CN1431506A - Immunity colloidal gold reagent for detecting IgG antibody of herpes simplex virus I type and its preparing method - Google Patents

Abstract

The reagent includes the sample pad, the binding pad, the cellulose nitrate film, the water uptake pad and the PVC back cover. The sample pad, the binding pad are adhered to the end with PVC back covered in sequence. The cellulose nitrate film is adhered to the middle. The water uptake pad is adhered to another end of the reagent. The binding pad is coated with the label of albumen A (or rabbit anti human IgG)-colloidal gold. The cellulose nitrate film is coated with the specifity surface film antigen of herpes simple virus I type and chickens anti albumen A (or sheep anti rabbit IgG). The reagent possesses the features of the high specificity and sensitivity, simple and fast as well as the low cost. The reagent is applicable to the detection in site within 30 min for the full testing procedure without need of professional training for the operation personnel.

Description

Detect the immune colloid gold reagent and the preparation method of herpes simplex virus I-type IgG antibody

Technical field

The present invention relates to a kind of immune colloid gold reagent that detects antibody, also relate to the preparation method of this reagent.

Background technology

The existing method that is used to detect the herpes simplex virus I-type IgG antibody mainly contains enzyme linked immunological absorption, radioimmunoassay, immune colloid gold percolation, western blot test (WBA) etc.The defective of enzyme linked immunological absorption (EIA) method is: need special instrument and equipment such as microplate reader to be used; The detecting operation personnel need pass through professional training; Operating process is relatively complicated, and it is long to detect required time; It is higher to detect required expense, can not realize that single part is detected.The defective of immune colloid gold percolation is: operating process is relatively complicated, can not realize single stepping; Reagent needs cryopreservation; It is higher to detect required expense; It is longer than colloidal gold immunity chromatography to detect required time.The defective of radioimmunoassay is: operating personnel need pass through professional training; Complicated operating process requires high; Reagent needs cryopreservation; Because test relates to radioactive isotope, test is not very safe, so seldom adopt; Testing cost is higher; The detection required time is long.The defective of western blot test (WBA) is: operating personnel need pass through professional training; Complicated operating process requires high; Reagent needs cryopreservation; Still the Western blot detection kit of not having at present commercial HSV serum antibody emerges, and this method only limits to carry out in some specialized laboratory; The testing cost height; The detection required time is long.

Summary of the invention

The objective of the invention is in order to overcome the defective that current techniques exists in promoting the use of, providing a kind of does not need the auxiliary detectable of particular instrument equipment, and can reduce the detection cost effectively, alleviates the burden that needs the testing staff.The preparation method of this reagent is provided simultaneously.

Detect the immune colloid gold reagent and the preparation method of herpes simplex virus I-type IgG antibody, this reagent comprises pad, nitrocellulose filter, adsorptive pads and PVC backing, PVC backing one end adheres to sample pad, pad successively, the middle nitrocellulose filter that adheres to, and the other end adheres to adsorptive pads.Bag is by albumin A (or rabbit anti-human igg)-colloid gold label thing on the pad.Bag is by herpes simplex virus I-type specific surfaces membranous antigen and the anti-albumin A of chicken (or goat anti-rabbit igg) on the nitrocellulose filter, and what specifically used by mark is that albumin A or rabbit anti-human igg decide.If come the mark collaurum, then be used as control line with the anti-albumin A of chicken with albumin A; If come the mark collaurum with the rabbit anti-human igg, then be used as control line with goat anti-rabbit igg.

The preparation method of this reagent may further comprise the steps: (1) preparation rabbit anti-human igg: extract human antiserum's immunizing rabbit, get rabbit anti-human igg's (if use albumin A, directly buying) behind the purifying; (2) preparation polyclonal antibody: use repeatedly immunizing rabbit of herpes simplex virus I-type specific surfaces membranous antigen, extract the antiserum immune goat, get goat anti-rabbit igg behind the purifying; (3) preparation collaurum: the colloid gold particle that gold chloride is reduced into 20nm～40nm with reductive agents such as trisodium citrates; (4) preparation monoclonal antibody colloid gold label: with collaurum and albumin A (or rabbit anti-human igg) in 1: 0.005～0.015 (ml/mg) ratio mixing, make collaurum and albumin A (or rabbit anti-human igg) form stable colloidal solid, concentrate by purifying and form albumin A (or rabbit anti-human igg)-colloid gold label thing; (4) albumin A (or rabbit anti-human igg) colloid gold label thing is coated on the collaurum pad, anti-herpes simplex virus I type specificity surface film antigen and the anti-albumin A of chicken (goat anti-rabbit igg) are coated on the detection zone and the control zone of nitrocellulose filter, fully dry.

Good effect of the present invention is: cheap, production procedure is simple, and cost is low, and the expense of detection is than using other detection method all to want considerably cheaper; Detection speed is fast, and overall process only needs 30 minutes, can realize that the oneself detects; Can on-the-spotly detect; Specificity is good, highly sensitive, good reproducibility; Easy and simple to handle, fast qualitative, the result is accurately, fast, and is easy and simple to handle, need not flushing process and standard control, can be in batches or single sample in time detect; Be easy to promote the use of, operating personnel need not professional training, and by specification gets final product complete operation.

Embodiment

PVC backing one end is adhered to sample pad, pad successively, the middle nitrocellulose filter that adheres to, the other end adheres to adsorptive pads.Bag is by albumin A (or rabbit anti-human igg)-colloid gold label thing on the pad.Bag is by herpes simplex virus I-type specific surfaces membranous antigen and the anti-albumin A of chicken (or goat anti-rabbit igg) on the nitrocellulose filter, and what specifically used by mark is that albumin A or rabbit anti-human igg decide.If come the mark collaurum, then be used as control line with the anti-albumin A of chicken with albumin A; If come the mark collaurum with the rabbit anti-human igg, then be used as control line with goat anti-rabbit igg.

Preparation according to the following steps: (1) preparation rabbit anti-human igg: extract human antiserum's immunizing rabbit, get rabbit anti-human igg's (, directly buying) behind the purifying if use albumin A; (2) preparation polyclonal antibody: use repeatedly immunizing rabbit of herpes simplex virus I-type specific surfaces membranous antigen, extract the antiserum immune goat, get goat anti-rabbit igg behind the purifying; (3) preparation collaurum: the particle that 100ml 0.01% chlorauride is reduced into the 40nm size with 0.9ml 1% trisodium citrate; (4) preparation albumin A--colloid gold label thing: use 0.1mol/LK ₂CO ₃The pH value of colloidal gold solution is transferred to about 6.5, colloidal gold solution and monoclonal antibody are mixed in the ratio that adds the 0.8mg albumin A in the 100ml colloidal gold solution, make collaurum and antibody form stable colloidal gold composite, again by repeatedly centrifugal, abandon supernatant, cleaning, concentrate formation rabbit anti-human igg-colloid gold label thing by purifying, refrigerate standby; (5) with Biodot point film machine albumin A colloid gold label thing is sprayed on the collaurum pad, anti-herpes simplex virus I type specificity surface film antigen and the anti-albumin A of chicken are sprayed on the detection zone and the control zone of nitrocellulose filter, fully dry; (6) nitrocellulose filter, collaurum pad, sample pad, adsorptive pads etc. are bonded on the PVC backing successively; (7) the PVC material that glues is cut into the reagent strip of certain width, promptly makes the immune colloid gold reagent that detects the herpes simplex virus I-type IgG antibody.

Before detection, earlier sample and reagent strip (plate) are placed on placement a period of time (10 minutes) under the room temperature condition, make it restore to room temperature; Take out the detectable bar from aluminium foil bag, by the direction shown in the arrow under the MARK line reagent strip is immersed in the sample solution, liquid level must not surpass the MARK line, takes out after 5 seconds～8 seconds, lies on the operator's console; If agent plate: from aluminium foil bag, take out the detectable plate, lie on the operator's console, in well, drip 3 (about 120ul) sample solutions (serum); Get final product judged result in 3 minutes～15 minutes, the result who judges after 30 minutes is invalid.The result judges: if there be " the herpes simplex virus I-type IgG antibody " that will detect to exist in the sample, then red stripes appears in the detection line place, red stripes also occurs on the nature controlling line simultaneously, and this moment, the result was positive; If " the herpes simplex virus I-type IgG antibody " that will not detect in the sample exists, then the detection line place does not have the band appearance, but occurs red stripes on the nature controlling line, and this moment, the result was negative.If there is not red stripes to occur on the nature controlling line, then product is invalid.

Claims (2)

1, detects the immune colloid gold reagent and the preparation method of herpes simplex virus I-type IgG antibody, this reagent comprises sample pad, pad, nitrocellulose filter, adsorptive pads and PVC backing, PVC backing one end adheres to sample pad, pad successively, the middle nitrocellulose filter that adheres to, the other end adheres to adsorptive pads, it is characterized in that bag is by albumin A (or rabbit anti-human igg)-colloid gold label thing on the pad, bag is by herpes simplex virus I-type specific surfaces membranous antigen and the anti-albumin A of chicken (or goat anti-rabbit igg) on the nitrocellulose filter.

2, the immune colloid gold reagent and the preparation method of detection herpes simplex virus I-type IgG antibody according to claim 1, the preparation method who it is characterized in that this reagent may further comprise the steps: (1) preparation rabbit anti-human igg: extract human antiserum's immunizing rabbit, get rabbit anti-human igg's (, directly buying) behind the purifying if use albumin A; (2) preparation polyclonal antibody: use repeatedly immunizing rabbit of herpes simplex virus I-type specific surfaces membranous antigen, extract the antiserum immune goat, get goat anti-rabbit igg behind the purifying; (3) preparation collaurum: the colloid gold particle that gold chloride is reduced into 20nm～40nm with reductive agents such as trisodium citrates; (4) preparation monoclonal antibody colloid gold label: with collaurum and albumin A (or rabbit anti-human igg) in 1: 0.005～0.015 (ml/mg) ratio mixing, make collaurum and albumin A (or rabbit anti-human igg) form stable colloidal solid, concentrate by purifying and form albumin A (or rabbit anti-human igg)-colloid gold label thing; (4) albumin A (or rabbit anti-human igg)-colloid gold label thing is coated on the collaurum pad, anti-herpes simplex virus I type specificity surface film antigen and the anti-albumin A of chicken (goat anti-rabbit igg) are coated on the detection zone and the control zone of nitrocellulose filter, fully dry.