CN101074959A

CN101074959A - Gold and silver combined inspection test paper for feline and canidae animals important pestilence pathogen and its production

Info

Publication number: CN101074959A
Application number: CN 200710055759
Authority: CN
Inventors: 夏咸柱; 杨松涛; 黄耕; 王承宇; 高玉伟; 王铁成; 侯小强; 高明华; 苏建青
Original assignee: Institute of Military Veterinary Academy of Military Medical Sciences PLA
Current assignee: Institute of Military Veterinary Academy of Military Medical Sciences PLA
Priority date: 2007-06-13
Filing date: 2007-06-13
Publication date: 2007-11-21

Abstract

A colloidal gold-silver united test paper used for detecting etiology of important disease on Felidae and Canidae animal is prepared mix-enveloping mouse source RV, CDV, RPV and CPV single-anti being labeled with colloidal gold on glass cellulose membrane to form combination pad; enveloping IgG of purified dog-resisting RV, CDV, FPV and CPV on R, D, F and P positions of detection line at nitric acid cellulose membrane and enveloping rabbit resisting mouse IgG on C position of quality control line at nitric acid cellulose membrane to from detection membrane.

Description

Dog, the important eqpidemic disease cause of disease of cats collaurum Unionpay close and detect test paper and technology of preparing thereof

Technical field:

The present invention relates to a kind of dog, the important eqpidemic disease cause of disease of cats collaurum Unionpay closes and detects test paper and preparation method thereof, be used for fast detecting, the diagnosis of important eqpidemic diseases such as dog, cats rabies, canine distemper, cat distemper heat and canine parvovirus disease, belong to animal epidemic detection technique field.

Background technology:

Dog, the important eqpidemic diseases of cats such as rabies, canine distemper, cat distemper heat and canine parvovirus disease be serious harm China fur economic animal industry not only, and the health of the uncommon wild animal of multiple treasure such as tiger, lion, wolf, the morbidity death that also can cause the people that has in serious threat.To fundamentally control these eqpidemic diseases, the generation that primary is to above-mentioned eqpidemic disease, popularly make in time, monitoring accurately.For the important eqpidemic disease of dog cats, cause of disease detection method commonly used in the world at present has methods such as viral separation, electron microscopic observation, PCR or RT-PCR, but these methods all need certain instrument and equipment and higher operative technique, have influenced its application aspect epidemiological surveillance.

Collaurum is a kind of detection technique that development in recent years is got up, at first be used for immuno-electron microscope by Faulk and Taylor (1971), has become additional greatly beyond fluorescein, radioactive isotope and the enzyme labeling now.Colloid gold test paper is to utilize antigen-antibody reaction and immunochromatography principle, with antigen or antibody with colloid gold label, when containing in the sample to be checked with the antibody of antigen or antibody specific bond or antigen, gathering will take place and produce macroscopic colour band, have easy, quick, visual result, advantage such as accurate.

Summary of the invention:

The invention provides a kind of dog, the detection test paper closes in the important eqpidemic disease cause of disease of cats collaurum Unionpay, can hydrophobin easy, that quickly and accurately dog, feline body are contained (RV), CDV (CDV), feline distemper virus (FPV) and canine parvovirus cause of diseases such as (CPV) make detection.

The invention also discloses the preparation technology of above-mentioned detection test paper, be suitable for suitability for industrialized production.

Solution of the present invention is the immunology ultimate principle according to antigen-antibody energy specific bond, utilize colloidal gold immunochromatographimethod technology and silver to dye the reinforcement technology, monoclonal antibodies such as mouse source RV, CDV, FPV and the CPV mixing of colloid gold label is coated on the plain film of glass fibre, makes pad; Respectively the IgG of purified dog-resisting RV, CDV, FPV and CPV etc. is coated on detection line R, D, F, the P place of nitrocellulose filter (NC), rabbit anti-mouse igg is coated on the nature controlling line C place of nitrocellulose filter, make the detection film; When containing corresponding virus in the test sample, can combine with corresponding gold mark mouse source monoclonal antibody earlier, then under the chromatography effect with the respective detection line on two resistive connections merge enrichment, strengthen the dyeing back through silver-colored dye liquor again and form macroscopic colour band, and then judge according to the result that develops the color.

Concrete preparation technology of the present invention is as follows: be by position shown in the accompanying drawing 1 respectively with corresponding reagent and material specking or bag by in plain film of glass fibre and nitrocellulose filter, form dog, the important eqpidemic disease cause of disease of cats joint-detection test paper.To place under the well by the sample pad that the plain film of glass fibre is made; Sample pad connects the pad of being made by the plain film of glass fibre, is coated with the monoclonal antibodies such as the anti-RV in mouse source, CDV, FPV and CPV of colloid gold label on the pad; Connect the detection film of making by the NC film behind the pad, detect on the film and can many detection lines and 1 nature controlling line be set with good grounds needs, wherein detection line R, D, F, P etc. are respectively IgG such as purified dog-resisting RV, CDV, FPV and CPV bag and are formed, and nature controlling line (C) be that the anti-mouse two of rabbit resists; Detect the film back and be connected with absorption pad; Down payment mark chromatographic test paper prepares requirement more at last, carries out adhesion, suppresses and cuts, and makes this antibody combined detection test paper.

The present invention has following advantage compared with prior art: detect easy, suitable, quick, economic, pollution-free, the easy to operate and interpretation of test paper, need be by Other Instruments equipment, and can detect multiple dog, the important eqpidemic disease cause of disease of cats simultaneously, be specially adapted to on-the-spot detection and epidemiology survey etc.Preparation method's science of the present invention, cost is low, stability is high, good reproducibility, is easy to make, storage and transport.

Description of drawings

Fig. 1 is the structural representation that the present invention detects test paper:

Fig. 2 is that the present invention detects the test paper sectional view.

1-plastic clip, 2-well, 3-viewport, 4-sample pad, 5-pad (are made by the plain film of glass fibre, on be coated with mouse source monoclonal antibodies such as gold mark RV, CDV, FPV and CPV), 6-detects film and (made by nitrocellulose filter, last specking has many detection lines and 1 nature controlling line, wherein R, D, F, P etc. are detection line, are coated with IgG such as the anti-RV of purifying dog, CDV, FPV and CPV respectively; C is a nature controlling line, and it is anti-to be coated with the anti-mouse of rabbit two), 7-absorption pad, 8-liner plate.

Embodiment

By following examples the present invention is described for example further, and do not limit the present invention in any way, under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.

Embodiment 1

According to shown in Figure 1, it is as follows that the present invention detects the test paper structure:

Liner plate 8 big envelopes are in plastic clip 1, and the surface of plastic clip 1 offers well 2 and viewport 3, are assembled with treated sample pad 4, pad 5 on the surface of liner plate 8, detect film 6 and absorption pad 7.Wherein, pad 5 is made by the plain film of glass fibre, on be coated with mouse source monoclonal antibodies such as gold mark RV, CDV, FPV and CPV; Detect film 6 and made by nitrocellulose filter, last specking has many detection lines and 1 nature controlling line, and R, D, F, P etc. are detection line, are coated with IgG such as the anti-RV of purifying dog, CDV, FPV and CPV respectively; C is a nature controlling line, is coated with the anti-mouse two of rabbit and resists; Detecting film is connected with absorption pad 7.

Embodiment 2

1) preparation of cause of disease monoclonal antibody such as RV, CDV, FPV and CPV and how anti-IgG: inoculate sensitive cells separately with cause of diseases such as RV, CDV, FPV and CPV respectively, cultivate each virus of propagation, immune Balb/c mouse of difference and domesticated dog after concentrating purification, prepare each viral monoclonal antibody and polyvalent antibody, and with this further its monoclonal antibody of preparation and how anti-IgG, standby in-30 ℃ of preservations.

2) preparation of cause of disease solid-phase immunity absorption carriers such as RV, CDV, FPV and CPV: use the how anti-IgG bag of prepared cause of diseases such as RV, CDV, FPV and CPV by solid phase carriers such as magnetic bead, latex particles, the efficient immunoadsorption carrier for preparing above-mentioned cause of disease respectively, and by the proper proportion mixing, make associating cause of disease solid-phase immunity absorption carrier, add antiseptic, standby in 4 ℃ of preservations.When sample pre-treatments, get sample 3-5ml to be checked, add an amount of solid-phase immunity absorption carrier, room temperature effect 20-30 minute, centrifugal or place magnetic bead frame precipitation magnetic bead, supernatant discarded adds 50-100ml antigen-antibody dissociation solution and is used for detecting in staying immunoadsorption carrier.

3) preparation of gold mark RV, CDV, FPV and CPV monoclonal antibody IgG: adopt collaurum protein labeling technology, use the prepared RV of colloid gold label, CDV, FPV and CPV monoclonal antibody IgG respectively, make the gold mark IgG of above-mentioned virus, mix by a certain percentage, make the antigen agent for capturing, adding preservative agent, standby in 4 ℃ of preservations.

4) rabbit anti-mouse igg preparation: prepare the anti-mouse serum of rabbit with Balb/c mouse IgG immunizing rabbit, and therefrom extract IgG, the preparation rabbit anti-mouse igg, adding preservative agent, standby in-30 ℃ of preservations.

5) pad preparation: made gold mark antigen agent for capturing is sprayed on the plain film of glass fibre, makes reaction film.4 ℃ of kept dry are standby.

6) detect film preparation: with how anti-IgG of prepared RV, CDV, FPV and CPV and rabbit anti-mouse igg, specking is made the detection film in detection line R, D, F, P place and the nature controlling line C place of nitrocellulose filter (NC) respectively.4 ℃ of kept dry are standby.

7) detect the test paper assembling: detect test paper by colloidal gold chromatographic and prepare requirement, as scheme above-mentioned prepared and material that choose is carried out adhesion, suppresses and cut into test strip, the test card of making in the plastic clip of packing into, the plastic pouch that add drying agent and packing, is closed in, 4 ℃ of kept dry.

8) preparation of silver-colored reinforcement corresponding reagent:

The preparation of activating solution: with the citrate buffer of deionized water preparation 0.05mol/L, PH3.8.

The preparation of silver dye liquor: silver acetate, p-dihydroxy-benzene, gum arabic are dissolved in the deionized water, make that its final concentration is respectively 0.11%, 0.35%-1.7% and 7%-10%.

The preparation of stop bath: 20% sodium thiosulfate solution.

Claims

1, the detection test paper closes in a kind of dog, the important eqpidemic disease antigen colloidal gold of cats Unionpay, it is characterized in that: liner plate 8 big envelopes are in plastic clip 1, the surface of plastic clip 1 offers well 2 and viewport 3, is assembled with treated sample pad 4, pad 5 on the surface of liner plate 8, detects film 6 and absorption pad 7.Wherein, pad 5 is made by the plain film of glass fibre, on be coated with mouse source monoclonal antibodies such as gold mark RV, CDV, FPV and CPV; Detect film 6 and made by nitrocellulose filter, last specking has many detection lines and 1 nature controlling line, and R, D, F, P etc. are detection line, are coated with IgG such as the anti-RV of purifying dog, CDV, FPV and CPV respectively; C is a nature controlling line, is coated with the anti-mouse two of rabbit and resists; Detecting film is connected with absorption pad 7.

2, according to the preparation method of the described detection test paper of claim 1, comprise the steps:

1) preparation of cause of disease monoclonal antibody such as RV, CDV, FPV and CPV and how anti-IgG: inoculate sensitive cells separately with cause of diseases such as RV, CDV, FPV and CPV respectively, cultivate each virus of propagation, immune Balb/c mouse of difference and domesticated dog after concentrating purification, prepare each viral monoclonal antibody and polyvalent antibody, and with this further its monoclonal antibody of preparation and how anti-IgG, standby in-30 ℃ of preservations;

2) preparation of cause of disease solid-phase immunity absorption carriers such as RV, CDV, FPV and CPV: use the how anti-IgG bag of prepared cause of diseases such as RV, CDV, FPV and CPV by solid phase carriers such as magnetic bead, latex particles, the efficient immunoadsorption carrier for preparing above-mentioned cause of disease respectively, and by the proper proportion mixing, make associating cause of disease solid-phase immunity absorption carrier, add antiseptic, standby in 4 ℃ of preservations; When sample pre-treatments, get sample 3-5ml to be checked, add an amount of solid-phase immunity absorption carrier, room temperature effect 20-30 minute, centrifugal or place magnetic bead frame precipitation magnetic bead, supernatant discarded adds 50-100ml antigen-antibody dissociation solution and is used for detecting in staying immunoadsorption carrier;

3) preparation of gold mark RV, CDV, FPV and CPV monoclonal antibody IgG: adopt collaurum protein labeling technology, use the prepared RV of colloid gold label, CDV, FPV and CPV monoclonal antibody IgG respectively, make the gold mark IgG of above-mentioned virus, make the antigen agent for capturing, adding preservative agent, standby in 4 ℃ of preservations;

4) rabbit anti-mouse igg preparation: prepare the anti-mouse serum of rabbit with Balb/c mouse IgG immunizing rabbit, and therefrom extract IgG, the preparation rabbit anti-mouse igg, adding preservative agent, standby in-30 ℃ of preservations;

5) pad preparation: made gold mark antigen agent for capturing is sprayed on the plain film of glass fibre, makes reaction film, kept dry is standby;

6) detect film preparation: with how anti-IgG of prepared RV, CDV, FPV and CPV and rabbit anti-mouse igg, specking is made the detection film in detection line R, D, F, P place and the nature controlling line C place of nitrocellulose filter (NC) respectively, and kept dry is standby;

7) detect the test paper assembling: detect test paper by colloidal gold chromatographic and prepare requirement, as scheme above-mentioned prepared and material that choose is carried out adhesion, suppresses and cut into test strip, the test card of making in the plastic clip of packing into, the plastic pouch that add drying agent and packing, is closed in, kept dry;

8) preparation of silver-colored reinforcement corresponding reagent:

The preparation of activating solution: with the citrate buffer of deionized water preparation 0.05mol/L, PH3.8;

The preparation of silver dye liquor: silver acetate, p-dihydroxy-benzene, gum arabic are dissolved in the deionized water, make that its final concentration is respectively 0.11%, 0.35%-1.7% and 7%-10%;

The preparation of stop bath: 20% sodium thiosulfate solution.