Summary of the invention
The present invention: one, provide a kind of biological specimen side direction reciprocation type immune chromatography method; Two, provide by the bottom of card, the diagnostic device that forms of Ka Gai and diagnosis test paper; Three, provide a kind of biological specimen side direction toward reciprocation type immunochromatography diagnosis test paper.
According to diagnosis test paper of the present invention, described diagnosis test paper comprises PVC board 11, and is pasted onto sample layer 7, mark layer 13, coated film 12 and the water accepting layer 10 in PVC board 11, it is characterized in that:
Described diagnosis test paper also comprises and is pasted onto damping fluid sample layer 14 in PVC board 11 and waterproof barrier film (6),
Wherein,
(I)
Water accepting layer 10 is pasted onto in PVC board 11, and its surface covers waterproof barrier film 6 and sample layer 7 successively, and waterproof barrier film 6 is easily extracted out from horizontal direction between sample layer 7 and water accepting layer 10;
(II)
Level along waterproof barrier film 6 extracts direction out, and damping fluid sample layer 14, mark layer 13, coated film 12 and water accepting layer 10 are pasted onto in PVC board 11 successively;
(III)
Sample layer (7), coated film (12), mark layer (13), damping fluid sample layer (14) stack gradually, and retain space between water accepting layer (10) and coated film (12); 2 ~ 7mm is spaced apart between preferred water accepting layer (10) and coated film (12).
Compared with the unidirectional immuno-chromatographic test paper strip shown in Fig. 1, the advantage of diagnosis test paper of the present invention is: sample layer 7 is arranged on water accepting layer 10, and centre waterproof water-separating film 6 is isolated.During chromatography, sample droplets is added on sample layer 7, antigen in sample or antibody enter coated film 12 through sample layer 7, with the antibody on coated film response line or antigen-reactive, and continue chromatography to nature controlling line, now level extracts waterproof barrier film 6 out, sample liquid contacts with water accepting layer 10, because the liquid absorption of water accepting layer is much larger than the liquid absorption of coated film, therefore there are 180 DEG C of conversions in the chromatography direction of sample, namely nature controlling line → response line direction is transformed into by original response line → nature controlling line direction, realize secondary chromatography, antigen in sample or antibody again with the antibody on response line or antigen-reactive, make the antibody in the antigen in sample or antibody and detection line or antigen-reactive abundant, ELISA test strip sensitivity can improve 50 ~ 100 times.
According to the present invention, each layer of composition reciprocation type immunochromatography diagnosis test paper is all pasted onto in PVC board, therefore, described by liner plate with cohesive material.
According to diagnosis test paper of the present invention, the liquid absorption of water accepting layer is 200 ~ 500 μ l, water accepting layer and coated film interval 2mm ~ 7mm, and described water accepting layer is thieving paper, thickness 0.3 ~ 1mm, and liquid absorption is 200 ~ 500 μ l.
Diagnosis test paper of the present invention, described coated film is nitrocellulose filter, PVDF membrane or PES (polyethersulfone resin membrane), thickness 50 ~ 200 μm, flow velocity 50 ~ 240S/4cm, preferable flow rate 120 ~ 140S/4cm, preferred film aperture membrane aperture 3 ~ 15 μm, more preferably membrane aperture 8 μm.
Diagnosis test paper of the present invention, described sample layer is nonwoven fabrics, non-woven paper, polyester film, glass fibre membrane, nylon membrane etc., thickness 0.2 ~ 1mm.
Diagnosis test paper of the present invention, the membrane material of described mark layer is nonwoven fabrics, non-woven paper, polyester film, glass fibre membrane or nylon membrane, thickness 0.2 ~ 1.0mm.
Diagnosis test paper of the present invention, described damping fluid sample layer is nonwoven fabrics, non-woven paper, polyester film, glass fibre membrane, nylon membrane, thickness 0.2 ~ 0.5mm.
Diagnosis test paper of the present invention, described mark layer label is collaurum, electroselenium, latex, quantum dot, up-converting phosphor (UCP), diacetylene vesica, styrene high molecular polymer or magnetic microsphere.
Biological specimen side direction reciprocation type immunochromatography diagnostic device of the present invention, by the bottom of card, Ka Gai and diagnosis test paper form, described diagnostic reagent srip is fixed at the bottom of card; Described diagnosis test paper comprises PVC board 11 and is pasted onto sample layer 7, mark layer 13, coated film 12 and the water accepting layer 10 in PVC board 11, it is characterized in that:
Described diagnosis test paper also comprises and is pasted onto damping fluid sample layer 14 in PVC board 11 and waterproof barrier film 6,
Wherein,
(I)
Water accepting layer 10 is pasted onto in PVC board 11, and its surface covers waterproof barrier film 6 and sample layer 7 successively, and waterproof barrier film 6 is easily extracted out from horizontal direction between sample layer 7 and water accepting layer 10;
(II)
Level along waterproof barrier film 6 extracts direction out, and damping fluid sample layer 14, mark layer 13, coated film 12 and water accepting layer 10 are pasted onto in PVC board 11 successively;
(III)
Sample layer 7, coated film 12, mark layer 13, damping fluid sample layer 14 stack gradually, retention gap between water accepting layer 10 and coated film 12; 2 ~ 7mm is spaced apart between preferred water accepting layer 10 and coated film 12.
Described Ka Gai corresponds to damping fluid sample layer 14, coated film 12 and sample layer 7 place and has window, the card window covered corresponding to sample layer is sample window 4, window corresponding to coated film is observation window 3, window corresponding to damping fluid sample layer is damping fluid window 2, at observation window, the position mark that response line is corresponding is " T ", and the position mark that nature controlling line is corresponding is " C "; Test strips to be fixed at the bottom of card in 17 by pickup groove 15,16; And waterproof barrier film 6 is easily extracted out from pulling and pushing horizontal direction groove 5.
Such as, in a specific embodiment of the present invention, detect the side direction reciprocation type immunochromatography colloidal gold strip of serum HIV1/2 antibody, be made up of the thieving paper that PVC board and PVC board are pasted successively, plastic sheet, sample layer, sale cellulose membrane coated film (NC film), colloid gold label layer, damping fluid sample layer, described nitrocellulose membrane coated film (NC film) is coated with goat anti-rabbit igg and HIV1/2 recombinant antigen.Mark layer scribbles colloid gold label rabbit igg and colloid gold label HIV1/2 recombinant antigen, and drying at room temperature.Described NC film is sprayed with goat anti-rabbit igg (C line) and HIV1/2 recombinant antigen (T line) with Membrane jetter.Described thieving paper is compression filter paper, long 20 ~ 35mm, wide 2.5mm ~ 10mm, thick 1mm.Described NC membrane material is tygon, thick 18mm ~ 35mm, preferred 20mm.Described plastics length of a film 30mm ~ 40mm, wide 25mm ~ 40mm, thick 0.1mm ~ 0.5mm.Described sample pad material material is non-woven paper, nonwoven fabrics, glass fibre membrane, polyester film etc., long 10mm ~ 30mm, wide 2.4mm ~ 8mm, thick 0.2mm ~ 1mm.Described colloidal gold pad material is nonwoven fabrics, glass fibre membrane, the long 2.5mm ~ 10mm of polyester film, wide 2.4mm ~ 8mm, thick 0.2mm ~ 0.5mm.Buffer layer material is glass fibre membrane, nonwoven fabrics, non-woven paper etc., long 18mm ~ 28mm, wide 2.4mm ~ 8mm, thick 0.2mm ~ 1mm.
This test strips is highly sensitive, simple and efficient, without the need to professional's operation, has boundless application prospect.
In a particular embodiment of the present invention, side direction reciprocation type immunochromatography colloidal gold strip is loaded in a kind of new shape card, this card of moulding is made up of lid and bottom, cover and be provided with sample window S and watch window 3 (having C, T to identify), damping fluid window B, three window linear array, wherein sample window and damping fluid window are square.View window oral pore is rectangle, long 14mm, wide 2.5mm ~ 7.8mm, has a wide 2.5mm ~ 8.2mm, the concave groove of high 0.5mm near Su Kagai side, sample window side, is convenient to waterproof barrier film 6 horizontal direction and extracts out, can fix test strips simultaneously.After extracting waterproof barrier film 6 out, the second time chromatography process of side direction reciprocation type immunochromatography colloidal gold strip of the present invention is accomplished.Through secondary chromatography, antigen-antibody reaction is more abundant, thus increases detection paper sensitivity.Mould at the bottom of card and play fixing test strips.
Biological specimen side direction reciprocation type chromatography method of the present invention comprises secondary chromatography,
Described diagnosis test paper comprises PVC board 11, and is pasted onto sample layer 7, mark layer 13, coated film 12 and the water accepting layer 10 in PVC board 11, it is characterized in that:
Described diagnosis test paper also comprises and is pasted onto damping fluid sample layer 14 in PVC board 11 and waterproof barrier film 6,
Wherein,
(I)
Water accepting layer 10 is pasted onto in PVC board 11, and its surface covers waterproof barrier film 6 and sample layer 7 successively, and waterproof barrier film 6 is easily extracted out from the gap horizontal direction between sample layer 7 and water accepting layer 10;
(II)
Level along waterproof barrier film 6 extracts direction out, and PVC board 11 is arranged in sequence with damping fluid sample layer 14, mark layer 13, coated film 12 and water accepting layer 10,
Described coated film 12 responds line (T) and nature controlling line (C), respectively envelope antigen or antibody, described response line near sample layer 7,
Sample droplets is added in sample layer 7, antigen in sample or antibody are analysed to coated film 12 through sample layer by layer, first with on response line wrap antibody or the antigen-reactive of quilt, and chromatography is to nature controlling line, now level pumps waterproof barrier film 6, sample liquid directly contacts with water accepting layer 10, because the liquid absorption of water accepting layer is much larger than the liquid absorption of coated film, therefore there are 180 DEG C of conversions in the chromatography direction of sample, namely nature controlling line → response line direction is transformed into by original response line → nature controlling line direction, realize secondary chromatography, antigen in sample or antibody again with the antibody on response line or antigen-reactive, antibody in antigen in sample or antibody and detection line or antigen are fully reacted.
After pumping waterproof barrier film 6, add label damping fluid to damping fluid sample layer 14, damping fluid is via cushion pad, the labelled antigen of release mark layer or antibody, via coated film chromatography, antibody or antigen-reactive with coated film wrapping quilt, show macroscopic detection line and nature controlling line.
Existing test strips then adopts disposable chromatographic theory, antigen in sample or antibody is antibody or antigen-reactive directly and in mark layer, again with antigen or the antibody response of T, C line on coated film, this unidirectional chromatography to decrease in sample antigen or antibody response time on antigen or antibody and label film and coated film, therefore Percentage bound is low, and sensitivity is also undesirable.Test strips of the present invention adopts secondary chromatographic theory, in sample, antigen or antibody are through secondary chromatography, extend and the antibody of coated film T, C line bag quilt or antigen-reactive time, add the Percentage bound of antigen-antibody, therefore sensitivity improves greatly, usually exceeds 50 ~ 100 times than the sensitivity of conventional test strips.
Embodiment
Embodiment 1
As shown in Fig. 2 .1 ~ 2.4, described device by the bottom of card 17, card lid 1 and diagnosis test paper 18 form.Described diagnosis test paper, based on PVC board 11, pastes water accepting layer 10 above, and surface covers waterproof barrier film 6 and sample layer 7 successively, and waterproof barrier film 6 is easily extracted out from groove 5 level that pulls and pushes between sample layer 7 and water accepting layer 10.Level along waterproof barrier film 6 extracts direction out, damping fluid sample layer 14, mark layer 13, coated film 12 and water accepting layer 10 are pasted onto in PVC board 11 successively, sample layer 7, coated film 12, mark layer 13, damping fluid sample layer 14 stack gradually, retention gap between water accepting layer 10 and coated film 12; Preferably, the spacing between water accepting layer 10 and coated film 12 is 2 ~ 7mm.On described coated film 12, response line 8 and nature controlling line 9 envelope antigen or antibody respectively, described response line 8 is near sample layer 7, wherein, the card window covered corresponding to sample layer is sample window 4, and the window corresponding to coated film is watch window 3, and the window corresponding to damping fluid sample layer is damping fluid window 2, in watch window, response line is designated " T ", and nature controlling line is designated " C ".Described diagnostic reagent srip is fixed at the bottom of card.Have 0.5mm × 5mm wide groove in card lid sample window side, card covers mark C (nature controlling line), T (response line), S (sample window), B (damping fluid window) printed words.More than design is with reference to the size of test strips, and card is built fixing test strips and controlled the effect in sample chromatography direction, and Ka Dize is mainly used in fixing test strips.
In this embodiment, the material of water accepting layer, coated film, mark layer, sample layer, damping fluid sample layer and parameter as shown in table 1.
Such as, first paste thieving paper in one end of PVC board, and then paste waterproof barrier film, sample pad (being pressed in 1mm on NC film), NC film successively, sample, once dropping, is just analysed to NC rete under rainbow suction, reaches C line through T line.Now level extracts waterproof barrier film out, and sample pad is directly contacted with thieving paper, and therefore changes the chromatography direction of sample on NC film, thus realizes the secondary chromatography of sample on NC film.Compared with conventional colloidal gold strip, because the antigen on T line and the antibody response time in sample are more abundant, therefore the detection sensitivity of colloid gold reagent bar have also been obtained and greatly improves.
Table 1
The preparation of the preparation of mark layer and coated film in above-described embodiment, sample pretreatment, antibody labeling method and mark damping fluid is laboratory routine techniques.For HIV1/2 side direction reciprocation type immunochromatography colloidal gold diagnosis test strips:
1, collaurum preparation:
0.01%HAuCl is prepared with distilled water
41000ml, boils.Add 1% citric acid three sodium solution 0.8 ~ 5ml according to colloid gold particle size, continue to boil and boil 10 minutes, existing purple or orange-red solution, colloidal solid size is 60 ~ 20mm.
2, colloid gold label:
Get colloidal gold solution 100ml, use 0.1mol/L K
2cO
3the pH value of colloidal gold solution is adjusted to 9.4 by liquid, get 0.5ml HIV1/2 recombinant antigen 2mg/ml, add in 100ml colloidal gold solution while stirring, continue stirring 15 ~ 60 minutes, add 1% skimmed milk power 1ml and continue stirring 5 ~ 10 minutes, centrifugal 30 ~ 50 minutes with 10000 ~ 12000 revs/min; Abandon supernatant, precipitation, precipitate with the PBS damping fluid resuspension containing 0.5%BSA, add 2 ~ 5ml0.05%NaN simultaneously
3, 4 DEG C of storages are for subsequent use.
Get colloidal gold solution 100ml, use 0.1mol/L K
2cO
3the pH value of colloidal gold solution is adjusted to 7.6 by liquid, get the goat anti-rabbit igg (5mg/ml) of 0.2ml purifying, add in 100ml colloidal gold solution while stirring, continue stirring 15 ~ 60 minutes, add 1% skimmed milk power 1ml and continue stirring 5 ~ 10 minutes, centrifugal 30 ~ 50 minutes with 10000 ~ 12000 revs/min; Abandon supernatant, precipitation, precipitate with the PBS damping fluid resuspension containing 0.5%BSA, add 2 ~ 5ml0.05%NaN simultaneously
3, 4 DEG C of storages are for subsequent use.
3., the preparation of coated film:
Prepare HIV1/2 recombinant antigen with 0.01M PBS (pH 7.2), concentration is 1.2mg/ml, and goat anti-rabbit igg antibody concentration is 2mg/ml.
4, side direction reciprocation type immunochromatography HIV colloidal gold test is prepared:
Above-mentioned gold mark HIV1/2 recombinant antigen and goat anti-rabbit igg antibody are coated on glass fibre membrane, polyester film, nonwoven fabrics, temperature 37 DEG C, drying 8 hours under humidity 20% condition; With Membrane jetter, HIV1/2 recombinant antigen and goat anti-rabbit igg antibody are sprayed on NC film simultaneously, form T line and C line, spacing 5mm, ambient temperature overnight, for subsequent use.Thieving paper, waterproof barrier film, sample pad, NC film, colloidal gold pad, collaurum cushion pad are pasted in PVC board successively, is cut into the slice of wide 2.5 ~ 8mm, be side direction reciprocation type immunochromatography HIV colloidal gold test.
5, detect
Sample droplets is added in sample layer 7, antibody in sample or antigen enter coated film 12 through sample layer, with the recombinant antigen on coated film response line or antibody response, then chromatography is to nature controlling line, now level pumps waterproof barrier film 6, sample liquid directly contacts with water accepting layer 10, because the liquid absorption of water accepting layer is much larger than the liquid absorption of coated film, therefore there are 180 DEG C of conversions in the chromatography direction of sample, namely nature controlling line → response line direction is transformed into by original response line → nature controlling line direction, realize secondary chromatography, antigen in sample or antibody again with the antibody on response line or antigen-reactive, antibody in antigen in sample or antibody and detection line or antigen are fully reacted.
After pumping waterproof barrier film 6, add label damping fluid to damping fluid sample layer 14, damping fluid is via cushion pad, the labelled antigen of release mark layer or antibody, via coated film chromatography, antibody or antigen-reactive with coated film wrapping quilt, show macroscopic detection line and nature controlling line.
Detect embodiment
Table 1 is depicted as 6 kinds of side direction reciprocation type immunochromatography colloidal gold tests, detects the antibody in serum, urine and saliva or antigen respectively, and contrast side direction unidirectional immunochromatography diagnosis test paper is commercially available, and testing result is as follows:
The detection of table 2-1 serum sample
Table 2-1 continues
Above list data display: obtain identical detection sensitivity, side direction reciprocation type immunochromatography diagnosis test paper is higher 100 times than side direction unidirectional immunochromatography diagnosis test paper to the dilutability of sample, thus the former more highly sensitive than the latter 100 times.
The detection of table 2-2 saliva sample
Table 2-2 continues
Above list data display: when saliva HIV1/2 antibody content improves 500 times, the indistinct wired G0.5 (critical value) of commercially available side direction unidirectional immunochromatography reagent, and HIV1/2 antibody content is when improving 1000 times, just manifest obvious response line G1 (the weak positive), continue to improve HIV1/2 antibody content 100000 times in saliva, test strips shows obvious response line G2 (the weak positive).And biological specimen side direction reciprocation type side direction immunochromatography diagnosis test paper of the present invention is G4 (generally positive) with saliva stoste display colour developing, when antibody titer brings up to 500 times, display strong positive (G6.5-7), therefore the detection sensitivity of biological specimen side direction reciprocation type side direction immunochromatography diagnosis test paper of the present invention exceeds 100 times than the unidirectional immunochromatography diagnosis test paper of commercially available side direction.
The detection of table 2-3 urine specimen
Table 2-3 continues
Above list data display: when urine HIV1/2 antibody content improves 1000 times, the indistinct wired G0.5 (critical value) of commercially available side direction unidirectional immunochromatography reagent, and HIV1/2 antibody content is when improving 10000 times, just manifest obvious response line G1 (the weak positive), further HIV1/2 antibody titer is improved 100000 times, then there is obvious response line G2 (the weak positive), and biological specimen side direction reciprocation type side direction immunochromatography diagnosis test paper urine extension rate of the present invention is when being 100,000 times, its detection sensitivity exceeds 100 times than the unidirectional immunochromatography diagnosis test paper of commercially available side direction.