CN1580778A - Colour latex chromato graphic analysis diagnositc test paper tape and its preparing method - Google Patents
Colour latex chromato graphic analysis diagnositc test paper tape and its preparing method Download PDFInfo
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- CN1580778A CN1580778A CN 200410027293 CN200410027293A CN1580778A CN 1580778 A CN1580778 A CN 1580778A CN 200410027293 CN200410027293 CN 200410027293 CN 200410027293 A CN200410027293 A CN 200410027293A CN 1580778 A CN1580778 A CN 1580778A
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Abstract
The present invention discloses a paper test strip for diagnosis by adopting colour latex chromatography. Said paper test strip is chracterized by that on the bottom liner the sample pad, glas fibre membrane coated with colour latex microparticle labeled protein, coating film and water-absorbing paper are successively mutually overlapped and adhered together to form a film strip. Said coating film has a detection zone and a control zone, the detection zone can be coated with antibody or antigen, and the control zone is coated with antirat antibody. Said invention has the advantages of quick and convenient detection and simple operation.
Description
Technical field
The invention belongs to field of medical examination, relate to a kind of diagnosis test paper that utilizes colored latex chromatography.
Background technology
It is to continue after the colloidal gold-labeled method that colored latex mark layer is analysed detection technique, on the basis of latex agglutination test, grow up, as a kind of immunological method, it is the combination of immune affine technology, engram technology, immunolabelling technique and chromatographic technique, but since its equally have quick, easy and simple to handle, stable reagent room temperature accumulating, be difficult for the pollution characteristics development rapidly with golden mark technology.
Colored latex labelling technique is with the colored latex microsphere of different-diameter scope 0.1 μ m～1 μ m and contains carboxyl, amino, ester class or other macromolecular substances covalent bond such as hydroxyl, the carboxyl that utilizes macromolecular substances to have, amino, group conjugated protein materials (antigen/antibody) such as hydroxyl, when the protein (antigen/antibody) of this colored latex mark with detect sample in enough corresponding antibodies/antigen combine the latex labeled complex of back formation, the antigen/antibody of bag quilt is caught on the tunicle, when colored latex label is built up in a large number at corresponding part place, form the naked eyes red color visible, aubergine, black, the band of blueness or other colors, thereby can be used in the qualitative or semiquantitative tachysynthesis detection method.
In immune detection, the main both at home and abroad test strips that adopts methods such as radioimmunoassay, enzyme linked immunosorbent assay (ELISA) and colloid gold label.All there is defective in various degree in these test strips.Immune colloidal gold chromatography method test strips with extensive employing at present is an example, has the following disadvantages:
(1) collaurum is a kind of nano particle, and preparation collaurum nano particle is difficult, and the difficult control of particle diameter evenly.
(2) use strong reductant in the colloid gold label process, the person's ion that will contact the heavy metal simultaneously has certain influence to health and safety.
(3) the colloid gold label process is a physical adsorption process, so less stable during liquid phase.
(4) can only be solid color---aubergine after the colour developing, can not require changes colour according to different kinds or difference.
Summary of the invention
The object of the present invention is to provide a kind of colored latex chromatography diagnosis test paper, can bring into play quick, easy, the easy to operate advantage of latex mark chromatography detection technique, avoid the shortcoming and defect of existing detection technique.
Colored latex chromatography diagnosis test paper of the present invention, this test strips is the mutual in turn film bar that overlaps glass fibre membrane, coated film and the thieving paper of ground stickup sample pad, coating coloured glue lactoconium labelled protein and form on end liner, coated film has detection zone and control zone, detection zone coated antibody or antigen, wrap by anti-mouse antibody the control zone.
On the described coated film, according to detection methods such as employing double antibody sandwich method, dual-antigen sandwich method, competition inhibition methods, bag is by corresponding antibody or antigen in detection zone; The anti-mouse antibody of quilt that wrap the control zone is that the IgG immune mouse with purifying obtains.
Another object of the present invention is to provide a kind of preparation method of colored latex chromatography diagnosis test paper.
The preparation method of colored latex chromatography diagnosis test paper of the present invention may further comprise the steps:
A. colored latex is covalent activated: ultrasonic Treatment latex microsphere body is after 30 seconds, and regulating the latex microsphere bulk concentration is 1.0 * 10
6～1.0 * 10
7/ ml, centrifugal 1～5 minute of 8000 * g, centrifugal back collecting precipitation thing be with distilled water or 50mM～200Mm pH6.0～7.0 sodium radio-phosphate,P-32 solutions dissolving, and ultrasonic Treatment 30 seconds; Add a certain amount of 20～100mg/mlEDC, mixing; Incubated at room 8000 * g after 30 minutes, centrifugal 1～5 minute, precipitation is with the citrate buffer solution dissolving of pH5.0～6.0 of 20mM～100mM, be positioned in 2～8 ℃ of environment standby, with labelled protein;
B. the preparation of coloured glue lactoconium labelled protein: with the colored latex ultrasonic Treatment after 30 seconds after the activation in the steps A, ratio according to the colored latex of 1 μ g～125 μ g albumen/100ul adds the protein that needs mark, the stirring at room reaction is 2 hours behind the mixing, 3 centrifuge washings, each 8000 * g, centrifugal 1～5 minute, precipitation is recovered certain volume with PBS-TBN dissolving and ultrasonic Treatment 30 seconds with phosphate buffer, the placement room temperature is standby, with the coated glass fiber film;
C. antibody or antigen coated to nitrocellulose filter and sealing: bag detection zone of quilt and control zone are that antigen/antibody and anti-mouse antibody are regulated finite concentration on the film, the film liquid measure is 20ul/35cm, be cushioned liquid dilution envelope antigen/antibody with bag, concentration is 6～15 μ g/ml, antigen/antibody and anti-mouse antibody are sprayed onto on the glass fibre membrane, two intervals should be careful even every 5mm, room temperature airing 20 minutes; 25 ℃～37 ℃ were soaked 60 minutes at confining liquid, took out rearmounted 25 ℃～37 ℃ oven dry and handled 2 hours, and envelope is used in order to pasting board;
D. on end liner, paste glass fibre membrane, coated film and the thieving paper of sample pad, coating coloured glue lactoconium labelled protein in turn mutually overlap joint, obtain test paper plate, cut into the test strips of different in width as requested.
Detection principle of the present invention is with the colored latex microsphere of different-diameter scope 0.1 μ m～1 μ m and contains carboxyl, amino, ester class or other biological macromolecular substances such as antigen/antibody covalent bond such as hydroxyl, when the protein (antigen/antibody) of this colored latex mark with detect sample in enough corresponding antibodies/antigen combine the compound of back formation, the antigen/antibody of bag quilt is caught on the tunicle, colored latex mark thing is when corresponding part is in a large amount of accumulation, form the naked eyes red color visible, aubergine, black, the band of blueness or other colors, thereby can be used in the qualitative or semiquantitative tachysynthesis detection.(sandwich method) formed detection band is a macroscopic colour developing band, visible colour developing band occurs and be containing in the sample destination protein make detect with on the colored latex particle that gathers be finding of naked eye, if it is invisible that detection is with, show in the sample that no destination protein or concentration are very low, thus detect with on the colored latex particle that gathers can not be seen by the naked eye.
Colored latex chromatography diagnosis test paper of the present invention is compared with radioimmunoassay, euzymelinked immunosorbent assay (ELISA), have handling safety (no radiation pollute), easy (one step of simple operations finishes), be fit to single/part detect (put exempt from, enzyme exempt to be not suitable for single/part or small amount of sample detect) and quick advantages such as (about 10 minutes the result can be arranged); Compare with the immuno-gold labeling test strips, the present invention has the protein labeling process need not contact the various advantage such as variable of heavy metal ion and color.
Test strips of the present invention can be used the test strips of detection methods such as double antibody sandwich method, dual-antigen sandwich method, competition inhibition method, applied range, can be used in individual event detection or the multinomial quick detection test paper bar of an inspection, can detect whole blood, serum, blood plasma, saliva, urine, food equal samples; Can be used in the detection of destination proteins such as pregnant detection, medical diagnosis on disease, drugs detect, bacterium detects.
Description of drawings
Fig. 1 is a structural representation of the present invention;
Fig. 2 is the synoptic diagram of the present invention's testing result when adopting the double antibodies sandwich method.
Fig. 3 is the synoptic diagram of the present invention's testing result when adopting competition inhibition method.
Fig. 4 is the synoptic diagram that the present invention is used for inspection testing result when multinomial.
Embodiment
Colored latex chromatography diagnosis test paper of the present invention, as shown in Figure 1, this test strips be on end liner 1 in turn mutually overlap joint ground paste sample pad 2, apply glass fibre membrane 3, coated film 4 and the thieving paper 5 of coloured glue lactoconium labelled protein and the film bar that forms, coated film 4 has detection zone 6 and control zone 7, detection zone 6 coated antibodies or antigen, control zone 7 bags are by anti-mouse antibody.
Test strips of the present invention can be used the test strips of detection methods such as double antibody sandwich method, dual-antigen sandwich method, competition inhibition method.Fig. 2 is the result schematic diagram when adopting double antibodies sandwich method test strips, shown in Fig. 2 (a), the antigen in the sample or the concentration of antibody can make the coloured glue lactoconium at detection zone (T district) but 6 places gather and form the naked eyes band, visible band appears in control zone (C district) 7, represents the sample positive; Shown in Fig. 2 (b) sees, detection zone (T district) but 6 places do not form the naked eyes band, visible band appears in control zone (C district) 7, expression sample feminine gender does not contain detected antigen or antibody.Fig. 3 is the result schematic diagram that adopts competition inhibition method test strips, and shown in Fig. 3 (a), antigen in the sample or antibody combine the antibody or the antigen of colored latex mark with antigen that is coated on detection zone or antibody competition.When the concentration of antigen in the sample or antibody during enough in conjunction with the antibody of latex mark or antigen, when the sample process is wrapped by line, the albumen and envelope antigen or the antibodies that do not have unnecessary colored latex mark, can not gather the formation band at detection zone (T district) 6, promptly band does not appear in detection zone.Shown in Fig. 3 (b), negative findings shows no purpose antigen or antibody in the sample, and detection zone (T district) 6 no bands occur.
As shown in Figure 4, the present invention can be used for the multinomial test item of an inspection, shown in Fig. 4 (a), when containing whole tested project in the sample, can occur two and detect band (represent two and detect indexs), and a control is with; Shown in Fig. 4 (b), when containing a certain test item in the sample, corresponding detection band and control band appear; Shown in Fig. 4 (c), when not containing any test item, a control band only appears; If any band do not occur, illustrate that detection kit lost efficacy.
Embodiment one: very early pregnancy HCG latex test strip (double antibody sandwich method)
Present embodiment very early pregnancy HCG latex test strip adopts the chromatography of double antibody sandwich method to detect principle, utilizes colored latex mark hCG monoclonal antibody, and detection zone on the coated film (T) is coated with α-hCG monoclonal antibody, and Quality Control district (C) wraps by anti-mouse antibody.Contain when urging parathyrine human chorionic (hCG) in detecting sample, the hCG antigen in the sample combines with the hCG monoclonal antibody of latex mark, forms Ag-Ab
*Latex compound under the chromatography effect, arrives the detection zone (T) that is coated with α-hCG monoclonal antibody, and reaction forms Ab-Ag-Ab
*Latex compound, hCG concentration is not less than 25mlU/ml in sample, and colored latex mark thing when a large amount of accumulation, forms the band of naked eyes red color visible, aubergine, black, blueness or other colors herein, thereby can the judgement sample positive, i.e. person under inspection's pregnancy.
As shown in Figure 3, behind the application of sample, react and to see in 1～2 minute on detection zone (T district) 6 and Quality Control district (C district) 7 corresponding positions and the aubergine band occurs, if the aubergine band all appears in C district 7 and T district 6, as Fig. 3 (a), the result is positive, and interpret sample contains hCG; The aubergine band does not appear in T district 6, and as Fig. 3 (b), the result is negative, and interpret sample does not contain hCG.The aubergine band does not appear in C district 7, illustrates that test strips lost efficacy.
The double antibody sandwich method that present embodiment adopts can also be applicable to ovulation (LH, interstitialcellstimulating hormone (ICSH)), hepatitis B surface antibody (HbsAg), and cardiac troponin (Tn-I, Tn-T), tumor marker (AFP, PSA, CEA, FOB), Chlamydia (CT) etc.
Embodiment two: acquired immune deficiency syndrome (AIDS) HIV1/2 type diagnosis test paper (dual-antigen sandwich method)
The chromatography of present embodiment acquired immune deficiency syndrome (AIDS) HIV1/2 type diagnose test paper strip adoption dual-antigen sandwich method detects principle, utilize colored latex mark HIVgp36, gp41 and gp27 recombinant antigen, detection zone on the coated film (T) bag is by certain density HIVgp36, gp41 and gp27 recombinant antigen, Quality Control district (C) wraps by anti-mouse antibody.When containing HIV antibody in detecting sample, the HIV antibody in the sample combines with the HIV antigen of latex mark, forms Ab-Ag
*Latex compound under the effect of test strips chromatography, arrives and is coated with HIVgp36, the detection zone of gp41 and gp27 (T), and reaction forms Ag-Ab-Ag
*Latex compound, the HIV antibody concentration is 1: 1280 in sample, and colored latex mark thing when a large amount of accumulation, forms the band of naked eyes red color visible, aubergine, black, blueness or other colors herein, thereby can the judgement sample positive, i.e. person under inspection's infected by HIV; Negative situation illustrates that the person under inspection does not have HIV antibody, and is still negative after 21 days, gets rid of the possibility that the person under inspection carries HIV.
The dual-antigen sandwich method that present embodiment adopts can also be applicable to hepatitis B surface antibody (HbsAb), hepatitis C (HCV) etc.
Embodiment three: drugs test strip (competition inhibition method)
Present embodiment drugs test strip is based upon on the principle of competition inhibition immunochromatography, utilizes colored latex mark principle to detect in the urine sample or whether contains Poison in other sample.(the perhaps bond of drugs-BTG), wrap by anti-mouse antibody in C district in control zone by drugs-BSA at the test section of each film bar (T) bag.Glass fibre membrane at the sticking coated with gold labeling antibody in the lower end of film bar.When not containing drugs in the urine, latex labelled antibody bond moves forward along film with urine under capillary action.When arrival was fixed with the test section (T) of drugs bond, the latex labelled antibody formed red line of compound demonstration with the drugs association reaction of pre-bag quilt, showed in the coloured band explanation urine sample at test section (T) and was negative, and did not contain drugs.When containing Poison in the urine, its will with the drugs competitiveness that is coated on the test section in conjunction with a certain amount of antibody.When drugs reach enough concentration in the urine, it will with complete combination of quantitative antibody of mark, thereby stoped the latex labelled antibody and be coated on combining of drugs bond on the detection zone in advance, therefore, test section (T) the positive result that do not develop the color.No matter in the sample No Poison is arranged at control zone (C), all colour developing illustrates that the check-out console performance is normal; If do not develop the color in control zone (C), this beta version performance failure is described then.
Drugs (comprising heroin, hemp, cocaine, methamphetamine, morphine, opium, head-shaking pill, amphetamine (amphetamine), barbital, Benzodiazepines, methadone, PCP), hepatitis C (HCV) that the indirect method principle that present embodiment adopts can be used for detecting.
The preparation method that colloidal gold chromatography of the present invention detects the test strips of blood HIV1/2 antibody sees following examples:
Embodiment four:
The preparation method of present embodiment may further comprise the steps:
A. colored latex is covalent activated: ultrasonic Treatment latex microsphere body is after 30 seconds, and regulating the latex microsphere bulk concentration is 1.0 * 10
6～1.0 * 10
7/ ml, centrifugal 1～5 minute of 8000 * g, centrifugal back collecting precipitation thing be with distilled water or 50mM～200Mm pH6.0～7.0 sodium radio-phosphate,P-32 solutions dissolving, and ultrasonic Treatment 30 seconds; Add a certain amount of 20～100mg/mlEDC, mixing; Incubated at room 8000 * g after 30 minutes, centrifugal 1～5 minute, precipitation is with the citrate buffer solution dissolving of pH5.0～6.0 of 20mM～100mM, be positioned in 2～8 ℃ of environment standby, with labelled protein;
B. the preparation of coloured glue lactoconium labelled protein: with the colored latex ultrasonic Treatment after 30 seconds after the activation in the steps A, ratio according to the colored latex of 1 μ g～125 μ g albumen/100ul adds the protein that needs mark, the stirring at room reaction is 2 hours behind the mixing, 3 centrifuge washings, each 8000 * g, centrifugal 1～5 minute, precipitation is recovered certain volume with PBS-TBN dissolving and ultrasonic Treatment 30 seconds with phosphate buffer, the placement room temperature is standby, with the coated glass fiber film;
C. antibody or antigen coated to nitrocellulose filter and sealing: bag detection zone of quilt and control zone are that antigen/antibody and anti-mouse antibody are regulated finite concentration on the film, the film liquid measure is 20ul/35cm, be cushioned liquid dilution envelope antigen/antibody with bag, concentration is 6～15 μ g/ml, antigen/antibody and anti-mouse antibody are sprayed onto on the glass fibre membrane, two intervals should be careful even every 5mm, room temperature airing 20 minutes; 25 ℃～37 ℃ were soaked 60 minutes at confining liquid, took out rearmounted 25 ℃～37 ℃ oven dry and handled 2 hours, and envelope is used in order to pasting board;
D. on end liner, paste glass fibre membrane, coated film and the thieving paper of sample pad, coating coloured glue lactoconium labelled protein in turn mutually overlap joint, obtain test paper plate, cut into the test strips of different in width as requested.
Embodiment five:
The preparation method of present embodiment and embodiment four are basic identical, and difference is:
Among the described step B, the preparation method of coloured glue lactoconium labelled protein is: the colored latex ultrasonic Treatment after steps A is activated is after 30 seconds, ratio according to the colored latex of 75 μ g albumen/100ul adds the protein that needs mark, stirring at room reaction is 2 hours behind the mixing, 3 centrifuge washings, each 8000 * g, centrifugal 1～5 minute, precipitation was with PBS-TBN dissolving and ultrasonic Treatment 30 seconds, recover certain volume with phosphate buffer, the placement room temperature is standby, with the coated glass fiber film.
Embodiment six:
The preparation method of present embodiment and embodiment four are basic identical, and difference is:
Among the described step C, antibody or antigen coated to nitrocellulose filter and enclosure method be: bag detection zone of quilt and control zone are that antigen/antibody and anti-mouse antibody are regulated finite concentration on the film, the film liquid measure is 20ul/35cm, be cushioned liquid dilution envelope antigen/antibody with bag, concentration is 10 μ g/ml, and antigen/antibody and anti-mouse antibody are sprayed onto on the glass fibre membrane, and two intervals are every 5mm, should be careful even, room temperature airing 20 minutes; 25 ℃～37 ℃ were soaked 60 minutes at confining liquid, took out rearmounted 25 ℃～37 ℃ oven dry and handled 2 hours, and envelope is used in order to pasting board.
Claims (5)
1, colored latex chromatography diagnosis test paper, it is characterized in that: this test strips be on end liner (1) in turn mutually overlap joint ground paste sample pad (2), apply glass fibre membrane (3), coated film (4) and the thieving paper (5) of coloured glue lactoconium labelled protein and the film bar that forms, coated film (4) has detection zone (6) and control zone (7), detection zone (6) coated antibody or antigen, wrap by anti-mouse antibody control zone (7).
2, colored latex chromatography diagnosis test paper according to claim 1 is characterized in that: the diameter range that described glass fibre membrane (3) is gone up the coloured glue lactoconium that applies is 0.1 μ m～1 μ m.
3, the preparation method of colored latex chromatography diagnosis test paper as claimed in claim 1 is characterized in that, may further comprise the steps:
A. colored latex is covalent activated: ultrasonic Treatment latex microsphere body is after 30 seconds, and regulating the latex microsphere bulk concentration is 1.0 * 10
6～1.0 * 10
7/ ml, centrifugal 1～5 minute of 8000 * g, centrifugal back collecting precipitation thing be with distilled water or 50mM～200Mm pH6.0～7.0 sodium radio-phosphate,P-32 solutions dissolving, and ultrasonic Treatment 30 seconds; Add a certain amount of 20～100mg/mlEDC, mixing; Incubated at room 8000 * g after 30 minutes, centrifugal 1～5 minute, precipitation is with the citrate buffer solution dissolving of pH5.0～6.0 of 20mM～100mM, be positioned in 2～8 ℃ of environment standby, with labelled protein;
B. the preparation of coloured glue lactoconium labelled protein: with the colored latex ultrasonic Treatment after 30 seconds after the activation in the steps A, ratio according to the colored latex of 1 μ g～125 μ g albumen/100ul adds the protein that needs mark, the stirring at room reaction is 2 hours behind the mixing, 3 centrifuge washings, each 8000 * g, centrifugal 1～5 minute, precipitation is recovered certain volume with PBS-TBN dissolving and ultrasonic Treatment 30 seconds with phosphate buffer, the placement room temperature is standby, with the coated glass fiber film;
C. antibody or antigen coated to nitrocellulose filter and sealing: bag detection zone of quilt and control zone are that antigen/antibody and anti-mouse antibody are regulated finite concentration on the film, the film liquid measure is 20ul/35cm, be cushioned liquid dilution envelope antigen/antibody with bag, concentration is 6～15 μ g/ml, antigen/antibody and anti-mouse antibody are sprayed onto on the glass fibre membrane, two intervals should be careful even every 5mm, room temperature airing 20 minutes; 25 ℃～37 ℃ were soaked 60 minutes at confining liquid, took out rearmounted 25 ℃～37 ℃ oven dry and handled 2 hours, and envelope is used in order to pasting board;
D. on end liner, paste glass fibre membrane, coated film and the thieving paper of sample pad, coating coloured glue lactoconium labelled protein in turn mutually overlap joint, obtain test paper plate, cut into the test strips of different in width as requested.
4, preparation method according to claim 3, it is characterized in that, among the described step B, the preparation method of coloured glue lactoconium labelled protein is: the colored latex ultrasonic Treatment after steps A is activated is after 30 seconds, ratio according to the colored latex of 75 μ g albumen/100ul adds the protein that needs mark, the stirring at room reaction is 2 hours behind the mixing, 3 centrifuge washings, each 8000 * g, centrifugal 1～5 minute, precipitation was with PBS-TBN dissolving and ultrasonic Treatment 30 seconds, recover certain volume with phosphate buffer, the placement room temperature is standby, with the coated glass fiber film.
5, preparation method according to claim 3, it is characterized in that, among the described step C, antibody or antigen coated to nitrocellulose filter and enclosure method be: bag detection zone of quilt and control zone are that antigen/antibody and anti-mouse antibody are regulated finite concentration on the film, the film liquid measure is 20ul/35cm, be cushioned liquid dilution envelope antigen/antibody with bag, concentration is 10 μ g/ml, antigen/antibody and anti-mouse antibody are sprayed onto on the glass fibre membrane, two intervals are every 5mm, should be careful even, room temperature airing 20 minutes; 25 ℃～37 ℃ were soaked 60 minutes at confining liquid, took out rearmounted 25 ℃～37 ℃ oven dry and handled 2 hours, and envelope is used in order to pasting board.
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| CNB2004100272939A CN100504390C (en) | 2004-05-21 | 2004-05-21 | Colour latex chromatography diagnosis test paper strip and its preparing method |
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|---|---|---|---|
| CNB2004100272939A CN100504390C (en) | 2004-05-21 | 2004-05-21 | Colour latex chromatography diagnosis test paper strip and its preparing method |
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| CN1580778A true CN1580778A (en) | 2005-02-16 |
| CN100504390C CN100504390C (en) | 2009-06-24 |
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