During detection, a sample is placed on the HIV antibody test paper, unrelated protein is washed and removed, the chromogenic system is utilized for a chromogenic purpose, and a result is determined by the immunity chromatography result identification instrument or naked eyes according to band chromogenic conditions. The human immunodeficiency virus antibody confirmation reagent has the advantages of being simple and fast to operate, being suitable for on-site detection, being economical and practical, etc.

Description

Human immunodeficiency virus antibody confirmations reagent

Technical field

The utility model relates to the biologic applications technical field, particularly relates to the external diagnosis reagent for a kind of human immunodeficiency virus antibody confirmations reagent.

Background technology

Since finding the eighties in 20th century, spread all over the whole world from acquired immune deficiency syndrome (AIDS) that human immunodeficiency virus (HIV) causes.According to acquired immune deficiency syndrome (AIDS) general administration of the United Nations (UNAIDS) report, to the end of the year 2010, global aids infection number reaches 3,700 ten thousand.The present nearly HIV the infected more than 70 ten thousand of China.There is no up to now effective medicine and vaccine and treat and prevent AIDS, thereby the HIV infection has become the most serious infectious disease and the public health problem of current whole world threat.Just due to this, the monitoring that HIV infects is subject to the great attention of countries in the world, and WHO, U.S. FDA and many developed countries have formulated the rules of the screening tests such as relevant blood donor, blood product in succession in 1985, and wherein the detection of HIV antibody is listed in routine.China has just promulgated " Provisions for Monitoring and Control of AIDS " in 1987, having formulated again relevant blood donor nineteen ninety and 1994 checks HIV antibody and strengthens blood supply person's blood sample test with the quality control of external immune diagnostic reagent, carried out the regulation of batch batch calibrating, guarantee that the HIV detection kit has stable quality, thereby effectively controlled the propagation that HIV infects.

Due to the changeableness of HIV virus, be much to seek at present effective vaccine, do not have active drug effectively to treat yet.The effective means that current control HIV virus is propagated is exactly to find as early as possible the virus carrier, as early as possible the isolated infection sources.

To HIV the infected's diagnosis mainly to detect antibody as main.Detection method has coacervation, indirect immunofluorescence, ELISA, gold marked reagent, Western blotting etc.First acquisition drugs approved by FDA is the ELISA kit of U.S. Abbott (doubly inferior) company for detection of the kit of HIV antibody, the ELISA kit of many countries also puts goods on the market in succession afterwards, and the ELISA kit that HIV antibody detects in China is appearance in 1989.Indirect immunofluorescence and coacervation be because operating process is loaded down with trivial details, influenced many factors, less employing.Colloidal gold method is the detection method commonly used that development in recent years is got up.Application at present most often adopts ELISA or collaurum crowd to carry out preliminary screening, then to there being the suspicious sample of infection to confirm with Western blot (Western Blot, WB) method, is called antibody recognize reagent.At present only have a few company to produce inside and outside WB France, and the reaction time is long, operates loaded down with trivial details.

Summary of the invention

The object of the invention is to provide a kind of human immunodeficiency virus antibody confirmations reagent, have easy and simple to handle, quick, be fit to Site Detection and the advantage such as economical and practical.

The invention provides a kind of human immunodeficiency virus antibody confirmations reagent, it is characterized in that, reagent comprises HIV antibody test paper cleansing solution, Color Appearance System, positive reference substance, negative control product and immunochromatography interpretoscope as a result; Being coated with 10 protein bands on described HIV antibody test paper, is respectively the human IgG band of HIV1gp160, gp120, P66, p51, gp41, p24, p17, HIV2gp36 and 2 variable concentrations; Anti-human IgG or the SPA of colloid gold label, or the Avidin Color Appearance System of biotin labeled anti-human IgG or SPA, colloid gold label.HIV antibody test paper structure is NC film, the NC film close-connected suction sample pad in below and loads the NC film and the plastic casing of suction sample pad; The NC film is since an end, and 10 coated protein bands are the high concentration human IgG successively, HIV1gp160, gp120, P66, p51, gp41, p24, p17, HIV2gp36 and low concentration human IgG.The collaurum diameter is 20-100nm.

Wherein, the reaction result of the anti-Confirmation reagent of described HIV antibody by immunochromatography as a result interpretoscope be that a kind of Systems for optical inspection is judged.Described human immunodeficiency virus antibody confirmations reagent, HIV antibody method for preparing test paper is as follows:

1, coated NC film: albumen is diluted to 0.001-2mg/ml with the phosphate buffer of 0.01M, be preferably, the high concentration human IgG is 0.05mg/ml, and the low concentration human IgG is 0.005mg/ml, gp160 is 1.0mg/ml, gp120 is 0.8mg/ml, and p66 is 0.8mg/ml, and p51 is 0.8mg/ml, gp41 is 0.8mg/ml, p24 is 0.5mg/ml, and p17 is 0.5mg/ml, and gp36 is 0.6mg/ml; The NC film is selected the film in 0.45um aperture, and width is 40mm, and length is 300mm; The antibody that dilution is good is loaded on Membrane jetter, press 1ul/cm and rule, each line be spaced apart 2-4mm, be preferably 3mm; After line is completed, the NC film is placed in humidity 30% below, drying is more than 4 hours.

2, preparation test paper: the NC film laterally is cut into the width of 3-8mm, is preferably 5mm, inhale the sample pad on the pad of NC film bottom, inhaling the sample pad is absorbent material, and water-intake capacity is not less than 2ml, in the sizeable plastic clip of then packing into, and the formation test paper.

Described human immunodeficiency virus antibody confirmations reagent, the preparation method of Color Appearance System is:

1, direct Color Appearance System: (1) prepares with reducing process the collaurum that concentration is 0.02% 20-100nm, is preferably the collaurum of 40nm; (2) use K ₂CO ₃Collaurum pH is adjusted to 8.0, by the anti-human IgG of 10ug or the every ml collaurum of SPA, albumen is added, mixing 30min, the centrifugal supernatant of abandoning with the phosphate buffer dissolving of solution with 1/10 volume, adds micro-antiseptic, namely becomes Color Appearance System.

2, biotin-avidin Color Appearance System: (1) prepares with reducing process the collaurum that concentration is 0.02% 20-100nm, is preferably the collaurum of 40nm; (2) with anhydrous DMSO preparation 10mg/ml biotin N-hydroxy-succinamide ester solution, with borate buffer solution (0.1mol/L, pH8.8) compound concentration is anti-human IgG or the SPA solution of 2mg/ml, ratio by 25ug/mg adds the biotin ester in antibody, mix and at room temperature hatch 4hr, antibody-solutions is dialysed with PBS, to remove unconjugated biotin, after being diluted to again suitable ratio, make chromogenic substrate 1; (3) use K ₂CO ₃Collaurum pH is adjusted to 8.0, by the every ml collaurum of 10ug Avidin, albumen is added, mixing 30min, the centrifugal supernatant of abandoning with the phosphate buffer dissolving of solution with 1/10 volume, adds micro-antiseptic, is chromogenic substrate 2; (4) chromogenic substrate 1 and 2 consists of Color Appearance System.

The anti-Confirmation reagent of described HIV antibody, positive reference substance behaviour HIV Positive Sera is made after treatment, the gp160 of its anti-HIV-1, gp120, p66, p51, gp41, p24, p17, the gp36 of anti-HIV-2 is all positive; Negative control product behaviour HIV negative antibody serum is made after treatment, and all kinds of antigens of anti-HIV-1 and HIV-2 are all negative.Cleansing solution is the phosphate buffer that contains the 0.01M of 0.5%Tween20.

The anti-Confirmation reagent of described HIV antibody, preservation condition is 2-8 ℃, keeps in Dark Place, the term of validity 12 months.

Described human immunodeficiency virus antibody confirmations reagent, detection method is as follows:

1, reagent and serum to be checked (slurry) are equilibrated to room temperature; The reagent of 2-8 ℃ of preservation needs the 20-30min balance to room temperature;

2, HIV antibody test paper is taken out, keep flat, drip serum to be checked (slurry) sample of 100-150ul on the NC film of test paper, sample will slowly infiltrate the NC film, enter at last suction sample pad, and this process is 2-3min approximately;

3, drip the 100-150ul cleansing solution on the NC film, wait cleansing solution to infiltrate after film, then drip the 100-150ul cleansing solution, repeated washing 2 times, this process is 5-8min approximately;

If 4 adopt direct Color Appearance System, drip the anti-human IgG of 100-150ul or SPA mark collaurum on the NC film, collaurum just slowly infiltrates the NC film, enters at last to inhale the sample pad, and this process is 2-3min approximately; If adopt the biotin-avidin Color Appearance System, drip the anti-human IgG of 100-150ul biotin labeling or SPA on the NC film, after the biotin labelled antibody slowly infiltrates the NC film, then drip the 100-150ul cleansing solution, with reference to step 3, wash 3 times, this process is 5-8min approximately, then adds the Avidin of 100-150ul colloid gold label, and collaurum will slowly infiltrate the NC film, enter at last and inhale the sample pad, this process is 2-3min approximately;

5, drip the 100-150ul cleansing solution on the NC film, wait cleansing solution to infiltrate after film, then drip the 100-150ul cleansing solution, repeated washing 1 time, this process is 4-6min approximately;

6, the red stripes of collaurum colour developing will occur on the NC film this moment, judge testing result with special-purpose detecting instrument or naked eyes.

Need the parallel positive and the negative control done simultaneously when carrying out above-mentioned detection, namely do simultaneously one-time detection with positive reference substance and negative control product.

The anti-Confirmation reagent of described HIV antibody, decision method is as follows as a result:

1, the decision method of band positive and negative:

Immunochromatography is interpretation registering instrument decision method as a result: reacted HIV antibody test paper is put into instrument, start setting program, instrument will demonstrate the testing result of each band automatically, the value of quantitative, detect effective value (detected value is not less than low concentration IgG band value) positive, on the contrary negative.

Testing result is with the naked eye interpretation method also: whether appointed area in have colour developing band occur, have band appearance and intensity to be not less than low concentration IgG intensity positive if directly observing, otherwise negative.

2, detection system is effectively judged, needs to satisfy following all conditions:

The band to some extent of positive control need be shown as the positive;

Two IgG lines of negative control need be shown as the positive, and remaining 10 bands all need be shown as feminine gender; Two IgG lines of sample to be checked need be shown as the positive, and the intensity of all Test paper middle and high concentration IgG bands needs higher than low concentration IgG band;

Otherwise it is invalid to detect, and needs again to detect.

3, the judgement of testing result yin and yang attribute needs to judge in the effective situation of detection system:

10 coated antigens of HIV antibody test paper are divided into following 4 classes: env antigen: gp160, gp120, gp41 and gp36 (HIV-2 type); Gag antigen: p24 and p17; Pol antigen: p66 and p51.

Negative: all HIV specific bands all are judged to be feminine gender;

HIV-1 is positive: have at least 2 env bands (gp41 and gp160/gp120) positive, or at least 1 env band and the p24 band simultaneously positive.

The doubtful positive of HIV-2: the gp36 band is positive.

Uncertain: as the special band of HIV antibody to occur, but do not satisfy positive criterion.

This patent and existing product or patent are relatively, following difference is arranged: the antigen part that (1) is selected is different, clearly select the HIV antigen of 10, make with restructuring or synthetic method, rather than make through cracking with the virus that HIV cultivates, reduce and make and judge complexity, improved the accuracy of product, but do not affected result of determination; (2) adopted the collaurum coloration method, compared with the enzyme development process, more simple, direct, stable; (3) obviously shortened detection time, and simple to operate, this reagent can be completed single detection in 60min, and 30 detection required times are no more than 90min, and other detection takes approximately 4 hours; (4) the available immunochromatography of result as a result the interpretation registering instrument judge, judge with naked eyes and compare, can detect fainter band, and potential mistake can avoid the naked eyes subjective determination time.Comprehensive above characteristics, this reagent is a kind of novel HIV antibody recognize reagent, has easy and simple to handle, quick, suitable Site Detection and the advantage such as economical and practical.

Description of drawings

Accompanying drawing 1 is the antigen coated schematic diagram of HIV antibody test paper

Specific embodiment

Embodiment 1: the manufacturing of human immunodeficiency virus antibody confirmations reagent

1, main material

1.1HIV specific antigen is the gp36 of gp160, gp120, p66, p51, gp41, p24, p17 and the HIV-2 of HIV-1: Military Medical Science Institute provides by Beijing, the HIV antigen of gene engineering expression; Human IgG and mouse-anti human IgG: luxuriant and rich with fragrance roc Bioisystech Co., Ltd provides by Shenzhen; NC film: U.S. Millipore company product; Bovine serum albumin(BSA) (BSA), polyglycol (PEG) 20000, protein hydrolysate: U.S. Sigma company product; Other reagent is for being conventional analysis pure chemistry reagent.

1.2 detecting instrument: immunochromatography knot interpretation registering instrument, model: NS3001, Newscen Coast Bio-Pharmaceutical Co., Ltd.'s product.

1.3 clinical serum: obtained in relevant hospital by company, 19 parts of HIV-1 positive sample, 1 part of HIV-2 positive sample detects conclusive evidence by Singapore MP reagent.20 parts of negative sample are picked up from Healthy People, and Comprehensive Clinical detects and is judged to be feminine gender.

2, main method

2.1HIV the preparation of antibody test paper: albumen is diluted to variable concentrations with the phosphate buffer of 0.01M, each albumen is all established 3 concentration: the high concentration human IgG is 0.02, 0.05 and 0.1mg/ml, the low concentration human IgG is 0.002, 0.005 and 0.01mg/ml, gp160 is 0.5, 1.0, 1.5mg/ml, gp120 is 0.4, 0.8, 1.2mg/ml, p66 is 0.4, 0.8, 1.2mg/ml, p51 is 0.4, 0.8, 1.2mg/ml, gp41 is 0.5, 1.0, 1.5mg/ml, p24 is 0.5, 1.0, 1.5mg/ml, p17 is 0.25, 0.5, 1.0mg/ml, gp36 is 0.6, 1.2, 1.8mg/ml, the NC film is selected the film in 0.45um aperture, width 30mm, length 300mm, the antibody that dilution is good is loaded on Membrane jetter, press the 1ul/cm line, each line be spaced apart 3mm, after line is completed, the NC film is placed in humidity below 30%, drying 4 hours.

After completing, drying with cutting cutter, the NC film laterally is cut into the width of 5mm, absorbent filter on the pad of NC film bottom, and the absorbent filter size is 15 * 30mm, 6 layers of overlapping putting of absorbent filter, water-intake capacity 2-2.5ml is in the sizeable plastic clip of then packing into, compress, form test paper.2.2 the preparation of Color Appearance System: prepare with gold chloride-trisodium citrate reduction method the colloidal gold solution that diameter is 40nm, get three parts of collaurums after preparation is completed, use respectively 0.2M K ₂CO ₃Solution is transferred to pH7.0, pH8.0 and pH9.0.Then solution is placed on magnetic stirring apparatus and slowly stirs, add 0.5mg, 1mg, 1.5mg mouse-anti human IgG by every 100ml solution, antibody is added drop-wise in colloidal gold solution, continue to stir 2 hours, be added dropwise to again final concentration and be 0.1% PEG2000 and 1% BSA and seal 20min, mark is centrifugal with 12000r/m after finishing, abandon supernatant, precipitation is redissolved to the collaurum working fluid (borate buffer solution of original volume 1/10 by original volume, pH8.0 contains BSA, sheep blood serum, sucrose, surfactant and antiseptic) in.

2.3 the preparation of cleansing solution: be the phosphate buffer of 0.01M pH7.4, contain 0.9%NaCl and 0.05%Tween20, preserve with 20 times of conc forms, be diluted to working concentration with purified water before using.

2.4 reference substance: make with clinical serum.Positive reference substance: select the HIV Positive Sera, several parts of serum merge, through suitably dilution, and deactivation is made, detect with the MP kit, the gp36 of the gp160 of HIV-1, gp120, p66, p51, gp41, p24, p17 and HIV-2 is all positive, the negative control product: be mixed with several parts of Healthy Human Serums, be verified as the HIV negative antibody with the MP kit.

2.5 detection method: the coated HIV antibody test paper of variable concentrations and the Color Appearance System of different pH marks are matched one by one, detect with positive reference substance and negative control product respectively, detection method is as described in instructions, testing result is put into immunochromatography interpretation registering instrument as a result, setup parameter, read the GOD value of each response line, determine the optimal parameter of product.

2.6 decision method as a result: judge as method as described in instructions.

3, result detects

3.1 determining of envelope antigen concentration

After the proteantigen of employing variable concentrations was coated, with the Color Appearance System test of pH8.0 mark, negative control product test result was all negative, and the positive reference substance test result is as follows:

Table 1: the detected value of different coating protein concentration (GOD)

Classification	Concentration 1	Concentration 2	Concentration 3
				The high concentration human IgG	2.6	4.5	7.4
gp160	2.3	4.8	7,5
				gp120	1.9	4.2	6.8
p66	2.8	5.0	7.2
				p51	2.3	4.4	6.4
gp41	4.2	6.5	7.9
				p24	4.5	7.1	8.4
p17	4.8	7.3	8.9
				gp36	4.9	6.8	8.3
The low concentration human IgG	0.2	0.4	0.8

Annotate: concentration 1,2, the 3rd, be coated with concentration from low to high, occurrence is seen the preparation of 2.1HIV antibody test paper.

With reference to the titre of antibody in positive reference substance, the testing result that the negative control product detect result and MP reagent, three coated concentration are preferably as follows: the high concentration human IgG is 0.05mg/ml, the low concentration human IgG is 0.005mg/ml, and gp160 is 1.0mg/ml, and gp120 is 0.8mg/ml, p66 is 0.8mg/ml, p51 is 0.8mg/ml, and gp41 is 0.5mg/ml, and p24 is 0.5mg/ml, p17 is 0.5mg/ml, and gp36 is 0.6mg/ml.Consider variation and the differences between batches of different batch protein antigenicities, above concentration is only reference concentration, and the concentration of different batches, different albumen can change between 0.001-2mg/ml to some extent.

3.2 determining of Color Appearance System reference

Adopt variable concentrations mouse-anti human IgG, after different pH marks, detect with the negative control product, observe the value of high and low concentration IgG, testing result is as follows:

Table 2: the detected value (GOD) of different labelled amounts, mark pH

Take into account the colour developing expectation value of high concentration and low concentration IgG, the preferred reference number of Color Appearance System is pH8.0, the collaurum (10ug/ml) of the every 100ml of the anti-human IgG of labelled amount 1mg.Consider variation and the differences between batches of different batch protein antigenicities, product labelling amount and the pH of different crowdes have certain variation.

Embodiment 2: clinical testing

1, main material

1.1 examination human immunodeficiency virus antibody confirmations reagent: press the reagent that embodiment 1 optimizes preparation.

1.2 contrast human immunodeficiency virus antibody confirmations reagent: Singapore MP company product.

1.3 clinical sample: obtained in relevant hospital for infectious diseases by company, 100 parts of HIV positive sample, wherein the HIV-1 positive sample is 95 parts, ELISA primary dcreening operation HIV antibody positive; 5 parts of HIV-2 positive sample are for confirming positive sample; 500 parts of negative sample are picked up from Healthy People, and Comprehensive Clinical detects and is judged to be feminine gender.

2, detection method

Undertaken by the method for product description separately, judge testing result, and analyze.

3, result: testing result is as follows:

Table 3: this kit and the contrast of MP testing result

Can find out from upper table testing result, the positive coincidence rate of this reagent is 98/99=99%, and negative match-rate is 493/495=99.6%, and total coincidence rate is 596/600=99.3%.The testing result height is consistent, meets the needs of proving conclusively as HIV antibody clinically.

Claims (3)

1. human immunodeficiency virus antibody confirmations reagent, it is characterized in that: reagent comprises HIV antibody test paper, cleansing solution, Color Appearance System, positive reference substance, negative control product and immunochromatography interpretoscope as a result; Being coated with 10 protein bands on described HIV antibody test paper, is respectively the human IgG band of HIV1gp160, gp120, P66, p51, gp41, p24, p17, HIV2 gp36 and 2 variable concentrations; Anti-human IgG or the SPA of colloid gold label, or the Avidin Color Appearance System of biotin labeled anti-human IgG or SPA, colloid gold label.

2. human immunodeficiency virus antibody confirmations reagent according to claim 1 is characterized in that: HIV antibody test paper structure is NC film, the NC film close-connected suction sample pad in below and loads the NC film and inhale the plastic casing of sample pad; The NC film is since an end, and 10 coated protein bands are the high concentration human IgG successively, HIV gp160, gp120, P66, p51, gp41, p24, p17, HIV2 gp36 and low concentration human IgG.

3. human immunodeficiency virus antibody confirmations reagent according to claim 1, it is characterized in that: the collaurum diameter is 20-100nm.

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