CN102507961B - Newborn ABO/Rh blood grouping reagent card and preparation method thereof - Google Patents
Newborn ABO/Rh blood grouping reagent card and preparation method thereof Download PDFInfo
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- CN102507961B CN102507961B CN201110322294.6A CN201110322294A CN102507961B CN 102507961 B CN102507961 B CN 102507961B CN 201110322294 A CN201110322294 A CN 201110322294A CN 102507961 B CN102507961 B CN 102507961B
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Abstract
The invention discloses a newborn ABO/Rh blood grouping reagent card and a preparation method thereof. Eight microcolumn gel tubes are provided, including: a gel tube containing a monoclonal anti-A antibody with an Ig (Immunoglobulin) M property, a gel tube containing a monoclonal anti-B antibody with the Ig M property, a gel tube containing a monoclonal anti-AB antibody with the Ig M property, a gel tube containing a monoclonal anti-D antibody with the Ig M property, a gel tube containing a monoclonal anti-D antibody with the Ig M property and an Ig G property, a gel tube which contains gel suspending medium and is used as negative control, a gel tube containing an anti-Ig G reagent and a gel tube containing the anti-Ig G reagent and an anti-C3d reagent. The reagent card disclosed by the invention has the advantages that standardization is provided for newborn ABO/Rh (D) blood grouping and conditions are created for accurate determination of ABO/Rh (D) blood group and early diagnosis and treatment of hemolytic diseases of newborn.
Description
Technical field
The present invention relates to a kind of neonate's blood group sizing and application thereof.In particular, the present invention relates to a kind of composition, preparation method and application thereof of ABO/Rh blood grouping reagent card, relate to early diagnosis and the blood transfusion medical domain of neonatal hemolytic disease.
Background technology
Neonatal hemolytic disease (being called for short HDN) is a kind of immune hemolysis disease that occurs in fetus and Newborns.This disease is closed and is caused by mother and baby's blood group incompatibility.When the dominant antigen being obtained by paternal inheritance when fetus just lacks for parent, this antigen enters parent by placenta when gestation, stimulates parent to produce corresponding immune antiboidy, and this antibody can obtain stimulating because undergoing another pregnancy to be strengthened.The immune antibody corresponding with fetal antigen existing in parent can enter in fetus body by placenta, and corresponding antigens is combined in fetal erythrocyte, causes fetal erythrocyte to be destroyed, and causes stillborn foetus, miscarriage or premature labor.After birth, show as anaemia, jaundice, seriously can cause heart failure and nuclear icterus even dead.Have data to show that China has 30% gestation to have blood group incompatibility, the incidence of disease of neonatal hemolytic disease is 11.9%.
The blood group antibody majority being produced by immunity is IgG antibody, and naturally occurring blood group antibody majority is IgM antibody.A large amount of immunohematologies experiment showed, to only have the IgG immune antiboidy of molecular weight to be entered in fetus body and to be caused neonatal hemolytic disease by placenta.Therefore neonatal hemolytic disease can occur in the blood group system that all energy immunity produce IgG antibody, but mainly occur in ABO and Rh system, wherein the neonatal hemolytic disease state of an illness of ABO system is generally lighter, the neonatal hemolytic disease state of an illness of Rh system is more serious, other blood group systems do not conform to Hemolysis also has report as Kidd, MN, Duffy etc., but seldom sees.Anti-P1, anti-Lea, Leb etc. belong to IgM character, can not pass through placenta, therefore can not produce HDN.
Neonatal hemolytic disease is one of neonatal common disease, as diagnoses and treatment early, cure rate is higher, the fewer nervous system sequelae of leaving over,, can there is severe complication nuclear icterus and can leave over the nervous system sequelae such as feeblemindedness, dysacousis, tic in not timely diagnoses and treatment person.Therefore diagnosing early, treating is the key of this disease.Therefore it is very important, neonatal hemolytic disease being done to early diagnosis in postpartum to take preventive measures in time.Accomplish that fetus early diagnosis just must do correct immune analysis and blood group serology inspection to neonate.
Neonate due to when birth ABO antigen also do not grow completely, until just reach full growth after 18 months, and Rh blood group antigens generally form at birth.When neonate is born, except minority has micro-IgM antibody, not yet form oneself IgG, IgA and IgE, most of baby will form blood group antibody after birth some months.So neonate's abo blood group detects, take positive definite form as main, compare with adult, a little less than antigen, antibody expression, have undetected risk, easily cause bracket for blood grouping and cross matching difficulty.Correct bracket for blood grouping is to guarantee safe neonate blood transfusion prerequisite and guarantee with selecting high sensitivity cross matching method.Contribute to making a definite diagnosis in time and treating of neonatal hemolytic disease.Traditional bracket for blood grouping, the method for cross matching be plectrum method, salt solution blood matching namely, operates loaded down with trivial details, easy undetected, false retrieval, can not be bundled into the correct of testing result, serious harm receptor's life security.Yet, at present domesticly there is no the kit that any neonatal hemolytic disease detects and sell, the detection method of various places hospital and Blood Center is multifarious, can not precise Identification can cause the antibody actual conditions of neonatal hemolytic disease.
At home and abroad, some produces house and produces the ABO/Rh blood typing detection reagent card for abo blood group sizing and Rh sizing, but its gel adopting is sephadex, and the diameter of this gel particle is greatly about 20-50 micron.In addition, existing card only carries out the detection of RhIgM antibody, does not carry out the mensuration of Rh IgG antibody, has reduced the accuracy of ABO/Rh blood typing, easily causes the mistake of ABO/Rh blood group.
Summary of the invention
The present invention puts object and is to provide neonate ABO/Rh bracket for blood grouping reagent card of a kind of highly sensitive, high specificity, good stability and preparation method thereof.
Technical solution of the present invention is:
A kind of neonate ABO/Rh bracket for blood grouping reagent card, it is characterized in that: there are 8 micro-column gel pipes, respectively: one manages the gel tube of the monoclonal anti-A antibodies for containing IgM character, one manages the gel tube of the monoclonal anti-B antibody for containing IgM character, one manages the gel tube of the monoclonal anti AB antibody for containing IgM character, one manages the gel tube of the monoclonal anti-D for containing IgM character, one manages the gel tube of the monoclonal anti-D for containing IgM character and IgG character, one pipe is the gel tube that contains gel suspending medium for negative control, gel tube and the pipe gel tube for contain anti-IgG reagent and anti-C3d reagent of one pipe for containing anti-IgG reagent.
A preparation method for neonate ABO/Rh bracket for blood grouping reagent card, is characterized in that: comprise the following steps:
1) preparation of gel buffer suspension liquid
Described buffer formulation is as follows:
The 0.15M phosphate buffer of 20mL
Sodium chloride 1.75g
Glycocoll 18g
Wherein the 0.15M phosphate buffer of 20mL is the 0.15MKH by 11.3mL
2pO
40.15M Na with 8.7mL
2hPO
4form;
Above reagent dissolves with 800 ml distilled waters, with 1M NaOH, regulates pH to 6.7, is finally settled to one liter;
Add methyl p-hydroxybenzoate 0.6g and propylparaben 0.13g, add bovine serum albumin(BSA), addition is that to make the final concentration of bovine serum albumin(BSA) be 3%; Obtain gel buffer suspension liquid;
2) preparation of gel
Select sephadex, grain size is 20-50 micron, by step 1) join gel buffer suspension liquid fully soak after again with this gel buffer suspension liquid washing 3-5 time, remove the damaged fragment of gel, collection obtains the complete gel spherical in shape of even particle size;
3) selection of antibody
Select the monoclonal anti-A antibodies of IgM character, tire >=128
Select the monoclonal anti-B antibody of IgM character, tire >=128
Select the monoclonal anti AB antibody of IgM character, tire >=128
Select the monoclonal anti-D of IgM character, tire >=64
Select the monoclonal anti-D of IgG character and IgM character, tire >=64
The selection of anti-IgG reagent: require according to the antihuman globulin reagent of volume ratio 1: 2 and volume ratio dilution in 1: 4, must be to having the visible aggegation of naked eyes according to the red blood cell of the anti-D IgG sensitization of volume ratio dilution in 1: 16;
The selection of anti-C3d reagent: requiring must have the visible aggegation of naked eyes to the red blood cell of C3d sensitization according to the anti-C3d reagent of volume ratio 1: 2 and volume ratio dilution in 1: 4, with the response intensity >=1+ of anti-C3d reagent stoste;
4) preparation of gel
By step 2) gel and step 3 prepared) selected each antibody mixes according to the ratio of volume 65%: 35%, is mixed with respectively the gel of the monoclonal anti-A antibodies that contains IgM character, the gel, the gel, the gel, the gel, the gel that contains anti-IgG reagent of monoclonal anti-D that contains IgG character and IgM character of monoclonal anti-D that contains IgM character of monoclonal anti AB antibody that contains IgM character of monoclonal anti-B antibody that contains IgM character; By step 2) gel and the step 3 prepared) anti-IgG reagent and anti-C3d reagent according to volume ratio, be mixed with the gel that contains anti-IgG reagent and anti-C3d reagent at 65%: 17.5%: 17.5%, by step 2) gel and step 1 prepared) in join gel buffer suspension liquid according to the ratio of volume 65%: 35%, mix, be mixed with the gel that contains gel suspending medium.
5) packing
By step 4) in each gel of obtaining of preparation be added to respectively in 8 microtrabeculae pipes of a blank card, form the neonate with 8 micro-column gel pipes and detect reagent card.
The invention has the advantages that:
1. use the buffer system with very strong surge capability, the buffer system that adopts phosphate and amino acid to form, the pH of system maintains 6.7 left and right.Compare with the citric acid buffer system that routine blood transfusion field is used, there is stronger surge capability, be conducive to guarantee that whole system is in suitable buffering range.
2. gel buffer liquid of the present invention has low ion concns system, with amino acid and sodium chloride as adjuvant, auxiliary micro-phosphate maintains low ionic strength environment, keeps gel particle to be ball particle abundant swelling, and the diameter of gel particle is controlled in certain scope.Low ionic strength salt solution can increase the gravitation between antigen-antibody simultaneously, makes originally in brine media, can not aggegation can occur the erythrocytic antibody of aggegation.Can fast reaction speed, shorten the time of insulation.
3. this damping fluid has lubricating system, adopt certain density bovine serum albumin(BSA), while making red blood cell pass through gel gap, obtain proper lubrication ability, make to have without the red blood cell of aggegation the ability in gel gap of passing through completely, the red blood cell of aggegation can not pass through.
4. there is antisepsis, lot of documents confirms to select organism benzoates as antiseptic, can effectively prevent the procreation of bacterium, with respect to Sodium azide, not only there is the storage life of longer time, and can avoid using Sodium azide chemical preservative to promote the ionic strength of gel suspending medium system and the harmful effect that the sensitivity of neonate ABO/Rh bracket for blood grouping reagent card and specificity are produced that causes.
Another aspect of the present invention in order to guarantee the quality of neonate ABO/Rh bracket for blood grouping reagent card of the present invention, needs to select suitable gel in gel set-up procedure, and existing selected gel particle diameter is 20-50 micron.By sephadex particle is carried out to abundant swelling, damaged gel particle and super large or extra small gel particle are removed in washing.The buffer suspension liquid gel that suspends for gel after washing.Except the particle of screening gel, also need in addition to select suitable antibody of tiring.
The storage life why neonate ABO/Rh bracket for blood grouping reagent card of the present invention has good specificity, sensitivity and reach 1 year half, it is the synergy that is the various compositions of whole system, gel particle is arranged on a card can complete neonate ABO/Rh bracket for blood grouping, buffer system can maintain the pH that reaction system needs, and less salt is done the system of being permitted can guarantee that gel particle obtains abundant swelling and gel particle diameter in needed scope.Lubricating system can guarantee the lubricating ability between gel particle.Organic anti-corrosive agent can prevent that gel or antibody lost efficacy because of bacterial contamination.Gel spherical in shape can guarantee gap suitable between gel particle.The detection that suitable antibody is antigen provides effective guarantee.
This neonate ABO/Rh bracket for blood grouping reagent card can be for the confirmation of ABO, Rh (D) blood group, and the positive reaction of generation is not less than 3+.Generally under 2-25 ℃ of condition, preserve the term of validity and be not less than 18 months.
The neonate of being embodied as ABO of the present invention, Rh (D) bracket for blood grouping provide standardization, various big hospital and Blood Transfusion Services can be with consistent neonate ABO/Rh (D) the bracket for blood grouping reagent cards of this standard, confirm accurately ABO/Rh (D) blood group, to neonatal hemolytic disease early diagnosis, condition has been created in treatment.
Below in conjunction with embodiment, the invention will be further described.
Embodiment
A preparation method for neonate ABO/Rh bracket for blood grouping reagent card, is characterized in that: comprise the following steps:
1) preparation of gel buffer suspension liquid
Described buffer formulation is as follows:
The 0.15M phosphate buffer of 20mL
Sodium chloride 1.75g
Glycocoll 18g
Wherein the 0.15M phosphate buffer of 20mL is the 0.15MKH by 11.3mL
2pO
40.15M Na with 8.7mL
2hPO
4form;
Above reagent dissolves with 800 ml distilled waters, with 1M NaOH, regulates pH to 6.7, is finally settled to one liter;
Add methyl p-hydroxybenzoate 0.6g and propylparaben 0.13g, add bovine serum albumin(BSA), addition is that to make the final concentration of bovine serum albumin(BSA) be 3%; Obtain gel buffer suspension liquid;
2) preparation of gel
Select sephadex, grain size is 20-50 micron, by step 1) join gel buffer suspension liquid fully soak after again with this gel buffer suspension liquid washing 3-5 time, remove the damaged fragment of gel, collection obtains the complete gel spherical in shape of even particle size;
3) selection of antibody
Select the monoclonal anti-A antibodies of IgM character, tire >=128
Select the monoclonal anti-B antibody of IgM character, tire >=128
Select the monoclonal anti AB antibody of IgM character, tire >=128
Select the monoclonal anti-D of IgM character, tire >=64
Select the monoclonal anti-D of IgG character and IgM character, tire >=64
The selection of anti-IgG reagent: require according to the antihuman globulin reagent of volume ratio 1: 2 and volume ratio dilution in 1: 4, must be to having the visible aggegation of naked eyes according to the red blood cell of the anti-D IgG sensitization of volume ratio dilution in 1: 16;
The selection of anti-C3d reagent: requiring must have the visible aggegation of naked eyes to the red blood cell of C3d sensitization according to the anti-C3d reagent of volume ratio 1: 2 and volume ratio dilution in 1: 4, with the response intensity >=1+ of anti-C3d reagent stoste;
4) preparation of gel
By step 2) gel and step 3 prepared) selected each antibody (or reagent) mixes according to the ratio of volume 65%: 35%, is mixed with respectively the gel of the monoclonal anti-A antibodies that contains IgM character, the gel, the gel, the gel, the gel, the gel that contains anti-IgG reagent of monoclonal anti-D that contains IgG character and IgM character of monoclonal anti-D that contains IgM character of monoclonal anti AB antibody that contains IgM character of monoclonal anti-B antibody that contains IgM character; By step 2) gel and the step 3 prepared) anti-IgG reagent and anti-C3d reagent according to volume ratio, be mixed with the gel that contains anti-IgG reagent and anti-C3d reagent at 65%: 17.5%: 17.5%, by step 2) gel and step 1 prepared) in join gel buffer suspension liquid according to the ratio of volume 65%: 35%, mix, be mixed with the gel that contains gel suspending medium.
5) packing
By step 4) in each gel of obtaining of preparation according to the amount of every pipe 40 microlitres, be added to respectively in 8 microtrabeculae pipes of a blank card after mixing, form the neonate with 8 micro-column gel pipes and detect reagent card.
6) semi-manufacture detect
Requirement will divide the micro-column gel pipe installing, and has with the red blood cell of antibody corresponding antigens and produces positive reaction, and red blood cell concentrates on gel upper surface, presents linear pattern.And produce negative reaction with the red blood cell that does not contain the corresponding antigen of antibody, red blood cell can all arrive microtubule bottom by gel, is deposited on microtubule bottom.And only do not contain red blood cell in the gel tube of gel suspending medium and gel mixture containing antibody, can all by gel, arrive microtubule bottom, be deposited on bottom microtubule, present negative reaction.
7) sealing
With aluminium-foil paper, pass through press mold mode by the sealing suitable for reading of microtrabeculae pipe.Label after mark in 2-25 ℃ of preservation.
8) preserve experiment
The above-mentioned ABO/Rh sizing completing detects reagent card and can preserve 18 months, and between storage life, this ABO/Rh blood grouping reagent jig has following testing result:
(1) outward appearance:
Neonate ABO/Rh blood typing detection reagent card is that 8 microtrabeculae pipes form, and gel is milky, 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 gel particle.
(2) sensitivity
In the micro-column gel pipe that contains antibody, have with the red blood cell of antibody corresponding antigens and produce positive reaction, and red blood cell concentrates on gel upper surface, present linear pattern.
(3) specificity
Containing in the micro-column gel pipe of antibody, have with the red blood cell of antibody corresponding antigens and produce positive reaction, red blood cell concentrates on gel upper surface, presents linear pattern.Produce negative reaction with the red blood cell that does not contain the corresponding antigen of antibody, red blood cell can all arrive microtubule bottom by gel, is deposited on microtubule bottom.And only do not contain red blood cell in the gel tube of gel suspending medium and gel mixture containing antibody, can all by gel, arrive microtubule bottom, be deposited on bottom microtubule, present negative reaction.
Above-mentioned neonate ABO/Rh blood typing detection reagent card using method:
ABO/Rh blood typing detection reagent card is comprised of 8 gel tubes, the gel of the monoclonal anti-A antibodies that wherein the first pipe contains IgM character, the gel of the monoclonal anti-B antibody that the second pipe contains IgM character, the gel of the monoclonal anti AB antibody that the 3rd pipe contains IgM character, the gel of the monoclonal anti-D that the 4th pipe contains IgM character, the gel tube of the monoclonal anti-D that the 5th pipe contains IgM character and IgG character, the 6th pipe is for the gel that contains gel suspending medium of negative control, the gel tube that the 7th pipe contains anti-IgG antibody, the gel tube of the monoclonal anti IgG+C3d antibody that the 8th pipe contains IgM character.
1, with LISS (LISS) or physiological saline (NS), person's red blood cell to be measured is mixed with to 0.8% (v/v), adds respectively in first, second, third, fourth, the 5th, the 6th, the 7th and the 8th microtubule, 50 μ L/ pipes.
2, incubated at room 10min, centrifugal with micro-column gel card special centrifugal machine, 900rpm2min, 1500rpm 3min, naked eyes result of determination after taking out, record.
3, result judgement
Positive findings: red blood cell suspension is in gel surface or glue.
Negative findings: red blood cell is deposited on the bottom of micro-column gel.
9) blood group judgement sees the following form
ABO series
| Pipe A | Pipe B | Pipe AB | Pipe (contrast) | ABO Group |
| 0 | 0 | 0 | 0 | O |
| + | 0 | + | 0 | A |
| 0 | + | + | 0 | B |
| + | + | + | 0 | AB |
Rh series
+: the positive
0: feminine gender
Alserver's solution comprises as follows:
| Citric acid trisodium | 23-25mmol | Potassium dihydrogen phosphate | 1.5mmol |
| Sodium hydrogen phosphate | 1mmol | Sodium chloride | 68-72mmol |
| Glucose | 50-55mmol | Sucrose | 19-22mmol |
| Adenine | 1.5-1.7mmol | Inosine | 4.5-4.8mmol |
| Neomycinsulphate | 0.033-0.040％ | Chloromycetin | 0.01-0.02％ |
This shows excellent effect of the present invention.
Claims (1)
1. a preparation method for neonate ABO/Rh bracket for blood grouping reagent card, is characterized in that: comprise the following steps:
1) preparation of gel buffer suspension liquid
Described buffer formulation is as follows:
The 0.15M phosphate buffer of 20mL
Sodium chloride 1.75g
Glycocoll 18g
Wherein the 0.15M phosphate buffer of 20mL is the 0.15MKH by 11.3mL
2pO
40.15M Na with 8.7mL
2hPO
4form;
Above reagent dissolves with 800 ml distilled waters, with 1M NaOH, regulates pH to 6.7, is finally settled to one liter;
Add methyl p-hydroxybenzoate 0.6g and propylparaben 0.13g, add bovine serum albumin(BSA), addition is that to make the final concentration of bovine serum albumin(BSA) be 3%; Obtain gel buffer suspension liquid;
2) preparation of gel
Select sephadex, grain size is 20-50 micron, with step 1) join gel buffer suspension immersion bubble after again with this gel buffer suspension liquid washing 3-5 time, remove gel breakage fragment, collection obtains the complete gel spherical in shape of even particle size;
3) selection of antibody
Select the monoclonal anti-A antibodies of IgM character, tire >=128
Select the monoclonal anti-B antibody of IgM character, tire >=128
Select the monoclonal anti AB antibody of IgM character, tire >=128
Select the monoclonal anti-D of IgM character, tire >=64
Select the monoclonal anti-D of IgG character and IgM character, tire >=64
The selection of anti-IgG reagent: require according to the antihuman globulin reagent of volume ratio 1:2 and volume ratio 1:4 dilution, must be to having the visible aggegation of naked eyes according to the red blood cell of the anti-D IgG sensitization of volume ratio 1:16 dilution;
The selection of anti-C3d reagent: requiring must have the visible aggegation of naked eyes to the red blood cell of C3d sensitization according to the anti-C3d reagent of volume ratio 1:2 and volume ratio 1:4 dilution, with the response intensity >=1+ of anti-C3d reagent stoste;
4) preparation of gel
By step 2) gel prepared mixes according to the ratio of volume 65%: 35% with selected each antibody of step 3), is mixed with respectively the gel of the monoclonal anti-A antibodies that contains IgM character, the gel, the gel, the gel, the gel, the gel that contains anti-IgG reagent of monoclonal anti-D that contains IgG character and IgM character of monoclonal anti-D that contains IgM character of monoclonal anti AB antibody that contains IgM character of monoclonal anti-B antibody that contains IgM character; By step 2) gel prepared and anti-IgG reagent and the anti-C3d reagent of step 3) is mixed with the gel that contains anti-IgG reagent and anti-C3d reagent according to volume ratio 65%:17.5%:17.5%, by step 2) gel prepared with in step 1), join gel buffer suspension liquid according to the ratio of volume 65%: 35%, mix, be mixed with the gel that contains gel suspending medium;
5) packing
Each gel that preparation in step 4) is obtained is added to respectively in 8 microtrabeculae pipes of a blank card, and the neonate that formation has 8 micro-column gel pipes detects reagent card.
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| CN104569443B (en) * | 2014-12-22 | 2016-05-11 | 合肥天一生物技术研究所 | A kind of Rh blood type test card |
| CN104597259A (en) * | 2015-01-04 | 2015-05-06 | 许明安 | Antihuman-globulin blood matching detection card with different formulas at primary side and secondary side |
| CN105606829A (en) * | 2015-12-30 | 2016-05-25 | 合肥天一生物技术研究所 | Newborn blood type identification card |
| CN106970233B (en) * | 2017-05-02 | 2019-06-14 | 临沂大学 | A kind of new life mule coltfoal kit for testing hemolytic disease and preparation method thereof |
| CN107561294A (en) * | 2017-08-16 | 2018-01-09 | 北京乐普医疗科技有限责任公司 | Neonate's ABO, RhD blood type test card and preparation method thereof |
| 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 |
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