CN1438486A - Method for detecting ABO blood type using immuno-chromatography, its prepared indicator paper and use - Google Patents
Method for detecting ABO blood type using immuno-chromatography, its prepared indicator paper and useInfo
- Publication number
- CN1438486A CN1438486A CN 02159979 CN02159979A CN1438486A CN 1438486 A CN1438486 A CN 1438486A CN 02159979 CN02159979 CN 02159979 CN 02159979 A CN02159979 A CN 02159979A CN 1438486 A CN1438486 A CN 1438486A
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- Prior art keywords
- coagulin
- antibody
- blood group
- trace particle
- nature controlling
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The method includes following steps. (1) Processing the specimen to be tested. (2) Preparing the testing line of the film of cellulose nitrate and the quality control line. (3) Preparing the pad for trace particle conjugate. (4) Assembling test paper pieces. Through the chromatography action, the sandwich structure is formed among the blood type antigen in the specimen, the antibody/condensation element marked by trace particle and the antibody/condensation element on the film of cellulose nitrate. The goal of testing ABO blood type is reached rapidly and simple by revealing color of the trace particles. The invention is applicable to the test of ABO blood type by using bloods, saliva, urine, bones, hairs and various black spots of body fluid.
Description
Technical field
The invention belongs to biomedicine field, disclose a kind of immunochromatography and detected the method for abo blood group and the preparation of test paper.
Background technology
Landsteiner had found the congealing reaction of the same race between human erythrocyte and serum in 1900, and human blood is divided into ABO three types.Student Decastelle and the Sturl of Landsteiner in 1902 continue to discover the AB type.Determined that like this human blood has four kinds of types.Blood group all is widely used at aspects such as clinical blood transfusion, histoorgan transplanting, anthropology, science of heredity, medical jurisprudence, archaeology.
The abo blood group principle of classification is: no A and Blood group antigen B on the red blood cell, and anti-A and anti-B antibody person are arranged in the serum, be called the O type; A antigen is arranged on the red blood cell, and the person that has the anti-B antibody in the serum is called the A type; B antigen is arranged on the red blood cell, and the person that has the anti-A antibody in the serum is called Type B; A and B antigen are arranged on the red blood cell, and nonreactive A and anti-B antibody person in the serum are called the AB type.The biosynthesizing of H antigen is prior to A and B antigen, so except that variant, all there are H antigen in A type, Type B, AB type and O type red blood cell.
A type, Type B and H antigen do not exist only in red blood cell, and are present in histocyte, and particularly content is abundant in various body fluid, for example serum, saliva, gastric juice, ovarian cyst liquid, seminal fluid, amniotic fluid, sweat, urine, tear, bile, milk etc.Also can determine blood group by the blood group antigens that detect in the body fluid.
The technology that detects at present abo blood group can be divided into two classes, and a class comprises red blood cell salt solution coagulation, neutralization test, absorption test, separates separating test and mixed agglutination reaction etc. for detecting on the cell membrane in conjunction with the immunological technique and the improvement technology thereof of blood group antigens.Another kind of is the immunological technique that directly detects free blood group antigens, comprises collaborative congealing reaction, radiommunoassay, enzyme linked immunosorbent assay (ELISA) etc.
Red blood cell salt solution coagulation is by the blood group antigens on known IgM type antibody test fresh red blood cell surface and makes red blood cell condense judged result.Red blood cell salt solution coagulation has been widely used in the clinical blood transfusion check, is the most conventional blood group authenticate technology.Red blood cell salt solution coagulation is with low cost, and is easy and simple to handle, and the result but can only detect the blood group of fresh red blood cell accurately and reliably, and reagent needs to preserve with refrigerator, and the blood group that can not be used for body fluid, blood stain, hair etc. detects.
The improving one's methods as neutralization test, absorption test, separate the blood group that separating test and mixed agglutination reaction can be used for body fluid, blood stain, hair etc. and detect of red blood cell salt solution coagulation.Neutralization test, absorption test need 15-18 hour, and be both time-consuming and sensitivity is low, and the result is difficult for observing; Separate separating test, condensation by mixing reaction, though the time is shorter, its technical requirement is higher, whenever does a test and need grope condition repeatedly, correctly could sentence read result up to standard control.More than experiment sensitivity is lower, and all needs fresh standard A BO red blood cell indication, and this new fresh cell is difficult for preserving, and is not portable, easily pollutes haemolysis, judges thereby influence the result.
Collaborative congealing reaction is the A albumen with IgG type antibody sensitized aureus surface, whether condenses judged result by staphylococcic.Collaborative alcohol coagulation test is easy and simple to handle, quick, susceptibility is high, has not only saved the time but also reduced loaded down with trivial details check program, does not more need high-precision instrument and equipment.But this method reagent normal temperature is preserved inconvenience, the on-the-spot inspection case of legal medical expert is used be subjected to certain limitation.
Radiommunoassay and enzyme linked immunosorbent assay (ELISA) are to condense usually with known antibody or specificity to detect free blood group antigens, and be highly sensitive, but reagent can't preserve by normal temperature, measures also inconveniently for single part, can't realize on-the-spot the detection.Radiommunoassay more has radioactive contamination, makes to use to be restricted.
The antibody type that detects free blood group antigens mainly contains IgG, IgM and IgA, the coagulin that detects free blood group antigens mainly contains anti-A coagulin (gland beans coagulin, snail coagulin etc.), anti-B coagulin (spindle tree coagulin, loach ovum coagulin, perch ovum coagulin etc.) and anti-H coagulin (chaste tree beans coagulin, Birdfoot coagulin, yellow eel coagulin etc.).
Immunochromatography technique of the present invention detects abo blood group, is to detect free blood group antigens with known antibody or specificity coagulin with sandwich method.The advantage that radiommunoassay and enzyme linked immunosorbent assay (ELISA) are therefore arranged.Simultaneously, the present invention has simply, fast, normal temperature preserves, single part of mensuration, single test are measured abo blood group, do not need the advantage of any instrument and equipment except that commercially available reagent, is particularly suitable for the field quick detection of on-the-spot abo blood group, also can be used for the self check of blood group.
Immunochromatography is the immunoassay mode that comes across a kind of uniqueness at the initial stage eighties, it is a solid phase with the fibre strip chromatographic material often, make sample solution swimming on chromatography strip by capillary action, and make the immune response that high special high-affinity takes place at the acceptor (as antibody or antigen) of determinand on determinand in the sample and the chromatographic material simultaneously, immune complex is by enrichment or be trapped in certain zone (detect band) of chromatographic material in the chromatography process, by enzyme reaction or directly the label (as collaurum) that can estimate of utilization obtain experimental phenomena (as colour developing) intuitively.Free label is then crossed and is detected band, reaches the purpose of separating automatically with binding label.The common trace particle of immunochromatography technique has collaurum, latex, electroselenium, gelatin etc., and wherein the most successful label of utilization is a collaurum.
Immunochromatography technique has been widely used in the clinical treatment detection at present.The test item of domestic and international existing supply of commodities and bibliographical information mainly comprises following aspect: forbidden drug detects (amphetamine, cocaine, hemp, morphine, heroin etc.), the infectious disease cause of disease detects (microspironema pallidum, gonococcus, genito-urinary system chlamydia trachomatis, helicobacter pylori etc.), hormone detection, parasite, tumor marker, cardiovascular disease detection mark etc.Immunochromatography technique is used to detect abo blood group and does not appear in the newspapers.
Summary of the invention
The objective of the invention is to set up a kind of immunochromatography detects the method for abo blood group and prepares abo blood group detection test paper.
Be the scheme that realizes that purpose of the present invention is taked:
1. the method for an immunochromatography detection abo blood group comprises the steps:
(1) processing of sample to be checked; (2) preparation of nitrocellulose filter detection line and nature controlling line; (3) preparation of trace particle bond pad; (4) assembling test strips; It is characterized in that
The preparation of nitrocellulose filter detection line and nature controlling line comprises the steps:
1. use the antibody that can combine or coagulin linear spotting as detection line with blood group antigens A and B;
2. use the antibody or the coagulin that can combine with blood group antigens H, the antibody linear spotting of the antiantibody of trace particle labelled antibody or trace particle mark coagulin is as nature controlling line.
2. the antibody of preparation detection line and coagulin are to select like this:
1. select IgG type antibody, IgM type antibody, IgA type antibody, gland beans coagulin, snail coagulin a kind of of blood group antigens A to be used to prepare a detection line;
2. select IgG type antibody, IgM type antibody, IgA type antibody, the spindle tree coagulin of blood group antigens B, loach ovum coagulin, perch ovum coagulin a kind of is used to prepare another detection line.
3. nature controlling line is selected one to two, and the antibody of preparation nature controlling line and coagulin are to select like this:
1. select IgG type antibody, IgM type antibody, IgA type antibody, the chaste tree beans coagulin of blood group antigens H, the Birdfoot coagulin, yellow eel coagulin a kind of is used to prepare a nature controlling line;
2. select the antiantibody of trace particle labelled antibody or the antibody linear spotting of trace particle mark coagulin to prepare another nature controlling line.
4, detection line and nature controlling line are preparations like this:
Nitrocellulose filter is cut out by the wide size of 10-35mm; To fully dialyse through normal saline buffer solution, concentration is adjusted into the anti-A of 0.01-0.3mg/ml blood group antibody and anti-B solution or A, respectively linear spotting is as detection line on film for the B coagulin, and detection line point sample position is from film base 4-16mm, and the distance between two detection lines is 2-12mm; Be adjusted into corresponding antibodies or the coagulin spraying nature controlling line of 0.01-0.3mg/ml then with concentration on the top of film, when selecting two nature controlling line for use, the distance between two nature controlling lines is 2-12mm; Nitrocellulose filter drying at room temperature 30min places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min, takes out suck dry moisture, hatches 30min in 37 ℃, puts dry place hermetically storing under the room temperature.
5. the preparation of trace particle bond pad comprises the steps:
1. select for use can with blood group antigens A, the antibody of B and H combination or coagulin mark trace particle;
2. the good trace particle of mark, be dispersed on the all-glass paper.
6. the antibody of preparation trace particle bond pad and coagulin selection are such:
1. select IgG type antibody, IgM type antibody, IgA type antibody, the gland beans coagulin of blood group antigens A, a kind of mark trace particle that is used for of snail coagulin;
2. select IgG type antibody, IgM type antibody, IgA type antibody, spindle tree coagulin, the loach ovum coagulin of blood group antigens B, a kind of mark trace particle that is used for of perch ovum coagulin;
3. select IgG type antibody, IgM type antibody, IgA type antibody, chaste tree beans coagulin, the Birdfoot coagulin of blood group antigens H, a kind of mark trace particle that is used for of yellow eel coagulin.
7. the antibody and the coagulin of the preparation of trace particle bond pad are combined as following a kind of:
1. disperse antigen A, antibody or the coagulin of antigen B and antigen H at trace particle bond pad;
2. the antibody or the coagulin that disperse antigen A and antigen B at trace particle bond pad.
8. the trace particle of preparation trace particle bond pad is following a kind of:
1. colloid gold particle;
2. latex particle;
3. electroselenium particle;
4. gelatin particle;
5. magnetic-particle.
9. assembling test strips, step is as follows:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add trace particle bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.
10, a kind of immunochromatography detects the test paper of abo blood group, it is characterized in that being prepared from immunochromatographic method of the present invention, can be applied to detect abo blood group quick, easy, delicately.
Concrete grammar:
The present invention realizes that preparation that immunochromatography detects the method for abo blood group and test paper comprises the steps: the processing of 1. samples to be checked; 2. the preparation of nitrocellulose filter detection line and nature controlling line
With the antibody that can combine or coagulin linear spotting with blood group antigens A and B as detection line, totally 2 of detection lines.
With the antibody or the coagulin that can combine with blood group antigens H, the antibody linear spotting of the antiantibody of trace particle labelled antibody or trace particle mark coagulin is as nature controlling line, totally 2 of nature controlling lines.3. the preparation of trace particle bond pad:
Select for use can with blood group antigens A, the antibody of B and H combination or coagulin mark trace particle.The good trace particle of mark, be dispersed on the all-glass paper, as trace particle bond pad.4. the assembling of test strips:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add trace particle bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.
Method can be subdivided into four parts:
1. the processing of sample to be checked; 2. the preparation of nitrocellulose filter detection line and nature controlling line; 3. the preparation of trace particle bond pad; 4. the assembling of test strips.1. the processing 1 of sample to be checked) blood sample
With distilled water diluting 10-10000 doubly, lysed erythrocyte directly uses.2) samples such as blood cake, seminal stain, salivary stain, vaginal fluid spot
Add an amount of distilled water, boiled 1-30 minute, cool to room temperature uses.3) saliva sample
With distilled water diluting 10-10000 doubly, directly use or boiled 1-30 minute, cool to room temperature uses.4) urine sample:
Get an amount of urine sample and directly test (the centrifugal or filtration of suggestion when urine sample is muddy).5) tissue sample
Tissue sample adds an amount of distilled water homogenate, and the centrifuging and taking supernatant is standby.6) sample bone
With iron file preparation bone meal, add an amount of distilled water, boiled 1-30 minute, cool to room temperature uses.7) sample of hair
Be cut into tiny fragment with scissors, add an amount of distilled water and grind, boiled 1-30 minute, cool to room temperature uses.2. the preparation of nitrocellulose filter detection line and nature controlling line
Nitrocellulose filter is cut out by the wide size of 10-35mm; To fully dialyse through normal saline buffer solution, concentration is adjusted into the anti-A of 0.01-0.3mg/ml blood group antibody and anti-B solution or A, respectively linear spotting is as detection line on film for the B coagulin, and detection line point sample position is from film base 4-16mm, and the distance between two detection lines is 2-12mm; At the top of film spraying nature controlling line, nature controlling line is following one or two then.Article one, with the antibody or the coagulin that can combine with blood group antigens H, another is with the antiantibody of trace particle labelled antibody or the antibody of trace particle mark coagulin.When selecting two nature controlling lines, the distance between two nature controlling lines is 2-12mm; Nitrocellulose filter drying at room temperature 30min places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min, takes out suck dry moisture, hatches 30min in 37 ℃, puts dry place hermetically storing under the room temperature.The preparation of trace particle bond pad (1) preparation of tracer particles and mark 1. collaurum colloidal sol prepare mark:
Collaurum colloidal sol preparation: get 0.01% tetrachloro Jinsui River solution 200ml, be heated to boiling, add 1-20ml 1% sodium citrate aqueous solution, boil 5min, occur orange redly, the colloid gold particle diameter is determined as 10-80nm by Electronic Speculum;
Colloid gold label antibody or coagulin: get the 50ml collaurum, (the pH value of IgG is with 8.0 to transfer pH with 0.1mol/L sal tartari; Chaste tree beans coagulin is with 6.3; The wheat germ coagulin is with 9.9), stir down collaurum colloidal sol is mixed with blood group antibody respectively, add the polyglycol aqueous solution again, making ultimate density is 0.05%.With the centrifugal 45min of semifinished product 6000g, precipitation is suspended to 1.5ml with physiological saline, 4 ℃ of preservations.2. electroselenium colloidal sol prepares and antibody labeling:
The preparation of electroselenium colloidal sol: add deionized water 550ml and polyacrylic acid 10g in four neck round-bottomed flasks of 1 liter of capacity, logical nitrogen stirring at room also adds hydrazine hydrate 69.5ml, continues to stir 20 minutes.Selenous acid 9.63g is dissolved in 280ml water, splashes in the reaction mixture under the stirring at room, obtains the red selenium sol of 120nm.
The electroselenium antibody labeling: IgG antibody is made into 4.6mg/ml concentration solution with 20mmol/L pH7.3 phosphate buffer, add 25ul in the 25ml of pH7.3 selenium sol, stirring at room adds 1%PEG8000 1ml after 10 minutes, mixing, in 4 ℃ with 5000rpm centrifugal 5 minutes, obtain soft red precipitate, be made into the 1ml suspension with the phosphate buffer that contains 0.05% NaN3.3. the preparation of latex and antibody labeling:
The color latex particle is available from Bangs LABOratories` company.The antibody labeling program is: the phosphate buffer with pH7.1 is diluted to 1% concentration with latex, stirs to add a certain amount of IgG solution down, and stirring at room in 37 ℃ of water-bath hatchings 60 minutes, was placed 4-5 hour for 4 ℃ after 30 minutes.Centrifugal 30 minutes of 15000rpm, resuspended with an amount of phosphate buffer.(2) preparation of trace particle bond pad:
The blood group antibody of mark trace particle or coagulin mixed in proportion being dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.4. the assembling of paper slip:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add trace particle bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.
Advantage of the present invention and range of application
The present invention compares with the technology that detected body fluid, blood stain, hair abo blood group in the past has following advantage: (1) is easy and simple to handle, quick, only needs 5-10 minute; (2) highly sensitive, still can measure blood group as saliva dilution more than thousand times; (3) need not special instruments and equipment (comprising microscope, oscillator, refrigerator etc.), except that commercially available reagent, do not need any instrument and equipment, spent low; (4) need not the fresh red blood cell indication, and directly by the color judged result; (5) room temperature preservation, single part of mensuration can be carried.
The invention belongs to biomedical sector, be mainly used in blood, body fluid, the abo blood group of bone, hair and various body fluid spots detects.The applying unit of test strips of the present invention is mainly hospital, army, department of public security organs, sportsman's drug-testing department etc., and the individual also can carry out the blood group self check.
Description of drawings
Fig. 1: immunochromatography detects the structure of abo blood group test strips and detects schematic diagram
Detecting test paper is to constitute like this: have sample pad 4 to be used for adsorption sample on liner 1 in order, behind the adding sample, have antigen A (18) in the sample, antigen B (19) and antigen H (20); The anti-A (14) that trace particle 17 marks are arranged on the trace particle bond pad 3, anti-B (15) and anti-H (16); Detection line 6 is arranged on the nitrocellulose filter 2, detection line 7, nature controlling line 8 and nature controlling line 9, anti-A or A antigen coagulin (10) are arranged on the detection line 6, anti-B or B antigen coagulin (11) are arranged on the detection line 7, nature controlling line 8 has anti-H or H antigen coagulin (12), and nature controlling line 9 has anti-A (14), the antiantibody (13) of anti-B (15) and anti-H (16); 5 is adsorptive pads.
It is such detecting principle: drip sample on sample pad 4, if antigen A (18) is arranged in the sample, antigen B (19) and antigen H (20), move to trace particle bond pad 3 through the chromatography effect, anti-A (14) with trace particle 17 marks, anti-B (15) and anti-H (16) combination, when moving to detection line 6, when detection line 7 and nature controlling line 8, antigen A (18), antigen B (19) and antigen H (20) again respectively with detection line 6, antibody on detection line 7 and the nature controlling line 8 or coagulin 10,11,12 in conjunction with colour developing.Unnecessary trace particle labelled antibody and the antiantibody (13) on the nature controlling line 9 are in conjunction with also colour developing.
Embodiment
Embodiment one:
The method of colloidal gold immunochromatographimethod detection abo blood group and the preparation of test paper also are used to detect blood spot sample
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of nitrocellulose filter; (3) preparation of collaurum bond pad; (4) assembling of test strips; (5) detection and result judge: the processing of (1) sample to be checked:
Blood cake 1-5 cubic millimeter adds an amount of distilled water 1ml, boils 15 minutes, and cool to room temperature is standby.(2) preparation of nitrocellulose filter: antibody or the coagulin of 1. selecting preparation detection line and nature controlling line:
Anti-A of blood group antibody and anti-B are used to prepare detection line, and spindle tree coagulin or loach ovum coagulin, perch ovum coagulin, gland beans coagulin, snail coagulin a kind of is used to prepare detection line, and antibody is IgM type or IgA type.2. prepare detection line and nature controlling line
Nitrocellulose filter is cut out by the wide size of 25mm.To fully dialyse through normal saline buffer solution, concentration is adjusted into the anti-A of 0.2mg/ml blood group antibody and anti-B solution, and linear spotting is as detection line respectively on film, and detection line point sample position is from film base 5mm, and the distance between two detection lines is 4mm.Spray a kind of of a chaste tree beans coagulin or Birdfoot coagulin, yellow eel coagulin then as nature controlling line on the top of film, drying at room temperature 30min, place the physiological saline of 1% bovine serum albumin(BSA) to soak 30min film, take out suck dry moisture, hatch 30min in 37 ℃, put dry place hermetically storing under the room temperature.(3) preparation of collaurum bond pad:
Getting the 50ml particle diameter respectively is the 30-50nm collaurum, transfers pH to 8.0 with 0.1mol/L sal tartari, and with collaurum colloidal sol and anti-A, anti-B and anti-H monoclonal antibody are mixed, and add the polyglycol aqueous solution again under stirring, and making ultimate density is 0.05%.With the centrifugal 45min of semifinished product 6000g, precipitation is suspended to 1.5ml with physiological saline, 4 ℃ of preservations.The antibody of mark colloid gold particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.(4) assembling of test strips:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add collaurum bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.(5) detection and result judge
Detect: get the sample 0.5ml that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad, stops about 5 seconds in sample, vertically takes out test paper, observes after 5 minutes.
The result judges as follows: the A line, and B line and H line place have red line to appear as the positive, and no red line appears as feminine gender
Embodiment two:
| ??A | ???B | H | The result judges |
| ??+/- | ???+/- | - | There are not blood group antigens in the unreliable or sample of result |
| ??+ | ???- | + | The A type |
| ??- | ???+ | + | Type B |
| ??+ | ???+ | + | The AB type |
| ??- | ???- | + | The O type |
The method of colloidal gold immunochromatographimethod detection abo blood group and the preparation of test paper also are used to detect body fluid spot (blood cake, seminal stain or salivary stain etc.) sample
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of nitrocellulose filter; (3) preparation of collaurum bond pad; (4) assembling of test strips; (5) detection and result judge the processing of (1) sample to be checked:
Body fluid spot 1-5 cubic millimeter or have the spot sample fiber a little, add distilled water 0.5-1ml, boiled 15 minutes, cool to room temperature is standby.(2) preparation of nitrocellulose filter: the antibody of 1. selecting preparation detection line and nature controlling line
Anti-A of blood group antibody and anti-B are used to prepare detection line, and antibody is mouse IgG type, and rabbit anti-mouse igg is used to prepare nature controlling line.2. prepare detection line and nature controlling line
Nitrocellulose filter is cut out by the wide size of 25mm; To fully dialyse through normal saline buffer solution, concentration is adjusted into the anti-A of 0.2mg/ml blood group antibody and anti-B solution, and linear spotting is as detection line respectively on film, and detection line point sample position is from film base 5mm, and the distance between two detection lines is 4mm; Then at the top of film rabbit anti-mouse igg of spraying as nature controlling line, drying at room temperature 30min places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min film, takes out suck dry moisture, hatches 30min in 37 ℃, puts dry place hermetically storing under the room temperature.(3) preparation of collaurum bond pad:
Getting the 50ml particle diameter respectively is the 30-50nm collaurum, transfers pH to 8.0 with 0.1mol/L sal tartari, stirs down collaurum colloidal sol is mixed with the not anti-A and the anti-B monoclonal antibody of homophyletic, adds the polyglycol aqueous solution again, and making ultimate density is 0.05%.With the centrifugal 45min of semifinished product 6000g, precipitation is suspended to 1.5ml with physiological saline, 4 ℃ of preservations.The antibody of mark colloid gold particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.(4) assembling of test strips:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add collaurum bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.(5) detection and result judge
Detect: get the sample 0.5ml that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad, stops about 5 seconds in sample, vertically takes out test paper, observes after 5 minutes.
The result judges as follows: the A line, and B line and nature controlling line place have red line to appear as the positive, and no red line appears as feminine gender
Embodiment three:
| The A line | The B line | Nature controlling line | The result judges |
| +/- | +/- | - | There are not blood group antigens in the unreliable or sample of result |
| + | - | + | The A type |
| - | + | + | Type B |
| + | + | + | The AB type |
| - | - | + | The O type |
Latex particle or gelatin particle immunochromatography detect the preparation of abo blood group test paper and are used to detect blood spot sample
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of nitrocellulose filter; (3) preparation of collaurum bond pad; (4) assembling of test strips; (5) detection and result judge the processing of (1) sample to be checked:
Blood cake 1-5 cubic millimeter adds distilled water 1ml, boils 15 minutes, and cool to room temperature is standby.(2) preparation of nitrocellulose filter: the antibody of 1. selecting preparation detection line and nature controlling line
Anti-A of blood group antibody and anti-B are used to prepare detection line, and the anti-H of blood group antibody is used to prepare nature controlling line, and antibody is the IgG type.2. prepare detection line and nature controlling line
Nitrocellulose filter is cut out by the wide size of 25mm; To fully dialyse through normal saline buffer solution, concentration is adjusted into the anti-A of 0.2mg/ml blood group antibody and anti-B solution, and linear spotting is as detection line respectively on film, and detection line point sample position is from film base 5mm, and the distance between two detection lines is 4mm; Then at the top of film anti-H of blood group antibody of spraying as nature controlling line, drying at room temperature 30min places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min film, takes out suck dry moisture, hatches 30min in 37 ℃, puts dry place hermetically storing under the room temperature.(3) preparation of latex or gelatin bond pad:
The color latex particle is available from Bangs Laboratories company, and gelatin particle is available from Zodolabs company, and color is blue look.Phosphate buffer with pH7.1 is diluted to 1% concentration with latex particle or gelatin particle; get 50ml respectively, stir down the latex solution and the anti-A of homophyletic not, anti-B and the mixing of anti-H monoclonal antibody; stirring at room in 37 ℃ of water-bath hatchings 60 minutes, was placed 4-5 hour for 4 ℃ after 30 minutes.Centrifugal 30 minutes of 15000rpm, resuspended with the 2ml phosphate buffer.The antibody of mark latex particle or gelatin particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.(4) assembling of test strips:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add collaurum bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.(5) detection and result judge
Detect: get the sample 0.5ml that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad, stops about 5 seconds in sample, vertically takes out test paper, observes after 5 minutes.
The result judges as follows: it is positive that blue colo(u)r streak bar appears in the relevant position, and no lines are negative
Embodiment four:
| A | B | H | The result judges |
| +/- | +/- | - | There are not blood group antigens in the unreliable or sample of result |
| + | - | + | The A type |
| - | + | + | Type B |
| + | + | + | The AB type |
| - | - | + | The O type |
The electroselenium immunochromatography detects the preparation of abo blood group test paper and is used to detect bone, tissue and hair sample
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of nitrocellulose filter; (3) preparation of collaurum bond pad; (4) assembling of test strips; (5) detection and result judge the processing of (1) sample to be checked:
Sample bone adds an amount of distilled water with iron file preparation bone meal, boils 1-30 minute, and cool to room temperature uses.
Sample of hair is cut into tiny fragment with scissors, adds an amount of distilled water and grinds, and boils 1-30 minute, and cool to room temperature uses.
Tissue sample adds an amount of distilled water homogenate, and the centrifuging and taking supernatant is standby.(2) preparation of nitrocellulose filter: the antibody of 1. selecting preparation detection line and nature controlling line
Anti-A of blood group antibody and anti-B are used to prepare detection line, and antibody is mouse IgG type monoclonal antibody, and rabbit anti-mouse igg is used to prepare nature controlling line.2. prepare detection line and nature controlling line
Nitrocellulose filter is cut out by the wide size of 25mm; To fully dialyse through normal saline buffer solution, concentration is adjusted into the anti-A of 0.2mg/ml blood group antibody and anti-B solution, and linear spotting is as detection line respectively on film, and detection line point sample position is from film base 5mm, and the distance between two detection lines is 4mm; Then at the top of film rabbit anti-mouse igg of spraying as nature controlling line, drying at room temperature 30min places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min film, takes out suck dry moisture, hatches 30min in 37 ℃, puts dry place hermetically storing under the room temperature.(3) preparation of electroselenium bond pad:
Electroselenium antibody labeling: anti-A, anti-B is made into 4.6mg/ml concentration solution with 20mmol/L pH7.3 phosphate buffer, getting 25ul respectively adds in the 25ml selenium sol, stirring at room adds 1%PEG8000 1ml after 10 minutes, mixing, in 4 ℃ with 5000rpm centrifugal 5 minutes, obtain soft red precipitate, be made into the 1ml suspension with the phosphate buffer that contains 0.05%NaN3.The antibody of mark electroselenium particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.(4) assembling of test strips:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add collaurum bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.(5) detection and result judge
Detect: get the sample 0.5ml that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad, stops about 5 seconds in sample, vertically takes out test paper, observes after 5 minutes.
The result judges as follows: the A line, and B line and nature controlling line place have red line to appear as the positive, and no red line appears as feminine gender
Embodiment five:
| The A line | The B line | Nature controlling line | The result judges |
| +/- | +/- | - | There are not blood group antigens in the unreliable or sample of result |
| + | - | + | The A type |
| - | + | + | Type B |
| + | + | + | The AB type |
| - | - | + | The O type |
The magnetic-particle immunochromatography detects the preparation of abo blood group test paper and is used to detect blood spot sample:
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of nitrocellulose filter; (3) preparation of magnetic-particle bond pad; (4) assembling of test strips; (5) detection and result judge the processing of (1) sample to be checked:
Blood cake 1-5 cubic millimeter adds distilled water 1ml, boils 15 minutes, and cool to room temperature is standby.(2) preparation of nitrocellulose filter: the antibody of 1. selecting preparation detection line and nature controlling line:
Anti-A of blood group antibody and anti-B are used to prepare detection line, and the anti-H of blood group antibody is used to prepare nature controlling line, and antibody is the IgG type.2. prepare detection line and nature controlling line
Nitrocellulose filter is cut out by the wide size of 25mm; To fully dialyse through normal saline buffer solution, concentration is adjusted into the anti-A of 0.2mg/ml blood group antibody and anti-B solution, and linear spotting is as detection line respectively on film, and detection line point sample position is from film base 5mm, and the distance between two detection lines is 4mm; Then at the top of film anti-H of blood group antibody of spraying as nature controlling line, drying at room temperature 30min places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min film, takes out suck dry moisture, hatches 30min in 37 ℃, puts dry place hermetically storing under the room temperature.(3) preparation of magnetic-particle bond pad:
Magnetic-particle is available from Bangs Laboratories company.Phosphate buffer with pH7.1 is diluted to 1% concentration with magnetic-particle, gets 50ml respectively, stirs down itself and the anti-A of homophyletic not, and anti-B and anti-H monoclonal antibody are mixed, and stirring at room in 37 ℃ of water-baths hatchings 60 minutes, was placed 4-5 hour for 4 ℃ after 30 minutes.Centrifugal 30 minutes of 15000rpm, resuspended with the 2ml phosphate buffer.The antibody of mark magnetic-particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.(4) assembling of test strips:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add magnetic-particle bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.(5) detection and result judge
Detect: get the sample 0.5ml that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad, stops about 5 seconds in sample, vertically takes out test paper, reads the instrument judged result with magnetic after 5 minutes.
The result judges as follows: it is positive that the magnetic enhancing appears in the relevant position, and nonmagnetic enhancing is negative
| A | B | H | The result judges |
| +/- | +/- | - | There are not blood group antigens in the unreliable or sample of result |
| + | - | + | The A type |
| - | + | + | Type B |
| + | + | + | The AB type |
| - | - | + | The O type |
Claims (10)
1. the method for an immunochromatography detection abo blood group comprises the steps:
(1) processing of sample to be checked; (2) preparation of nitrocellulose filter detection line and nature controlling line; (3) preparation of trace particle bond pad; (4) assembling test strips; It is characterized in that
The preparation of nitrocellulose filter detection line and nature controlling line comprises the steps:
1. use the antibody that can combine or coagulin linear spotting as detection line with blood group antigens A and B;
2. use the antibody or the coagulin that can combine with blood group antigens H, the antibody linear spotting of the antiantibody of trace particle labelled antibody or trace particle mark coagulin is as nature controlling line.
2. detect the method for abo blood group according to the described a kind of immunochromatography of claims 1, the antibody and the coagulin that it is characterized in that preparing described detection line are to select like this:
1. select IgG type antibody, IgM type antibody, IgA type antibody, gland beans coagulin, snail coagulin a kind of of blood group antigens A to be used to prepare a detection line;
2. select IgG type antibody, IgM type antibody, IgA type antibody, spindle tree coagulin, loach ovum coagulin, perch ovum coagulin a kind of of blood group antigens B to be used to prepare another detection line.
3. detect the method for abo blood group according to claims 1 described a kind of immunochromatography, it is characterized in that one to two of described nature controlling line selection, the antibody of preparation nature controlling line and coagulin are to select like this:
1. select IgG type antibody, IgM type antibody, IgA type antibody, chaste tree beans coagulin, Birdfoot coagulin, yellow eel coagulin a kind of of blood group antigens H to be used to prepare a nature controlling line;
2. select the antiantibody of trace particle labelled antibody or the antibody linear spotting of trace particle mark coagulin to prepare another nature controlling line.
4. detect the method for abo blood group according to claims 1 described a kind of immunochromatography, it is characterized in that described detection line and nature controlling line are preparations like this:
Nitrocellulose filter is cut out by the wide size of 10-35mm; To fully dialyse through normal saline buffer solution, concentration be adjusted into the anti-A of 0.01-0.3mg/ml blood group antibody and anti-B solution or A, B coagulin on film respectively linear spotting as detection line, detection line point sample position is from film base 4-16mm, and the distance between two detection lines is 2-12mm; Be adjusted into corresponding antibodies or the coagulin spraying nature controlling line of 0.01-0.3mg/ml then with concentration on the top of film, when selecting two nature controlling line for use, the distance between two nature controlling lines is 2-12mm; Nitrocellulose filter drying at room temperature 30min places the physiological saline of 1% bovine serum albumin(BSA) to soak 30min, takes out suck dry moisture, hatches 30min in 37 ℃, puts dry place hermetically storing under the room temperature.
5. detect the method for abo blood group according to claims 1 described a kind of immunochromatography, it is characterized in that the preparation of described trace particle bond pad comprises the steps:
1. select for use can with blood group antigens A, the antibody of B and H combination or coagulin mark trace particle;
2. the good trace particle of mark, be dispersed on the all-glass paper.
6. detect the method for abo blood groups according to the described a kind of immunochromatographies of claims 5, it is characterized in that preparing that the antibody of described trace particle bond pad and coagulin select is such:
1. select IgG type antibody, IgM type antibody, IgA type antibody, the gland beans coagulin of blood group antigens A, a kind of mark trace particle that is used for of snail coagulin;
2. select IgG type antibody, IgM type antibody, IgA type antibody, spindle tree coagulin, the loach ovum coagulin of blood group antigens B, a kind of mark trace particle that is used for of perch ovum coagulin;
3. select IgG type antibody, IgM type antibody, IgA type antibody, chaste tree beans coagulin, the Birdfoot coagulin of blood group antigens H, a kind of mark trace particle that is used for of yellow eel coagulin.
7. detect the method for abo blood group according to claims 6 described a kind of immunochromatographies, it is characterized in that the antibody and the coagulin of described trace particle bond pad preparation is combined as following a kind of:
1. disperse antigen A, antibody or the coagulin of antigen B and antigen H at trace particle bond pad;
2. the antibody or the coagulin that disperse antigen A and antigen B at trace particle bond pad.
8. detect the method for abo blood groups according to the described a kind of immunochromatographies of claims 5, the trace particle that it is characterized in that preparing described trace particle bond pad is following a kind of:
1. colloid gold particle;
2. latex particle;
3. electroselenium particle;
4. gelatin particle;
5. magnetic-particle.
9. claims 1 described a kind of immunochromatography detects the method for abo blood group, it is characterized in that described assembling test strips, and step is as follows:
1. on liner, add the nitrocellulose filter of detection line and nature controlling line; 2. on liner, add trace particle bond pad; 3. on liner, add sample pad; 4. on liner, add adsorptive pads; Be assembled into test strip.
10, a kind of immunochromatography detects the test paper of abo blood group, it is characterized in that being prepared from immunochromatographic method of the present invention, can be applied to detect abo blood group quick, easy, delicately.
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| CNB021599793A CN1186636C (en) | 2002-12-31 | 2002-12-31 | Method for detecting ABO blood type using immuno-chromatography, its prepared indicator paper and use |
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| CNB021599793A CN1186636C (en) | 2002-12-31 | 2002-12-31 | Method for detecting ABO blood type using immuno-chromatography, its prepared indicator paper and use |
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| CN1186636C CN1186636C (en) | 2005-01-26 |
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| CN1300586C (en) * | 2003-09-30 | 2007-02-14 | 江西中德生物工程有限公司 | Fabrication method and application for patulin immune chromatography detection test paper |
| WO2009149615A1 (en) * | 2008-06-10 | 2009-12-17 | 英科新创(厦门)科技有限公司 | A method of quickly determining human abo/rh/mn blood type and a test kit thereof |
| CN1690711B (en) * | 2004-04-23 | 2010-04-14 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatographic test paper bar based on up conversion luminescence technology |
| CN1816746B (en) * | 2003-07-09 | 2010-05-05 | 麦迪奥诊断产品股份公司 | Device and method for simultaneously identifying blood group antigens |
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2002
- 2002-12-31 CN CNB021599793A patent/CN1186636C/en not_active Expired - Fee Related
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| CN106483303A (en) * | 2015-08-24 | 2017-03-08 | 北京中检安泰诊断科技有限公司 | Human blood types detection kit and preparation method thereof |
| CN107290520B (en) * | 2017-06-14 | 2019-08-02 | 邵超鹏 | Measure the immuno-chromatographic test paper strip of pregnant woman IgG anti-A and anti-blood group antibody B or human blood type reverse type |
| CN107290520A (en) * | 2017-06-14 | 2017-10-24 | 邵超鹏 | Determine the immuno-chromatographic test paper strip of the anti-A of pregnant woman IgG and anti-blood group antibody B or human blood type reverse type |
| CN115598356A (en) * | 2022-09-30 | 2023-01-13 | 杭州瑞测生物技术有限公司（Cn） | A cat blood type rapid detection card and its detection method |
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Address after: 330046 Jiangxi city in Nanchang Province, the provincial government compound Dongsilu dragon building C, Room 308 Patentee after: Jiangxi Sino German bioengineering Limited by Share Ltd Address before: 330029 Jianchang high tech Industrial Park, Overseas Building, Nanchang, Jiangxi, China, South China, 410 Patentee before: Jiangxi Zodogenes Biotechnology Co., Ltd. |
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