CN106970219A - One kind is based on HIV in colloidal gold method detection urine（1+2）Antibody reagent - Google Patents

The present invention relates to a kind of external diagnosis reagent and its application method, more particularly to a kind of detection method for detecting HIV (1+2) antibody in human urine.One kind detects the preparation method of HIV (1+2) antibody reagent in urine based on colloidal gold method, comprises the following steps：Colloidal gold conjugate pad is prepared with the SPA coupling anti-human IgG antibodies of colloid gold label, as colour developing probe, mixing coating gp 41 Antigens and gp36 antigens, are used as the detection line of detection HIV1 types antibody and HIV2 type antibody on nitrocellulose filter.

One kind is based on HIV (1+2) antibody reagent in colloidal gold method detection urine

Technical field

It is more particularly to a kind of to detect HIV (1+ in human urine the present invention relates to a kind of external diagnosis reagent and its application method 2) detection method of antibody.

Background technology

Existing HIV urines antibody test reagent, is ELISA, and this method is only limitted to detect HIV-1 antibody, and Special instrument and equipment is needed, detection time is long, it is necessary to operated by professional.

HIV (1+2) urine antibody test reagent a kind of quick and that self-inspection can be used for is researched and developed to be necessary.

The content of the invention

The present invention provides a kind of preparation method that HIV (1+2) antibody reagent in urine is detected based on colloidal gold method, with colloid The SPA coupling anti-human IgG antibodies of gold mark prepare colloidal gold conjugate pad, as colour developing probe, are mixed on nitrocellulose filter Coating gp 41 Antigens and gp36 antigens are closed, the detection line of the detection type antibody of HIV 1 and the type antibody of HIV 2 is used as.

The preparation method of colloidal gold conjugate pad therein is as follows：

1) SPA is marked, adjust collaurum PH with 0.2M potassium carbonate adds in the ratio of 8.2 or so, 10~30 μ g/ml gold SPA, after reacting 30~60 minutes, is closed with BSA or PEG, final concentration 1~10mg/ml, the 10000rpm centrifugation of confining liquid 10~60 minutes, abandon supernatant, precipitate with the redissolution of 10mM Tris-Hcl (pH8.2) buffer solution containing 2mg/ml BSA to original Volume.

2) in gold mark SPA coupling anti-human IgG antibodies, the gold mark SPA solution after above-mentioned redissolution, added in every milliliter of solution The μ g of anti-human IgG antibodies 0.1~10, react 60min, and 10000rpm is centrifuged 10~60 minutes, abandon supernatant, precipitation is with containing 0.5% BSA, 5% sucrose, 0.1%Triton X-100,0.03%proclin-300 10mM Tris-Hcl (pH8.2) buffer solution Redissolve to original volume.

3) it is freeze-dried, after above-mentioned gold mark solution paving gold, -50 DEG C of pre-freezes vacuumize progress distillation and freezed after 2 hours, Prepare colloidal gold conjugate pad.

Colloidal gold conjugate pad therein, is coupled anti-human IgG antibodies by gold mark SPA, makes anti-human IgG antibodies' orderly Property enhancing, i.e. Fab ends are unanimously outside, increase the quantity of potent antibodies, strengthen its ability with reference to floating preteins in urine, together When, SPA is marked by gold and is coupled anti-human igg, the arm of extension gold mark probe strengthens its respond, lifts the sensitive of product Degree.Wherein, coated gp 41 Antigens concentration is mixed on nitrocellulose filter for 0.5~2.5mg/ml, gp36 antigen concentrations are 0.1~0.5mg/ml.

The present invention further provides a kind of detection architecture, the detection architecture mainly includes, SPA, collaurum, BSA, PEG, Tris-Hcl buffer solutions, anti-human igg, sucrose, the composition such as Triton X-100, proclin-300.

Detection architecture of the present invention is that card is detected described in embodiment, and detection card includes nitrocellulose filter, gold standard pad, Blotting paper, offset plate and plastics get stuck, and detection is arranged with detection line display portion and sample application hole, gold standard pad therein according to It is prepared by the inventive method.

Other required articles such as aluminium foil bag, plastic bottle, drier, specification fall within scope of the invention.The present invention's is excellent Point is：

1st, SPA is marked by gold and is coupled anti-human IgG antibodies, making the order of antibody strengthens, i.e. Fab ends are unanimously outside, increased Plus the quantity of potent antibodies, strengthen its ability with reference to floating preteins in urine.Anti-human IgG antibodies are coupled by gold mark SPA, prolonged The arm of long gold mark probe, strengthens its respond, lifts the sensitivity of product.

2nd, detection efficiency is improved, and compared with enzyme exempts from urinalysis reagent, detection time significantly shortens, as a result 15min interpretations；

It is technical solution of the present invention and prior art implementation result comparison sheet below：

Accompanying drawing

Fig. 1 production technological process

The judgement of Fig. 2 testing results

20 testing results of Fig. 3 positive urines：

Fig. 4 feminine gender 20 testing results of urine：

Fig. 5 uniformity test results：

Fig. 6 present invention detection card schematic diagrames

Embodiment

The present invention is further illustrated by the following examples, but not as limitation of the present invention.

Embodiment 1

The committed step of specific implementation is as follows：

Step one：100ml 40nm colloidal gold solutions are taken, gold concentration 0.03% adds 1ml 0.2M potassium carbonate, in stirring 4ml SPA (5mg/ml) are added, after reacting 60 minutes, 1ml 20%BSA is added, reacts 60 minutes, 10000rpm centrifuges 30 points Clock, abandons supernatant, and precipitation is redissolved to 100ml with 10mM Tris-Hcl (pH8.2) buffer solution containing 2mg/ml BSA.Step 2： In gold mark SPA solution after above-mentioned redissolution, the μ l (10mg/ml) of anti-human IgG antibodies 100 are added, 60min, 10000rpm is reacted Centrifugation 30 minutes, abandons supernatant, precipitation is with containing 0.5%BSA, 5% sucrose, 0.1%Triton X-100,0.03%proclin- 300 10mM Tris-Hcl (pH8.2) buffer solution is redissolved to 100ml.

Step 3：Freeze-drying, solution is marked by 30ml/ glass fibre membrane (20cm × 30cm) paving gold by above-mentioned gold ,- 50 DEG C of pre-freezes vacuumize progress distillation and freezed, freeze-drying time 18 hours prepares colloidal gold conjugate pad after 2 hours.

Step 4：Mixing coating gp 41 Antigens and GP36 antigens, wherein gp41 on nitrocellulose filter detection line position Antigen concentration is 1.5mg/ml, and gp36 antigen concentrations are 0.2mg/ml, and Quality Control line position is coated with rabbit anti-mouse igg, and coating concentration is 1mg/ml。

Other detailed process are produced by production technological process, see Fig. 1.

Detection process is as follows：

【Sample requirement】

This product detection sample is urine.Urine specimen is collected in urine cup that is clean, drying, and the urine within one day is equal Detectable, gynecologic menstrual should leave and take midstream urine.Urine sample must not have obvious blood pollution (such as gynecologic menstrual) and germ contamination.

Same day detection is advisable after collecting sample, can not such as detect on the same day, can be preserved one week at 2-8 DEG C；Extension is such as needed to store It time, need to be frozen below -20 DEG C, should fully melt before freezing Samples detection and recover room temperature, shake up.

【The method of inspection】

The detection of the present invention is placed on clean flat table top, vertical about 80 μ L (the about 3 drop) sample that is added dropwise is in sample-adding In hole.Test result was observed at 15~20 minutes, judges invalid after 20 minutes.

Embodiment 2, operation instruction of the invention

【Product function】

HIV-1 and (or) HIV-2 type antibody in this product application immunochromatographic method qualitative detection human urine, it is adaptable to people The auxiliary diagnosis of para-immunity defective virus infection.

The chronic poison category of human immunodeficiency virus (HIV) category Retroviridae, HIV is mainly through blood, sexual intercourse, Mu Yingsan Kind of approach is propagated, and body is infected to occur serious immune deficiency, i.e., acquired immunodeficiency syndrome (AIDS, AIDS), it is a kind of high chronic infectious disease of case fatality rate.The standard method of HIV diagnosis is antibody test [1], except blood Outside sample, (urine, saliva, seminal fluid, tear) contains HIV antibody in a variety of body fluid of HIV person.To avoid HIV from examining Propagation during controlling, the demand for meeting self detection and bio-safety risk that may be present during sample transport is reduced, In recent years, researcher starts to research and develop Noninvasive HIV antibody detection method.There is document report, with traditional blood method ratio Compared with the sensitivity and specificity of urine antibody test is respectively reached 97.28% and 99.09% [2].In view of in the side such as safe and convenient Face has more advantage, and urine detection has turned into a kind of brand-new means that AIDS (AIDS) is detected.

【Inspection principle and yin and yang attribute decision method】

This product uses immunochromatography technique principle, and (HIV1+2) antibody in human urine sample is detected using indirect method. The two groups of HIV recombinant antigens (gp41, gp36) for different binding sites prepared by genetic engineering are used as envelope antigen. With staphylococcus aureus protein A (SPA), anti-human IgG antibodies' mark collaurum, rabbit anti-mouse igg is used as nature controlling line.When detected Urine is acted on by chromatography when being migrated in test strips, if containing HIV-1 or HIV-2 antibody, then in detection line position in urine One aubergine band of appearance is put, is determined as the positive；If not containing HIV antibody in urine, detection line position occurs without band It is determined as feminine gender.No matter whether contain HIV antibody in urine, an aubergine band can all occur in Quality Control line position.Specifically Result of determination is shown in accompanying drawing 2.

【Main constituents】

Detection card is by HIV gene recombinant antigens (gp41, gp36), the coated nitrocellulose filter of rabbit anti-mouse igg, golden yellow Staphylococcal protein A (SPA), anti-human IgG antibodies prepare gold standard pad, and glass fibre, blotting paper, offset plate and plastics get stuck composition.

Other required article aluminium foil bag, plastic bottle, drier, specifications

【Preparation method】

The 1st, sample pad, blotting paper are cut into 1.8cm × 30cm specifications are standby, golden conjugate pad is cut into 6mm × 30cm Specification is standby.

2nd, offset plate, nitrocellulose filter, sample pad, blotting paper, golden conjugate pad are subjected to lamination joint, assemble big plate.

3rd, the big plate assembled is cut, slitting width 4mm.

4th, the bar cut is assembled into card together with getting stuck, be fitted into together with one bag of drier in aluminium foil bag, sealed.

【The method of inspection】

Reagent card and sample are recovered to room temperature using preceding.

Aluminum foil sack is torn, detection is taken out and is placed on clean flat table top, it is vertical that about 70 μ L (about 3 drop) are added dropwise Sample is in well.

3. observing test result at 10~15 minutes, judge invalid after 20 minutes.

【The judgement of testing result】

See Fig. 2

Embodiment 3

Detection example is shown in accompanying drawing 3-5.

Bibliography：

Tian Yu, Shen Changxin, Jiang Yan urine HIV-1 antibody test technical progress【J】China's AIDS venereal disease, 2016 (7)： 579-581.

Feng Xia, Wang Jibao, Tian Yu, wait the Clinical application evaluation of urine HIV-1 antibody tests【J】China's AIDS Disease, 2016 (4)：241-243.

《National AIDS inspection specifications》(revised edition in 2015), CDC.