CN1032926A - Reagent box for determination of enzyme-labelled blood group and preparation method thereof - Google Patents
Reagent box for determination of enzyme-labelled blood group and preparation method thereof Download PDFInfo
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- CN1032926A CN1032926A CN 88107238 CN88107238A CN1032926A CN 1032926 A CN1032926 A CN 1032926A CN 88107238 CN88107238 CN 88107238 CN 88107238 A CN88107238 A CN 88107238A CN 1032926 A CN1032926 A CN 1032926A
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- blood group
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Abstract
The present invention is the application that blood group serology and Enzyme-multiplied immune technique are measured at the forensic abo blood group.It mainly is the ABH monoclonal anti body and function spot ELISA (enzyme-linked immunosorbent assay) of marker enzyme to be carried out abo blood group measure, and colour-change occurs and judges blood group by being combined in zymolyte on the antibody.Its advantage is simple to operate, highly sensitive, takes shortlyer, need not special instruments and equipment, and the test kit made from this method is specially adapted to basic unit's public security organs, procuratorial organs and people's court and carries out blood group determination.
Description
The present invention is the application that blood group serology and Enzyme-multiplied immune technique are measured at the forensic abo blood group.
At present, the blood group determination of forensic (body fluid, blood stain, hair etc.) most with neutralization test, absorption test, separate separating test and mixed agglutination reaction.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, mixed agglutination reaction, though the time is shorter, its technical requirements is higher, whenever does a sample and need grope condition repeatedly, correctly could judged result up to standard control.More than experiment all needs fresh standard A, B, the indication of O red corpuscle, and this new fresh cell is difficult for preserving, and is not portable, easily pollutes haemolysis, judges thereby influence the result.Foreign literature: J.Michael Cecka etc:Direct Blood Group Typing of Forensic Samples Using A Simple Monoclonal Antibody Assay.Forensic Science International 1987:34,205-216. report spot ELISA(enzyme-linked immunosorbent assay) indirect method, promptly add and use second antibody, not directly with enzyme labelling on the ABH monoclonal antibody, its operation is more numerous, required time is longer, about 4 hours, and need second antibody, its expense is increased, owing to increased second antibody, then increased a reaction conditions, need find out the required the best two anti-concentration of reaction, time etc., and its nonspecific reaction also increases than direct method.
In order to overcome above-mentioned shortcoming, the object of the present invention is to provide a kind of simple and efficient test kit that carries out blood group determinations such as body fluid, blood stain, hair and preparation method thereof.
The present invention is done in such a way that to combine with the ABH monoclonal antibody with horseradish peroxidase (HRP) and makes monoclonal antibody linked with peroxidase, forms reagent box for determination of enzyme-labelled blood group with antigenic dilution, confining liquid, washings, substrate and damping fluid, reaction film.Utilization spot ELISA method is used for abo blood group with monoclonal antibody linked with peroxidase to be measured, and colour-change occurs and judge blood group by being combined in zymolyte on the antibody.
The purification of ABH monoclonal antibody and mark enzyme are divided into nine steps, and existing division is as follows:
(1) get anti-A, anti-B, anti-H monoclonal antibody ascites 5ml, centrifugal (3000 rev/mins, 30 minutes) are got and are reset and added 0.001M PH7.0-7.2 phosphate buffered saline buffer (PBS) and be diluted to 15ml;
(2) aforesaid liquid is added the affine layer of anti-mouse IgG folding post respectively, go into post after, clamp react 60 minutes, washed liquid with PBS then, detected till the no albumen outflow;
(3) with the 3M KCNS 100-150ml antibody that dissociates, when adding the 50ml left and right sides, clamp 20 minutes dissociates with the 2ml/ component velocity again, when beginning KCNS to occur, collects dissociation solution, till not containing albumen;
(4) this liquid is removed KCNS with Sephadex G-25 post, flow velocity 3ml/ branch is collected the effluent liquid (gel-filtration) contain monoclonal antibody protein, surveys absorbancy (OD) value of this liquid when the 280nm, calculating protein content (OD with ultraviolet spectrophotometer
280nmValue ÷ 1.45=mg/ml);
(5) said monoclonal antibody is concentrated into the above concentration of 5mg/ml, concentrated solution centrifugal (3000 rev/mins, 15 minutes) is abandoned precipitation;
(6) take by weighing horseradish peroxidase (HRP) 12mg,, add freshly prepared sodium periodate (NaIO with the dissolving of 1ml distilled water
4) 0.2M 1ml, put 20 ℃-22 ℃ 20 minutes, continuous jog;
(7) add 0.4ml ethylene glycol, 20 ℃-22 ℃ effects five minutes, continuous jog, the dress dialysis tubing spends the night with 4 ℃ of cold insulation 0.001M PH4.2 acetate buffer solution dialysis equilibriums in advance, changes liquid 4-5 time;
(8) above-mentioned hydroformylation HRP is moved into test tube, add the monoclonal antibody 20mg(volume 3-4ml that has surveyed concentration) mixing, use the PH9.6 sodium carbonate buffer, transfer PH7-12, put 20 ℃-22 ℃ 2-3 hour, shake with battle array, add fresh preparation boron hydracid potassium liquid 0.3ml, put 4 ℃ 2-3 hour, use 0.2M NaH
2PO
4Liquid is transferred PH7.0-7.4, and this liquid is crossed Sephadex G-200 gel column, collects filtrate;
(9) collect liquid to each pipe, survey OD
280nmAnd OD
403nmValue, compute mark rate, A
403nm/ A
280nm, merge mark rate at each pipe more than 0.5, will merge pipe and survey OD again
280nmAnd OD
403nmValue is calculated mark rate, and it is anticorrosion to add equivalent calf serum and Thiomersalate, adds 50% glycerine mixing again, and packing is preserved or needn't the glycerol adding freeze-drying be preserved.
The prescription of reagent box for determination of enzyme-labelled blood group following (but never being limited to this):
Antigenic dilution: sodium-chlor 0.85g
Water adds to 100ml
Confining liquid: potassium primary phosphate 0.2106g
Sodium phosphate dibasic 3.0271g
Sodium-chlor 8.0000g
Repone K 0.2000g
Calf serum 300ml
Distilled water 700ml
Washings: potassium primary phosphate 0.2106g
Sodium phosphate dibasic 3.6271g
Sodium-chlor 8.0000g
Repone K 0.2000g
Polysorbas20 0.5000ml
Water is to 1000ml
Substrate and damping fluid 0.1M Tris 50ml
0.1N HCl 38.5ml
DAB-HCl 70mg
30% H
2O
230μl
Water 11.5ml
Enzyme labelled antibody: anti-A 200 μ l
Anti-B 200 μ l
Anti-H 200 μ l
Taking-up is dissolved back (then not needing of glycerol adding) with distilled water,
Promptly can be used for measuring with after 30 times of the confining liquid dilutions.
The volume ratio of reagent box for determination of enzyme-labelled blood group prescription is: antigenic dilution: confining liquid: enzyme labelled antibody: washings: substrate and damping fluid were generally 1: 0.05: 1: 0.5: 0.5, the mentioned reagent consumption can change in ± 50% scope.
Working method:
1. drop on the reaction film after determinand being diluted with antigenic dilution reagent; (three minutes)
2. reaction film is put into confining liquid; (five minutes)
3. reaction film is put into enzyme labelled antibody reagent; (ten minutes)
4. reaction film is put into washings; (two minutes)
5. the taking-up reaction film drips substrate and damping fluid reagent; (one minute)
6. washing;
7. the result observes: by the color judged result, have color person positive, colourless person is negative; But the filing of reaction film prolonged preservation also can be taken a picture.
Synoptic diagram:
A B O AB
Anti-A ● 00 ●
A B O AB
Anti-B zero ● zero ●
A B O AB
Anti-H ● ● ● ●
The present invention compares with the technology of surveying body fluid, blood stain, hair abo blood group in the past has following advantage: (1) is easy and simple to handle, quick, only needs more than 20 minute; (2) highly sensitive, still can measure blood group such as saliva dilution more than ten thousand times, nonsecreting type saliva also can be measured blood group; (3) need not special instruments and equipment (comprising microscope, vibrating machine, refrigerator etc.), spent low; (4) need not the fresh red blood cell indication, and directly by the color judged result, be specially adapted to public security organs, procuratorial organs and people's court and carry out blood group determination.
Claims (4)
1, a kind of reagent box for determination of enzyme-labelled blood group and preparation method thereof is characterized by:
(1) combines with the ABH monoclonal antibody with horseradish peroxidase (HRP) and make monoclonal antibody linked with peroxidase, form with antigenic dilution, confining liquid, washings, substrate and damping fluid, reaction film.
(2) utilization spot ELISA method is used for the mensuration of abo blood group with monoclonal antibody linked with peroxidase, and colour-change occurs and judge blood group by being combined in zymolyte on the antibody.
2, the preparation method of reagent box for determination of enzyme-labelled blood group according to claim 1, the purification and the mark enzyme that it is characterized by the ABH monoclonal antibody are divided into nine steps:
(1) get anti-A, anti-B, anti-H monoclonal antibody ascites 5ml, centrifugal (3000 rev/mins, 30 minutes) are got and are reset and added 0.001M PH7.0-7.2 phosphate buffered saline buffer (PBS) and be diluted to 15ml;
(2) aforesaid liquid is added the affine layer of anti-mouse IgG folding post respectively, go into post after, clamp react 60 minutes, washed liquid with PBS then, detected till the no albumen outflow;
(3) with the 3M KCNS 100-150ml antibody that dissociates, when adding the 50ml left and right sides, clamp 20 minutes dissociates with the 2ml/ component velocity again, when beginning KCNS to occur, collects dissociation solution, till not containing albumen;
(4) this liquid is removed with Sephadex G-25 post get KCNS, flow velocity 3ml/ branch is collected the effluent liquid (gel-filtration) that contains monoclonal antibody protein, surveys absorbancy (OD) value of this liquid when the 280nm, calculating protein content (OD with ultraviolet spectrophotometer
280nmValue ÷ 1.45=mg/ml);
(5) said monoclonal antibody is concentrated into the above concentration of 5mg/ml, concentrated solution centrifugal (3000 rev/mins, 15 minutes) is abandoned precipitation;
(6) take by weighing horseradish peroxidase (HRP) 12mg,, add freshly prepared sodium periodate (NaIO with the dissolving of 1ml distilled water
4) 0.2M 1ml, put 20 ℃-22 ℃ 20 minutes, continuous jog;
(7) add 0.4ml ethylene glycol, 20 ℃-22 ℃ effects five minutes, continuous jog, the dress dialysis tubing spends the night with 4 ℃ of cold insulation 0.001M PH4.2 acetate buffer solution dialysis equilibriums in advance, changes liquid 4-5 time;
(8) above-mentioned hydroformylation HRP is moved into test tube, add the monoclonal antibody 20mg(volume 3-4ml that has surveyed concentration) mixing, use the PH9.6 sodium carbonate buffer, transfer PH7-12, put 20 ℃-22 ℃ 2-3 hour, shake with battle array, add fresh preparation canopy hydracid potassium liquid 0.3ml, put 4 ℃ 2-3 hour, use 0.2M NaH
2PO
4Liquid is transferred PH7.0-7.4, and this liquid is crossed Sephadex G-200 gel column, collects filtrate;
(9) collect liquid to each pipe, survey OD
280nmAnd OD
403nm, calculate mark rate, A
403nm/ A
280nm, merge mark rate at each pipe more than 0.5, will merge pipe and survey OD again
280nmAnd OD
403nmValue is calculated mark rate, and it is anticorrosion to add equivalent calf serum and Thiomersalate, adds 50% glycerine mixing again, and packing is preserved or needn't the glycerol adding freeze-drying be preserved.
3, reagent box for determination of enzyme-labelled blood group according to claim 1, the volume ratio that it is characterized by the prescription of test kit is: antigenic dilution: confining liquid: enzyme labelled antibody: washings: substrate and damping fluid were generally 1: 0.05: 1: 0.5: 0.5, the mentioned reagent consumption can change in ± 50% scope.
4, reagent box for determination of enzyme-labelled blood group according to claim 1 is characterized by confining liquid and is made up of potassium primary phosphate, Sodium phosphate dibasic, sodium-chlor, Repone K, calf serum, distilled water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 88107238 CN1014550B (en) | 1988-10-15 | 1988-10-15 | Reagent box for determination of enzyme-labelled blood group and its preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 88107238 CN1014550B (en) | 1988-10-15 | 1988-10-15 | Reagent box for determination of enzyme-labelled blood group and its preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1032926A true CN1032926A (en) | 1989-05-17 |
| CN1014550B CN1014550B (en) | 1991-10-30 |
Family
ID=4834575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 88107238 Expired CN1014550B (en) | 1988-10-15 | 1988-10-15 | Reagent box for determination of enzyme-labelled blood group and its preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1014550B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103487590A (en) * | 2013-09-03 | 2014-01-01 | 华南农业大学 | Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody |
| CN104714034A (en) * | 2013-12-17 | 2015-06-17 | 天津德祥生物技术有限公司 | Blood type detection method based on membrane structure |
| CN104950114A (en) * | 2014-03-31 | 2015-09-30 | 天津德祥生物技术有限公司 | Screening method for serum (plasma) antibody based on membrane structure and preparation method of screening and detecting kit |
| CN104950115A (en) * | 2014-03-31 | 2015-09-30 | 天津德祥生物技术有限公司 | Reverse typing detecting method for human ABO blood type based on membrane structure |
-
1988
- 1988-10-15 CN CN 88107238 patent/CN1014550B/en not_active Expired
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103487590A (en) * | 2013-09-03 | 2014-01-01 | 华南农业大学 | Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody |
| CN103487590B (en) * | 2013-09-03 | 2015-04-08 | 华南农业大学 | Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody |
| CN104714034A (en) * | 2013-12-17 | 2015-06-17 | 天津德祥生物技术有限公司 | Blood type detection method based on membrane structure |
| CN104950114A (en) * | 2014-03-31 | 2015-09-30 | 天津德祥生物技术有限公司 | Screening method for serum (plasma) antibody based on membrane structure and preparation method of screening and detecting kit |
| CN104950115A (en) * | 2014-03-31 | 2015-09-30 | 天津德祥生物技术有限公司 | Reverse typing detecting method for human ABO blood type based on membrane structure |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1014550B (en) | 1991-10-30 |
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