CN101852799A - Immunochromatography assay detection reagent and preparation method thereof - Google Patents
Immunochromatography assay detection reagent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an immunochromatography assay reagent and a preparation method thereof. The immunochromatography assay reagent comprises a PVC sheet on which a sample adding zone, a first reaction zone, a second reaction zone and a water adsorption zone are successively arranged, wherein the sample adding zone is a glass fiber layer, the first reaction zone is a polyester membrane on which at least two emulsion antibody binding substances are arranged, the second reaction zone is a nitrocellulose membrane, the water adsorption zone is water adsorption filter paper, and the different emulsion antibody binding substances are marked with different colors; and the nitrocellulose membrane is provided with multiple antibodies the same as the antibodies in the emulsion antibody binding substances on the polyester membrane and an antigen, and the multiple antibodies and the antigen are respectively in a strip shape and are arranged on the nitrocellulose membrane in parallel. The preparation method of the immunochromatography assay reagent comprises the following four steps: utilizing color emulsions to mark the antibodies, carrying out film dispensing, carrying out emulsion dispensing and assembling. Therefore, the invention can simultaneously detect multiple substances, and can display the multiple substances through different colors.
Description
Technical field
The present invention relates to a kind of medical testing product and preparation method thereof, relate in particular to a kind of immunochromatographyassay assay reagent and preparation method thereof.
Background technology
Immunochromatographic method is a kind of quick diagnosis technology based on immune colloidal solid technology of rising nineteen nineties, its principle is a certain district band that special antibody is fixed in nitrocellulose membrane earlier, after this dry nitrocellulose filter one end immerses sample, because capillarity, sample will move forward along this film, when moving to when being fixed with antibody regional, corresponding antigen promptly combines with this antibody generation specificity in the sample, if can make this zone show certain color with immune colloidal solid, thereby realize specific immunodiagnosis.The detection species of samples is many: both can be used for having a blood test, can be used for inspection urine or saliva again, thereby be fit to various crowds' inspection.Therefore this technology has obtained development fast since invention, has been applied in a plurality of detection ranges now.Product based on this technology has been comprised that vast basic medical unit and household person user accept and use.Immunochromatographyassay assay principle of the prior art as shown in Figure 1, Fig. 2 is the chromatography process flow diagram.This technology is compared with routine diagnostic method in the past has following outstanding advantage:
1). fast: all testing process only needs 3-20 minute;
2). easy: as not need other any instrument and equipment, operate also extremely simply, can carry out whenever and wherever possible;
3). can single part of detection: can detect in batch sample, again can single part of detection, the patient can take the result at once, needn't wait for;
4). good stability: stable reagent, but long preservation.
But along with the further expansion of user's scope, through the user of special training particularly the personal user when using this product, also exposed certain problem.The colloidal solid that is commonly used at present as developer is a collaurum, and collaurum can only show a kind of color-redness.Therefore nature controlling line and the p-wire that shows based on the immunochromatography reagent of collaurum is redness.But for some users, they can not distinguish correctly which bar is a nature controlling line, and which bar is a p-wire, when particularly having many red lines to represent different analyte respectively on the diverse location on a test strips, the user can not correctly judge the pairing analyte of red line, causes erroneous judgement easily.
Summary of the invention
Technical matters to be solved by this invention is at above-mentioned the deficiencies in the prior art, provide a kind of and can know simultaneously and show detection zone and Quality Control district by different colours exactly, and immunochromatographyassay assay reagent that also can be by the different detection zone of different color demonstrations and preparation method thereof.
For solving the problems of the technologies described above, the technical solution used in the present invention is: a kind of method for making of immunochromatographic method reagent may further comprise the steps:
1). the diameter of at least two kinds of colors of usefulness is that the polystyrene colloid particle of 200-500nm is distinguished at least two kinds of antibody of mark: a as the color latex particle, PBS with 0.01M PH7.4 is diluted to 1% final concentration (mass percent) to the color latex particle, and adding antibody or antigen again, to make its final concentration be 0.1mg/ml; B places 4 ℃ to react and spend the night above-mentioned solution; C adds final concentration and is 1% BSA and sealed 1 hour in the solution that step b is obtained; D carries out the solution among the step c centrifugal, obtains the sediment of latex antibody conjugates; E, the PBS with the 0.01M PH7.4 that contains 0.1%BSA redissolves with the sediment in the steps d, and the concentration after the redissolution is 1% (mass percent), and places 4 ℃ with standby;
2). the some film: the PBS that uses 0.01M PH7.4 is respectively with a kind of antigen and at least a antibody dilution, described antigen is specificity at the antigen of an antibody at least two kinds of antibody described in the step 1), remaining antibody and step 2 in the step 1)) described at least a antibody identical; Concentration after the dilution is to 0.5-3.0mg/ml, with a film machine dilution of every kind of antibody and antigen is put zones of different on nitrocellulose filter respectively, then nitrocellulose filter was placed under 37 ℃ the temperature oven dry 2 hours, good seal also places room temperature standby;
3). the some latex: the PBS with the 0.01M PH7.4 that contains 0.1%BSA dilutes the step 1) latex antibody conjugates that mark is good in the step, concentration after the dilution is 0.2%-0.6% (mass percent), with a latex machine specking on polyester film, then polyester film was placed under 37 ℃ the temperature oven dry 2 hours, good seal also places room temperature standby;
4). assembling: glass fibre, the polyester film that point is good, nitrocellulose filter and the absorbent filter that point is good are sticked on the PVC sheet material respectively in order.
After according to the method described above that glass fibre, polyester film, point that point is good is good nitrocellulose filter and absorbent filter stick on the PVC sheet material respectively in order, be cut into respective width again and pack into stand-by in the plastic clip.During use, sample to be measured is added on the glass fibre, sample to be measured under capillary action to absorbent filter end chromatography, in the chromatography process, sample to be measured redissolves the latex antibody conjugates on the polyester film earlier, potpourri after the redissolution continues chromatography to nitrocellulose filter, and antibody and antigen generation immune response with bag quilt on the nitrocellulose filter show corresponding colored lines.
Invent further improved technical scheme as this institute, in step 1), with two kinds, three kinds of latex particle marks or four kinds of antibody of two kinds, three kinds or four kinds different colours, a kind of antibody of latex particle mark of each color; In step 2) in, the PBS that uses 0.01M PH7.4 is respectively with a kind of, two kinds or three kinds of antibody and a kind of antigen diluent.Adopt the different antibody of latex mark of different colours, during test, the zone at various antibody and antigen place can show different colors.
A kind of immunochromatographyassay assay reagent, comprise the PVC sheet material, be provided with sample application zone on the PVC sheet material successively, first reaction zone, second reaction zone and suction zones, sample application zone, first reaction zone, second reaction zone and suction zones are joined successively and are arranged on the PVC sheet surface, described sample application zone is a glass layer, described first reaction zone is a polyester film, described second reaction zone is a nitrocellulose filter, described suction zones is an absorbent filter, described nitrocellulose filter is provided with identical antibody and a kind of antigen of antibody in the latex antibody conjugates on multiple and the polyester film, and described multiple antibody and a kind of antigen are banded respectively and are arranged in parallel on nitrocellulose filter; Described polyester film is provided with at least two kinds of latex antibody conjugates, described every kind of latex antibody conjugates is marked with different colors, latex particle in the described glue antibody conjugates is the color latex particle, and described color latex particle is that diameter is the polystyrene colloid particle of 200-500nm.
Invent further improved technical scheme as this institute, described polyester film is provided with two kinds, three kinds or four kinds of latex antibody conjugates, and is corresponding, described nitrocellulose filter is provided with a kind of, two kinds or three kinds of antibody and a kind of antigen.
The present invention adopts the latex particle of different colours to substitute the colour developing particle of colloid gold particle as immunochromatography reagent.The color latex particle of using among the present invention is that diameter is the polystyrene colloid particle of 200-500nm, modifies the latex particle that different pigments promptly can be made into different colours at particle surface.Only need with the different antibody of different color marks, and corresponding antibody and antigen are set on nitrocellulose filter, when containing determinand in the sample, the test section just can demonstrate corresponding color on the nitrocellulose filter; If do not contain determinand in the sample, the test section just can not demonstrate corresponding color on the nitrocellulose filter; No matter whether contain the antigen of antibody to be measured in the sample, because chromatography effect, on the polyester film with nitrocellulose filter on the corresponding antibody of antigen also can chromatography to nitrocellulose filter, and with the antigen generation specific reaction on the nitrocellulose filter, therefore point has the zone of antigen also can demonstrate different colors on the nitrocellulose filter, and this zone can be used as the Quality Control district.Therefore the present invention can show with the Quality Control district test section simultaneously by different colors, also different test sections can be shown by different colors.In a word, the present invention is simple in structure, and is with low cost, easy to use, can detect more than one material simultaneously.
Description of drawings
Fig. 1 is an immunochromatographyassay assay principle schematic in the prior art.
Fig. 2 is the chromatography process flow diagram of immunochromatographic method in the prior art.
Fig. 3 is an immunochromatographyassay assay reagent structural representation in the embodiment of the invention 1.
Fig. 4 is an immunochromatographyassay assay reagent structural representation among the embodiment of the invention 2 and the embodiment 3.
Embodiment
Referring to Fig. 3, the method for making of this immunochromatographic method reagent may further comprise the steps:
1) with blue and red two kinds of color latex particles difference mark anti-biotin antibodies and two kinds of antibody of anti-very early pregnancy antibody:
1.1). with blue latex particles mark anti-biotin antibodies: a, with the PBS of 0.01M PH7.4 blue latex particles is diluted to the final concentration of 1% (mass percent), adding anti-biotin antibodies again, to make its final concentration be 0.1mg/ml; B places 4 ℃ to react and spend the night above-mentioned solution; C adds final concentration and is 1% BSA and sealed 1 hour in the solution that step b is obtained; D carries out the solution among the step c centrifugal, obtains the sediment of latex antibody conjugates; E, the PBS with the 0.01M PH7.4 that contains 0.1%BSA redissolves with the sediment in the steps d, and the concentration after the redissolution is 1% (mass percent), and places 4 ℃ with standby;
1.2). with red latex particle mark anti-very early pregnancy antibody: a, with the PBS of 0.01M PH7.4 the red latex particle is diluted to the final concentration of 1% (mass percent), adding anti-very early pregnancy antibody again, to make its final concentration be 0.1mg/ml; B places 4 ℃ to react and spend the night above-mentioned solution; C adds final concentration and is 1% BSA and sealed 1 hour in the solution that step b is obtained; D carries out the solution among the step c centrifugal, obtains the sediment of latex antibody conjugates; E, the PBS with the 0.01M PH7.4 that contains 0.1%BSA redissolves with the sediment in the steps d, and the concentration after the redissolution is 1% (mass percent), and places 4 ℃ with standby;
2). the some film: the PBS that uses 0.01M PH7.4 is respectively with biotin and anti-very early pregnancy antibody dilution, concentration after the dilution is 0.5-3.0mg/ml, with the film machine Quality Control district 4-1 and the first test section 4-2 on nitrocellulose filter with biotin dilution and anti-very early pregnancy antibody diluent point respectively, then nitrocellulose filter is placed 37 ℃ of oven dry 2 hours, the nitrocellulose filter good seal that point is good also places room temperature standby;
3). some latex: with the PBS of the 0.01M PH7.4 that the contains 0.1%BSA latex antibody conjugates dilution that mark in the step 1) is good, concentration after the dilution is 0.2%-0.6% (mass percent), then with some latex machine with each dilution specking on polyester film, then polyester film is placed 37 ℃ of oven dry 2 hours, good seal places room temperature standby.
4). assembling: glass fibre, the polyester film that point is good, nitrocellulose filter and the absorbent filter that point is good are sticked on respectively on the PVC sheet material in order, be cut into respective width again and pack into stand-by in the plastic clip.
During test, urine specimen is added to sample application zone 2, urine specimen is the absorbent filter end chromatography in suction zones 5 under capillary action, and latex antibody conjugates in the polyester film redissolved and continues chromatography to nitrocellulose filter.If contain very early pregnancy hormone (HCG) in the sample, then form red latex * very early pregnancy antibody-HCG-very early pregnancy antibody complex at the first test section 4-2, the band of a redness appears; If do not contain HCG in the sample, then can not form compound, red stripes can not appear.No matter whether contain HCG in the sample, the biotin of Quality Control district 4-1 all can be caught blue latex * anti-biotin antibodies, thereby the band of a blueness occurs.
Referring to Fig. 4, the method for making of this immunochromatographic method reagent may further comprise the steps
1). use red, blue and green three kinds of color latex particles mark anti-biotin antibodies, anti-rotavirus (ROTA) antibody and three kinds of antibody of anti-adenovirus (ADENO) antibody respectively:
1.1). with red latex particle mark anti-biotin antibodies: a, with the PBS of 0.01M PH7.4 red glue particle is diluted to the final concentration of 1% (mass percent), adding anti-biotin antibodies again, to make its final concentration be 0.1mg/ml; B places 4 ℃ to react and spend the night above-mentioned solution; C adds final concentration and is 1% BSA and sealed 1 hour in the solution that step b is obtained; D carries out the solution among the step c centrifugal, obtains the sediment of latex antibody conjugates; E, the PBS with the 0.01M PH7.4 that contains 0.1%BSA redissolves with the sediment in the steps d, and the concentration after the redissolution is 1% (mass percent), and places 4 ℃ with standby;
1.2). with blue latex particles mark anti-rotavirus (ROTA) antibody: a, with the PBS of 0.01M PH7.4 blue latex particles is diluted to 1% final concentration, adding anti-rotavirus (ROTA) antibody again, to make its final concentration be 0.1mg/ml; B places 4 ℃ to react and spend the night above-mentioned solution; C adds final concentration and is 1% BSA and sealed 1 hour in the solution that step b is obtained; D carries out the solution among the step c centrifugal, obtains the sediment of latex antibody conjugates; E, the PBS with the 0.01M PH7.4 that contains 0.1%BSA redissolves with the sediment in the steps d, and the concentration after the redissolution is 1% (mass percent), and places 4 ℃ with standby;
1.3). with the anti-adenovirus of green latex particle mark (ADENO) antibody: a, with the PBS of 0.01M PH7.4 green latex particle is diluted to 1% final concentration, adding anti-adenovirus (ADENO) antibody again, to make its final concentration be 0.1mg/ml; B places 4 ℃ to react and spend the night above-mentioned solution; C adds final concentration and is 1% BSA and sealed 1 hour in the solution that step b is obtained; D carries out the solution among the step c centrifugal, obtains the sediment of latex antibody conjugates; E, with the 0.01M PH7 that contains 0.1%BSA of the sediment in the steps d, 4 PBS redissolves, and the concentration after the redissolution is 1% (mass percent), and places 4 ℃ with standby;
2). the some film: the PBS that uses 0.01M PH7.4 is respectively with biotin, anti-ROTA antibody and anti-ADENO antibody dilution, concentration after the dilution is 0.5-3.0mg/ml, with a film machine respectively with in biotin dilution, anti-ROTA antibody diluent and anti-ADENO antibody diluent point Quality Control district 4-1, the first test section 4-2 and the second test section 4-3 on nitrocellulose filter, then nitrocellulose filter is placed 37 ℃ of oven dry 2 hours, good seal also places room temperature standby;
3). some latex: with respectively that mark in the step 1) is the good latex antibody conjugates dilution of the PBS of the 0.01M PH7.4 that contains 0.1%BSA, concentration after the dilution is 0.2%-0.6%, then with some latex machine with each dilution specking on polyester film, then polyester film is placed 37 ℃ of oven dry 2 hours, good seal places room temperature standby;
4). assembling: glass fibre, the polyester film that point is good, nitrocellulose filter and the absorbent filter that point is good are sticked on respectively on the PVC sheet material in order, be cut into respective width again and pack into stand-by in the plastic clip.
During test, sample is added to sample application zone 2, the absorbent filter end chromatography of sample in suction zones 5 under capillary action, and latex antibody conjugates in the polyester film redissolved and continue chromatography to nitrocellulose filter, at this moment:
(1). if contain ROTA in the sample, then form blue latex * ROTA antibody-ROTA-ROTA antibody complex, the band of a blueness occurs at the first test section 4-2; If do not contain ROTA in the sample, then can not form compound, band appears.
(2). if contain ADENO in the sample, then form green latex * ADENO antibody-ADENO-ADENO antibody complex, the band of a green occurs at the second test section 4-3; If do not contain ADENO in the sample, then can not form compound, band appears.
(3) no matter. whether contain ROTA or ADENO in the sample, the biotin of Quality Control district 4-1 all can be caught red latex * anti-biotin antibodies, thereby the band of a redness occurs.
Referring to Fig. 4, this immunochromatographyassay assay reagent comprises PVC sheet material 1, is provided with sample application zone 2, first reaction zone 3, second reaction zone 4 and suction zones 5 on the PVC sheet material 1 successively; Sample application zone 2, first reaction zone 3, second reaction zone 4 and suction zones 5 are joined successively and are arranged on PVC sheet material 1 surface, described sample application zone 2 is a glass layer, described first reaction zone 3 is a polyester film, described second reaction zone 4 is a nitrocellulose filter, described suction zones 5 is an absorbent filter, described polyester film is provided with at least two kinds of latex antibody conjugates, is marked with different colors on the described latex antibody conjugates; Described nitrocellulose filter is provided with the identical antibody of antibody in the latex antibody conjugates on multiple and the polyester film, and described multiple antibody is banded respectively and is arranged in parallel on nitrocellulose filter.Two kinds, three kinds or four kinds of latex antibody conjugates can be set on the described polyester film; Accordingly, described nitrocellulose filter is provided with a kind of, two kinds or three kinds of antibody, and a kind of antigen.
Three kinds of latex antibody conjugates are set on the polyester film in the present embodiment, be respectively anti-biotin antibodies, anti-rotavirus (ROTA) antibody and three kinds of antibody of anti-adenovirus (ADENO) antibody, accordingly, adopt redness, blueness and Green Marker corresponding antibody respectively.First reaction zone on the nitrocellulose filter, second reaction zone and Quality Control district have put biotin, anti-rotavirus (ROTA) antibody and anti-adenovirus (ADENO) antibody respectively.
This immunochromatographyassay assay reagent is rotavirus/adenovirus two link detection reagents, during detection, at first sample to be measured is added on sample application zone 2, under capillary action, sample to be measured, during through first reaction zone 3 redissolves the latex antibody conjugates in the polyester film to suction zones 5 chromatographies, continue chromatography then to the nitrocellulose filter of second reaction zone 4, at this moment:
(1). if contain ROTA in the sample, then form blue latex * ROTA antibody-ROTA-ROTA antibody complex, the band of a blueness occurs at the first test section 4-2; If do not contain ROTA in the sample, then can not form compound, band does not appear.
(2). if contain ADENO in the sample, then form green latex * ADENO antibody-ADENO-ADENO antibody complex, the band of a green occurs at the second test section 4-2; If do not contain ADENO in the sample, then can not form compound, band does not appear.
(3) no matter. whether contain ROTA or ADENO in the sample, the biotin of Quality Control district 4-1 all can be caught red latex * anti-biotin antibodies, thereby the band of a redness occurs.
Claims (4)
1. the method for making of an immunochromatographyassay assay reagent may further comprise the steps:
1). the diameter of at least two kinds of colors of usefulness is that the polystyrene colloid particle of 200-500nm is distinguished at least two kinds of antibody of mark: a as the color latex particle, PBS with 0.01M PH7.4 is diluted to 1% final concentration (mass percent) to the color latex particle, and adding antibody or antigen again, to make its final concentration be 0.1mg/ml; B places 4 ℃ to react and spend the night above-mentioned solution; C adds final concentration and is 1% BSA and sealed 1 hour in the solution that step b is obtained; D carries out the solution among the step c centrifugal, obtains the sediment of latex antibody conjugates; E, the PBS with the 0.01M PH7.4 that contains 0.1%BSA redissolves with the sediment in the steps d, and the concentration after the redissolution is 1% (mass percent), and places 4 ℃ with standby;
2). the some film: the PBS that uses 0.01M PH7.4 is respectively with a kind of antigen and at least a antibody dilution, described antigen is specificity at the antigen of an antibody at least two kinds of antibody described in the step 1), remaining antibody and step 2 in the step 1)) described at least a antibody identical; Concentration after the dilution is to 0.5-3.0mg/ml, with a film machine dilution of every kind of antibody and antigen is put zones of different on nitrocellulose filter respectively, then nitrocellulose filter was placed under 37 ℃ the temperature oven dry 2 hours, good seal also places room temperature standby;
3). the some latex: the PBS with the 0.01M PH7.4 that contains 0.1%BSA dilutes the step 1) latex antibody conjugates that mark is good in the step, concentration after the dilution is 0.2%-0.6% (mass percent), with a latex machine specking on polyester film, then polyester film was placed under 37 ℃ the temperature oven dry 2 hours, good seal also places room temperature standby;
4). assembling: stick on glass fibre, the polyester film that point is good, nitrocellulose filter and the absorbent filter that point is good on the PVC sheet material respectively in order;
2. immunochromatographyassay assay reagent according to claim 1, it is characterized in that: in step 1), with two kinds, three kinds of latex particle marks or four kinds of antibody of two kinds, three kinds or four kinds different colours, a kind of antibody of latex particle mark of each color; In step 2) in, the PBS that uses 0.01M PH7.4 is respectively with a kind of, two kinds or three kinds of antibody and a kind of antigen diluent.
3. immunochromatographyassay assay reagent according to the method for making made of the immunochromatographic method reagent of claim 1 or 2, comprise PVC sheet material (1), be provided with sample application zone (2) on the PVC sheet material (1) successively, first reaction zone (3), second reaction zone (4) and suction zones (5), sample application zone (2), first reaction zone (3), second reaction zone (4) and suction zones (5) are joined successively and are arranged on PVC sheet material (1) surface, described sample application zone (2) is a glass layer, described first reaction zone (3) is a polyester film, described second reaction zone (4) is a nitrocellulose filter, described suction zones (5) is an absorbent filter, described nitrocellulose filter is provided with identical antibody and a kind of antigen of antibody in the latex antibody conjugates on multiple and the polyester film, and described multiple antibody and antigen are banded respectively and are arranged in parallel on nitrocellulose filter; It is characterized in that: described polyester film is provided with at least two kinds of latex antibody conjugates, described every kind of latex antibody conjugates is marked with different colors, latex particle in the described glue antibody conjugates is the color latex particle, and described color latex particle is that diameter is the polystyrene colloid particle of 200-500nm.
4. immunochromatographyassay assay reagent according to claim 1, it is characterized in that: described polyester film is provided with two kinds, three kinds or four kinds of latex antibody conjugates, accordingly, described nitrocellulose filter is provided with a kind of, two kinds or three kinds of antibody and a kind of antigen.
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Application publication date: 20101006 |