CN101718783B - Method for preparing broad spectrum antihumanglobulin cards - Google Patents
Method for preparing broad spectrum antihumanglobulin cards Download PDFInfo
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- CN101718783B CN101718783B CN2009102346060A CN200910234606A CN101718783B CN 101718783 B CN101718783 B CN 101718783B CN 2009102346060 A CN2009102346060 A CN 2009102346060A CN 200910234606 A CN200910234606 A CN 200910234606A CN 101718783 B CN101718783 B CN 101718783B
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Abstract
The invention relates to a method for preparing broad spectrum antihumanglobulin cards. Six micro-column gel tubes are arranged on the cards, and the six micro-column gel tubes are gel tubes containing an antibody for resisting IgG+C3d. The method comprises the following processes of preparation of a gel suspension medium, preparation of a gel, selection of the antibody, and preparation and sub-package of the gel. The gel suspension medium comprises the reagents of (5.5-6.5)*10-4g/ml methyl- p-hydroxybenzoate, (1.0-1.5)*10-4g/ml propyl p-hydroxybenzoate, (1.6-1.9)*10-2g/ml glycine, (1.7-1.9)*10-3g/ml sodium chloride, (2.1-2.4)*10-4g/ml monopotassium phosphate, (4.6-4.8)*10-4g/ml disodium hydrogen phosphate and less than or equal to 2 percent of bovine serum albumin. The reagents are dissolved by using distilled water; and the pH value is adjusted into 6.6-6.8. The broad spectrum antihumanglobulin cards prepared by the method have the advantages of high sensitivity, favorable specificity and stable quality.
Description
(1) technical field
The present invention relates to a kind of micro-column gel blood group card and application thereof that is used for blood group cross matching, irregular antibody examination, irregular antibody evaluation.In particular, the present invention relates to a kind of composition, preparation method and application thereof of broad spectrum antihumanglobulin cards, relate to the blood transfusion medical domain.
(2) background technology
Micro-column gel antiglobulin card is an immune detection new method of rising gradually recently; It is the red blood cell of incomplete antibody and corresponding antigens; The test of the antihuman globulin that in containing the antihuman globulin gel media, carries out, this method are the products that gel column combines with antihuman globulin experiment detection method, and French Dr.YvesLapierre at first invented the method in 1984; Compare with traditional test tube method antiglobulin test; Omitted the loaded down with trivial details washing procedure of traditional test tube liquid medium, also saved the red blood cell that must in the negative findings test tube, add incomplete antibody sensitization, with the step of confirming that negative findings is whether correct; Therefore have the sample consumption few, easy and simple to handle, save time, human factor is little, be easy to standardization and robotization, but the result is prone to advantages such as interpretation and long preservation, is suitable for the detection of sample in enormous quantities.
Micro-column gel antiglobulin card has become that routine is widely used in that blood group is identified, irregular antibody detects and various blood types serological test such as cross matching abroad.In recent years, the domestic detection that has begun to adopt screening that import micro-column gel antiglobulin sticks into capable irregular antibody and evaluation, sensitized erythrocyte etc., but because its price is high, apparatus expensive, has a strong impact on it and popularize at home.The domestic production that micro-column gel albumen card is also arranged in addition, but the gel that adopts both at home and abroad is a sephadex, the grain size of this gel is that particle is bigger more than 70 nanometers, the slit between the gel is bigger, thereby makes the sensitivity of its detection reduce.
(3) summary of the invention
The objective of the invention is to overcome above-mentioned deficiency, provide a kind of highly sensitive, specificity good and the preparation method of stay-in-grade broad spectrum antihumanglobulin cards.
The objective of the invention is to realize like this: a kind of preparation method of broad spectrum antihumanglobulin cards, have 6 micro-column gel pipes on the said card, said 6 micro-column gel pipes are all and contain anti-IgG+C
3The gel tube of d antibody, said method comprises following technological process:
The preparation of step 1, gel suspending medium
Said gel suspending medium prescription is following:
Methyl p-hydroxybenzoate (5.5-6.5) * 10
-4G/ml
Propylparaben (1.0-1.5) * 10
-4G/ml
Glycocoll (1.6-1.9) * 10
-2G/ml
Sodium chloride (1.7-1.8) * 10
-3G/ml
Potassium dihydrogen phosphate (2.1-2.4) * 10
-4G/ml
Sodium hydrogen phosphate (4.6-4.8) * 10
-4G/ml
Bovine serum albumin(BSA)≤2%,
Above reagent is used dissolved in distilled water, and adjust pH is 6.6-6.8;
The preparation of step 2, gel
Select the Sephacryl gel for use; Grain size is the 30-60 nanometer; Wash 3-5 time with this gel suspending medium again after the gel suspending medium immersion with the step 1 preparation; Remove the gel particle of damaged fragment of gel and gathering, collect and obtain even particle size and the complete gel that is suitable for spherical in shape.
The selection of step 3, antibody
Select broad spectrum antihumanglobulin reagent (anti-IgG+C
3D),
The sensitivity of wherein anti-IgG composition: the red blood cell that requires to contrast the anti-D IgG sensitization of volume ratio dilution in 1: 16 according to the IgG anti-humanglobulin serum that volume ratio is diluted with volume ratio at 1: 2 at 1: 4 has the visible aggegation of naked eyes,
Anti-C
3The sensitivity of d composition: require: according to antihuman globulin reagent (the anti-IgG+C of volume ratio 1: 2 and volume ratio dilution in 1: 4
3D) must be to C
3The red blood cell of d sensitization has the visible aggegation of naked eyes, with the response intensity>=1+ of said broad spectrum antihumanglobulin reagent stoste,
Broad spectrum antihumanglobulin reagent (anti-IgG+C
3D) produce positive reaction with the anti-D sensitized erythrocyte of IgG character, with C
3The d sensitized erythrocyte produces positive reaction, and does not only have IgG antibody but also do not have C
3The red blood cell reaction of d antibody sensitized is negative;
The preparation of step 4, gel
The antibody of the gel of step 2 preparation and step 3 selection according to 2: 1～3: 1 mixed of volume ratio, is mixed with and contains broad spectrum antihumanglobulin reagent (anti-IgG+C
3D) gel,
Step 5, packing
According to the amount of every pipe 22～28 microlitres, the gel that step 4 is prepared joins in six microtrabeculae pipes of a blank card, forms the broad spectrum antihumanglobulin cards with 6 micro-column gel pipes.
The invention has the beneficial effects as follows:
First aspect of the present invention has proposed a kind of standardized gel suspending medium system, is used for the washing and the suspension of gel, and can keep the stable of antibody and gel for a long time, and its characteristics are:
1, designed the buffer system with very strong surge capability, the buffer system that adopts potassium phosphate, sodium salt and amino acid to form makes the pH value of system maintain 6.6-6.8.Compare with the single citric acid buffer system that adopt usually in the blood transfusion field, have the stronger characteristics of surge capability, help keeping whole system to be in the buffering range of requirement, guaranteed the ionic strength of whole gel suspending medium simultaneously.
2, has more effectively low salt concn system; Can find out from its composition; Gel suspending medium of the present invention adopts amino acid and sodium chloride as adjuvant; Assist and keep the LIS environment, keep gel particle to be ball particle and abundant swelling, and keep the diameter (30-60 nanometer) in desired scope of gel particle with the phosphate of trace.
3, the present invention has unique lubricating system; Adopt certain density bovine protein, methyl p-hydroxybenzoate and propylparaben; Make and obtain the proper lubrication ability when red blood cell passes through the gel gap; Make the red blood cell of no aggegation have the ability of passing through the gel particle gap fully, the red blood cell of aggegation then can not pass through.
4, the present invention has superior protective system; Select the organism benzoates as antiseptic; Adopt methyl p-hydroxybenzoate and the propylparaben synergy (difference of the side carbochain length in the various P-hydroxybenzoic acid monoesters; Thereby its ability in permeates cell membranes is different, and its antibacterial action site is also just different, so various monoesters is just different to the inhibition ability of different types of microorganisms.Several kinds of esters compound has better antiseptic power.A large amount of practical application has both at home and abroad also confirmed the good antimicrobial effect of compound p-hydroxybenzoate than single p-hydroxybenzoate); Effectively prevent the procreation of bacterium; Obtain the storage life of long period; Not only avoid the use of simultaneously the Sodium azide chemical preservative thereby the ionic strength of gel suspending medium system is promoted sensitivity and the harmful effect that specificity produces to broad spectrum antihumanglobulin cards, also avoid the use of of the harmful effect of the antibiotics intermedium that institute's metabolism produces in the preservation process the specificity generation of broad spectrum antihumanglobulin cards.
Another aspect of the present invention; In order to guarantee the quality of broad spectrum antihumanglobulin cards of the present invention; In the preparation process, also need screen, that is, at first will select suitable gel gel; The condition that generally should satisfy is: selecting particle diameter is the 30-60 nanometer, through the sephadex after the propylene acidylate.The gel raw material that screening is obtained need carry out swelling, washing, suspension; Purpose is to let the abundant swelling of gel particle, and gel particle, internal diameter super large or other impurity component beyond extra small gel particle and the gel beyond the 30-60 nanometer of damaged gel particle, gathering removed in washing.After washing is accomplished with the gel suspending medium gel that suspends.
One side more of the present invention in order to guarantee the quality of broad spectrum antihumanglobulin cards of the present invention, is also screened the antibody raw material that mixes with gel in preparation process.
In sum; The storage life why broad spectrum antihumanglobulin cards of the present invention can have good specificity, sensitivity and reach 1 year; Be the synergy that is the various compositions of whole system, buffer system can be kept the pH that micro-column gel card reaction system needs; The low salt concn system can guarantee that gel particle obtains abundant swelling and gel particle diameter in needed scope.Lubricating system can guarantee lubricating ability suitable between the gel particle.Ester class antiseptic can prevent that gel or antibody lost efficacy because of bacterial reproduction.The gel of propylene acidylate can guarantee the suitable gap between the gel particle.Standardized antibody can ensure effectively detecting of antigen.Suitable bayonet socket encapsulant and air-proof condition have guaranteed that broad spectrum antihumanglobulin cards can not cause broad spectrum antihumanglobulin cards to lose efficacy because the liquid evaporation initiated gel is withered in the preservation process.
This broad spectrum antihumanglobulin cards is generally preserved the term of validity and was not less than 12 months under 18-25 ℃ of condition.
In a word; Enforcement of the present invention provides standardized broad spectrum antihumanglobulin cards product; Each hospital and Blood Transfusion Services can directly obtain the broad spectrum antihumanglobulin cards of conformance to standard from the production and supply merchant, accurately carry out blood group evaluation, cross matching, irregular antibody and detect and guarantee that safe transfusion created condition for clinical.
(4) embodiment
Below specify enforcement of the present invention and the beneficial effect that had through specific embodiment, be intended to help the reader better to understand spirit of the present invention and essence, can not constitute any qualification to practical range of the present invention.
Embodiment 1:
The preparation of step 1, gel suspending medium
Said gel suspending medium prescription is following:
Methyl p-hydroxybenzoate (5.5-6.5) * 10
-4G/ml
Propylparaben (1.0-1.5) * 10
-4G/ml
Glycocoll (1.6-1.9) * 10
-2G/ml
Sodium chloride (1.7-1.8) * 10
-3G/ml
Potassium dihydrogen phosphate (2.1-2.4) * 10
-4G/ml
Sodium hydrogen phosphate (4.6-4.8) * 10
-4G/ml
Bovine serum albumin(BSA)≤2%,
Above reagent is used dissolved in distilled water, and adjust pH is 6.6-6.8.
The preparation of step 2, gel
Select the Sephacryl gel for use, grain size is the 30-60 nanometer.Wash 3-5 time with this gel suspending medium again after the gel suspending medium immersion with the step 1 preparation, remove the gel particle of damaged fragment of gel and gathering, collect and obtain even particle size and complete suitable gel spherical in shape.
The selection of step 3, antibody
Select broad spectrum antihumanglobulin reagent (anti-IgG+C
3D): produce positive reaction with the anti-D sensitized erythrocyte of IgG character, with C
3The d sensitized erythrocyte produces positive reaction, and does not only have IgG antibody but also do not have C
3The red blood cell reaction of d antibody sensitized is negative.
The sensitivity of wherein anti-IgG composition: the red blood cell that requires to contrast the anti-D IgG sensitization of volume ratio dilution in 1: 16 according to the IgG anti-humanglobulin serum that volume ratio is diluted with volume ratio at 1: 2 at 1: 4 has the visible aggegation of naked eyes;
Wherein anti-C
3The sensitivity of d composition: require: according to antihuman globulin reagent (the anti-IgG+C of volume ratio 1: 2 and volume ratio dilution in 1: 4
3D) must be to C
3The red blood cell of d sensitization has the visible aggegation of naked eyes.Response intensity >=1+ with said broad spectrum antihumanglobulin reagent stoste;
Broad spectrum antihumanglobulin reagent (anti-IgG+C
3D) produce positive reaction with the anti-D sensitized erythrocyte of IgG character, with C
3The d sensitized erythrocyte produces positive reaction, and does not only have IgG antibody but also do not have C
3The red blood cell reaction of d antibody sensitized is negative.
The preparation of step 4, gel
The antibody of the gel of step 2 preparation and step 3 selection according to 2: 1～3: 1 mixed of volume ratio, is mixed with and contains broad spectrum antihumanglobulin reagent (anti-IgG+C
3D) gel.
Step 5, packing
According to the amount of every pipe 28 microlitres, the gel that step 4 is prepared joins in six microtrabeculae pipes of a blank card, forms the broad spectrum antihumanglobulin cards with 6 micro-column gel pipes.
Step 6, semi-manufacture are measured
Require broad spectrum antihumanglobulin cards (anti-IgG+C
3D) anti-D that detects is tired and is not less than the indirect antihuman globulin method mensuration of test tube result; Broad spectrum antihumanglobulin cards (anti-IgG+C
3D) the anti-Fy that detects
aAntibody titer is not less than the indirect antihuman globulin method of test tube and measures the result; Broad spectrum antihumanglobulin cards (anti-IgG+C
3D) with the red blood cell response intensity>=1+ of complement sensitization.Require to produce positive agglutinating reaction with the positive red blood cell of O type RhD, and negative with the negative red blood cell reaction of O type RhD; With O type Fy
aPositive red blood cell reaction is positive, and with O type Fy
aNegative red blood cell reaction is negative; With C
3The reaction of the red blood cell of d sensitization is positive, and does not only have IgG antibody but also do not have C
3The red blood cell reaction of d sensitization is negative.
Step 7, seal
Pass through the press mold mode with the sealing suitable for reading of microtrabeculae pipe with aluminium-foil paper.Label behind the mark in 18-25 ℃ of preservation.
Step 8, preservation test
Above-mentioned broad spectrum antihumanglobulin cards was preserved more than 1 year, and between this storage life, this broad spectrum antihumanglobulin cards has following testing result:
(1) outward appearance:
It is green that gel on the micro-column gel card in the microtubule should be even pale blue, and there is the as clear as crystal liquid of 1～2mm glue face upper end, and bubble and foreign matter do not have between the gel particle.
(2) sensitivity:
The sensitivity of anti-IgG gel
Require broad spectrum antihumanglobulin cards (anti-IgG+C
3The anti-D of the Rh system that d) detects is tired and the anti-Fy of Duffy system
aAntibody titer all is not less than the indirect antihuman globulin method of test tube and measures the result.
Anti-C
3D sensitivity
Require broad spectrum antihumanglobulin cards (anti-IgG+C
3D) with the red blood cell response intensity>=1+ of complement sensitization.
(3) specificity:
The specificity of anti-IgG composition
Detect IgG character anti-D, require to produce positive agglutinating reaction with the positive red blood cell of O type RhD, and negative with the negative red blood cell reaction of O type RhD; Detect the anti-Fy of IgG character
aAntibody requires and O type Fy
aPositive red blood cell reaction is positive, and with O type Fy
aNegative red blood cell reaction is negative.
Anti-C
3The specificity of d composition
Require and C
3The red blood cell reaction of d sensitization is positive.With not only do not have IgG antibody but also do not have C
3The red blood cell reaction of d sensitization is negative.
Claims (1)
1. the preparation method of a broad spectrum antihumanglobulin cards has 6 micro-column gel pipes on the said card, and said 6 micro-column gel pipes are all and contain anti-IgG+C
3The gel tube of d antibody is characterized in that said method comprises following technological process:
The preparation of step 1, gel suspending medium
Said gel suspending medium prescription is following:
Methyl p-hydroxybenzoate (5.5-6.5) * 10
-4G/ml
Propylparaben (1.0-1.5) * 10
-4G/ml
Glycocoll (1.6-1.9) * 10
-2G/ml
Sodium chloride (1.7-1.8) * 10
-3G/ml
Potassium dihydrogen phosphate (2.1-2.4) * 10
-4G/ml
Sodium hydrogen phosphate (4.6-4.8) * 10
-4G/ml
Bovine serum albumin(BSA)≤2%,
Above reagent is used dissolved in distilled water, and adjust pH is 6.6-6.8;
The preparation of step 2, gel
Select the Sephacryl gel for use; Grain size is the 30-60 nanometer; Wash 3-5 time with this gel suspending medium again after the gel suspending medium immersion with the step 1 preparation; Remove the gel particle of damaged fragment of gel and gathering, collect and obtain even particle size and complete gel spherical in shape;
The selection of step 3, antibody
Select broad spectrum antihumanglobulin reagent,
The sensitivity of wherein anti-IgG composition: the red blood cell that requires to contrast the anti-D IgG sensitization of volume ratio dilution in 1: 16 according to the IgG anti-humanglobulin serum that volume ratio is diluted with volume ratio at 1: 2 at 1: 4 has the visible aggegation of naked eyes,
Anti-C
3The sensitivity of d composition: the antihuman globulin reagent that requires with volume ratio 1: 4 to dilute at 1: 2 according to volume ratio is to C
3The red blood cell of d sensitization has the visible aggegation of naked eyes, with the response intensity>=1+ of said broad spectrum antihumanglobulin reagent stoste,
The anti-D sensitized erythrocyte of broad spectrum antihumanglobulin reagent and IgG character produces positive reaction, with C
3The d sensitized erythrocyte produces positive reaction, and does not only have IgG antibody but also do not have C
3The red blood cell reaction of d antibody sensitized is negative;
The preparation of step 4, gel
The antibody of the gel of step 2 preparation and step 3 selection according to 2: 1～3: 1 mixed of volume ratio, is mixed with and contains broad spectrum antihumanglobulin reagent (anti-IgG+C
3D) gel;
Step 5, packing
According to the amount of every pipe 22～28 microlitres, the gel that step 4 is prepared joins in six microtrabeculae pipes of a blank card, forms the broad spectrum antihumanglobulin cards with 6 micro-column gel pipes.
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|---|---|---|---|
| CN2009102346060A CN101718783B (en) | 2009-11-25 | 2009-11-25 | Method for preparing broad spectrum antihumanglobulin cards |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102346060A CN101718783B (en) | 2009-11-25 | 2009-11-25 | Method for preparing broad spectrum antihumanglobulin cards |
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| CN101718783A CN101718783A (en) | 2010-06-02 |
| CN101718783B true CN101718783B (en) | 2012-11-14 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692510A (en) * | 2012-06-07 | 2012-09-26 | 北京金豪制药股份有限公司 | Broad spectrum antihuman globulin reagent assay card and preparation thereof |
| EP3298407B1 (en) * | 2015-05-19 | 2020-11-18 | Bio-Rad Europe GmbH | Use of a low-density immiscible compound in a device for detecting an analyte in a sample |
| CN105606829A (en) * | 2015-12-30 | 2016-05-25 | 合肥天一生物技术研究所 | Newborn blood type identification card |
| CN108693355B (en) * | 2017-09-25 | 2021-02-09 | 广东睿碁生物科技有限公司 | Anti-human globulin detection reagent card and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282871A (en) * | 2000-03-29 | 2001-02-07 | 长春生物制品研究所 | Technique and reagent kit for investigating blood platelet, blood type, antigen and antibody by microcolumn gel method |
| CN101101293A (en) * | 2006-06-22 | 2008-01-09 | 基立福有限公司 | Suspension medium for red blood cells |
-
2009
- 2009-11-25 CN CN2009102346060A patent/CN101718783B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282871A (en) * | 2000-03-29 | 2001-02-07 | 长春生物制品研究所 | Technique and reagent kit for investigating blood platelet, blood type, antigen and antibody by microcolumn gel method |
| CN101101293A (en) * | 2006-06-22 | 2008-01-09 | 基立福有限公司 | Suspension medium for red blood cells |
Non-Patent Citations (1)
| Title |
|---|
| Y. LAPIERRE et al.The gel test: a new way to detect red cell antigen-antibody reactions.《TRANSFUSION》.1990,第30卷(第2期),109-113. * |
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Address after: 214434 Chengjiang Middle Road, Jiangsu, China, No. 159, No. Patentee after: Jiangsu Bo medicine biotechnology Limited by Share Ltd Address before: 214434 Chengjiang Middle Road, Jiangsu, China, No. 159, No. Patentee before: JIANGYIN LIBO MEDICINE BIOTECHNOLOGY CO., LTD. |