CN107014992B - A kind of foundation of quantitative detection human blood type antibody concentration method - Google Patents
A kind of foundation of quantitative detection human blood type antibody concentration method Download PDFInfo
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- CN107014992B CN107014992B CN201611006650.2A CN201611006650A CN107014992B CN 107014992 B CN107014992 B CN 107014992B CN 201611006650 A CN201611006650 A CN 201611006650A CN 107014992 B CN107014992 B CN 107014992B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Abstract
The invention discloses a kind of with the anti-A of immunoturbidimetry quantitative detection people, the method for anti-blood group antibody B concentration, for clinical vitro detection reagent technique field.Reagent applied in this method includes reagent R1, reagent R2, reagent R3, standard items 1, standard items 2.Method particularly includes: Antigen of human blood group specificity trisaccharide is coupled on KLH, it is prepared into KLH-A or KLH-B protein dry powder, purity unicity, the permanence of preservation of blood group antigens can effectively be improved, and the reagent R1 (effective component KLH-A) and reagent R2 (effective component KLH-B) being prepared into through the PBS dissolution with 0.01M pH7.4 have accurate sample concentration, finally it is mixed with reagent R3 and test sample, with the concentration that can quantify blood group antibody in sample after standard control.Through being found compared with clinically used blood group antibody detection method, heretofore described method can will test result quantization, have the characteristics that intuitive and accurate, and the influence to testing result such as reagent stability, human factor can be effectively excluded, thus popularization and application of the blood group antibody quantitative detecting method for being conducive to be illustrated in the present invention in terms of clinical detection.
Description
Technical field
The present invention relates to a kind of with anti-A in immunoturbidimetry quantitative detection human peripheral, the side of anti-blood group antibody B concentration
Method belongs to clinical vitro detection reagent technique field.
Background technique
Blood group antigens are not distributed only on erythrocyte membrane as one of big antigen of human body three, exist in the groups such as liver, kidney
Knit cell and Surface of Vascular Endothelial Cells.According to the similarities and differences of antigenic substance on erythrocyte membrane, the blood group that International Society of Blood Transfusion announces is divided
Just up to 30 kinds of type system, wherein ABO blood group system is widely used in human body as main and common blood grouping method
Blood transfusion, organ transplant etc..
ABO blood group system includes four kinds of blood groups, i.e. A type, Type B, AB type and O-shaped.Four kinds of different blood group crowds are anti-in blood group
Difference in terms of former and blood group antibody essentially consists in: blood group A antigen is expressed on the erythrocyte membrane of A type blood, bone-marrow-derived lymphocyte expresses blood
Type B antigen-antibody, blood plasma prestore certain density blood group B antigen-antibody；Blood group B antigen, B are expressed on the erythrocyte membrane of Type B blood
Expressions In Lymphocytes blood group A antigen antibody prestores certain density blood group A antigen antibody in blood plasma；The erythrocyte membrane of AB type blood
Upper expression blood group A antigen and blood group B antigen, bone-marrow-derived lymphocyte do not express blood group antigens antibody, the blood group antigens that nothing prestores in blood plasma
Antibody；Blood group antigens are not expressed on the erythrocyte membrane of O-shaped blood, bone-marrow-derived lymphocyte expresses blood group A antigen antibody and blood group B antigen is anti-
Body prestores certain density blood group A antigen antibody and blood group B antigen-antibody in blood plasma.
Currently, waiting the patient numbers of organ transplant can not have always been high any more both at home and abroad, the main reason is that groups compatible
The height of organ lacks.Based on this, the incompatible organ transplant of abo blood group (ABO blood group incompatibility
Organ transplantation, ABOi-OT) in global successful development, become and solves an organ origin weight in short supply
Want approach.And prestore blood group antibody in preoperative removing recipient's body and control postoperative blood group antibody concentration rebound, becoming influences ABOi-
The major obstacle of OT.Therefore, the concentration of monitoring and quantitative detection ABOi organ transplant front and back blood group antibody is performed the operation as ABOi-OT
The important evidence of solution formulation.
Currently, the method for clinical labororatory's detection blood group antibody mainly has high performance liquid chromatography (HPLC) and brine media
Method, antiglobulin medium method, low ion polybrene medium method, micro-column gel detection card etc..Wherein, although HPLC is quantitative detection
Method, but because the device is complicated and expensive for needs, it is difficult to it is widely used in clinical labororatory；Remaining detection method --- especially
It is that clinically used micro-column gel detection blocks at present, is semi-quantitative detection method, it is difficult to provide quantitative as a result, and to detection
As a result judgement is influenced vulnerable to reagent stability, human factor etc..And the blood group antibody immunoturbidimetry used in the present invention
It is to be reacted based on the specific binding of blood group antibody-blood group antigens, the immune complex formed has absorption peak in 340nm wavelength
Value.This method does not need expensive equipment, and can realize automation, high-volume Samples detection, meets in clinical detection application
Basic demand.In addition, artificial synthesized blood group antigens specificity trisaccharide is coupled to keyhole limpet hemocyanin in the present invention, it can be ensured that
Detection purity unicity, the certainty of concentration, the permanence of preservation of reagent, more improves the method in terms of clinical detection
Popularization and application.
Summary of the invention
For the deficiency of current clinical human blood type antibody concentration measuring method, the present invention provides a kind of quantitative detection blood groups
The method for detecting antibody concentration, compared with micro-column gel detection card, as a result more objectivity is conducive to be illustrated in the present invention
Popularization and application of the blood group antibody quantitative detecting method in terms of clinical detection.
1. agents useful for same of the present invention and composition
A kind of human blood type antibody quantitative detecting method based on immunoturbidimetry, which is characterized in that include reagent R1, reagent
R2, reagent R3, calibration object 1 and calibration object 2.Wherein reagent R1 group becomes the PBS buffer solution of 0.01M pH 7.4,0.008-
The keyhole limpet hemocyanin of 0.012mg/ml is coupled blood group A antigen (KLH-A)；Reagent R2 group is slow as the PBS of 0.01M pH 7.4
Keyhole limpet hemocyanin coupling blood group B antigen (KLH-B) of fliud flushing, 0.008-0.012mg/ml；Reagent R3 group becomes 0.01M pH
7.4 PBS buffer solution, 0.15M NaCl, 40-60g/L PEG-8000.
KLH-A, KLH-B of the present invention are Alberta Innovates Techenology Futures company
Synthesis, calibration object 1 (the anti-human blood group A antigen antibody reagent of mouse) and calibration object 2 (the anti-human blood group B antigen-antibody reagent of mouse) are purchased from
Abcam company.
2. blood group antibody quantitative detecting method is established in the present invention
1. the specific binding site of Antigen of human blood group is the specific trisaccharide on erythrocyte membrane, this side chain is glycosyl transfer
Enzymatic specificity trisaccharide group is coupled to albumen on cell membrane, such as Band 3,4.5 albumen of Band on erythrocyte membrane, kidney
VWF, PLVAP, PECAM1 albumen of tissue.It therefore, is the purity and antigenicity that guarantee antigen, artificial synthesized blood group in the present invention
After A and B antigentic specificity trisaccharide, it is coupled on KLH albumen (KLH-A, KLH-B).
2. detecting common Antigen of human blood group antibody is the anti-human blood group antigens antibody plasma of mouse, contain various kinds of cell culture
Liquid Proteins.Therefore, it is anti-human that PAGE electrophoresis combination western-blotting technology is used in the present invention, identify and analyzes mouse
Blood group A, B antibody concentration, concrete operations and result are as follows:
The identification of blood group antibody effective ingredient in practice
By the anti-human blood group A antigen antibody plasma of 1 μ l mouse or the 4 anti-human blood group B antigen-antibody blood plasma of μ l mouse through 8% gum concentration
After PAGE electrophoresis, 300mA constant current ice bath electricity turns 90min, and TBS washes film 5min × 3 time, sheep anti mouse IgM-HRP (1: 2000) room temperature
It is incubated for 40min, TBS washes film 10min × 2 time, ECL luminesceence analysis.
As a result as shown in Figure 1, being deposited in the anti-human blood group A antigen antibody plasma of mouse and the anti-human blood group B antigen-antibody blood plasma of mouse
In a variety of non-IgM molecules, and there was only the anti-human blood group A antigen antibody of mouse or the anti-human blood plasma B antigen-antibody of mouse in two parts of plasma antibodies
With sheep anti mouse IgM-HRP association reaction.
The analysis of effective ingredient concentration in blood group antibody reagent kit
Using BSA as standard items, 0.5 μ g, 1 μ g, 2 μ g, 4 μ g, 8 μ gBSA albumen, Cowes brilliant blue dye are separated by electrophoresis by PAGE
Color and after sufficiently decolourizing, with each concentration BSA band gray value in PDQuest software analysis polyacrylamide gel, and is established
Protein content-gray value standard curve.On the basis of secondary, the anti-human blood group A antigen antibody of 1 μ l mouse or the 1 anti-human blood group B of μ l mouse are taken respectively
Antigen-antibody analyzes it after the PAGE electrophoresis of 8% gum concentration and sufficiently coomassie brilliant blue staining decoloration with PDQuest software
Gray value, and establishing criteria curve calculates the concentration of mouse anti-human blood group A antigen antibody and the anti-human blood group B antigen-antibody of mouse.Every group
Data are repeated three times.
As a result as shown in Fig. 2, BSA protein content-gray value standard curve related coefficient is 0.9952, Dependence Results can
Letter.Be computed analysis, the concentration of the anti-human blood group A antigen antibody of mouse and the anti-human blood group B antigen-antibody of mouse be respectively 0.90 μ g/ μ l and
0.57μg/μl。
3. on the basis of the above results, with the immunoturbidimetry analysis anti-human blood group B antigen-antibody of mouse and KLH-B antigen
Association rate curve, with the optimal detection time of determination, detecting instrument used is BioPhotometer Plus nucleic acid-protein
Instrument (λ of the present invention340This model instrument is used in the measurement of nm light absorption value), concrete operations and result are as follows:
Under the conditions of verifying current experiment, optimal light absorption value minute point.The present invention chooses practical R2 and calibration object 2
As measure object, to determine the optimum detection time point of the invention that should be taken.
Firstly, taking 5 small test tubes, physiological saline 0.1ml is added in every pipe, and 0.1ml standard items 2 are added in the 1st pipe, is mixed
0.1ml is drawn afterwards, and the 2nd pipe is added, and so on, until the 5th pipe, finally discards 0.1ml.Then, it is returned to zero with the reagent R2 of 100 μ l
Afterwards, the reagent R2 of 99 μ l is mixed with the sample after 1 μ l dilution, since 3min, the sample of detection mixing per minute is in λ340nm
Light absorption value, until 10min.Every group of Data duplication three times, takes its average value to establish the anti-human blood group antigens antibody of mouse and antigen
Association rate curve.
As a result as shown in figure 3, the association rate of the anti-human blood group B antigen-antibody of mouse and KLH-B reach maximum value in 5min,
Association rate variation later gradually decreases.It therefore, is the combination situation of the better reacting antigen-antibody of energy, the present invention will take
As detection time point when 5min.Taken detection time point is consistent with relevant report.
4. further by formulating the anti-human blood group A antigen antibody of mouse and KLH-A, the anti-human blood group B of mouse with immunoturbidimetry
The antibody concentration of antigen-antibody and KLH-B-light absorption value standard curve (antigen-antibody binding curve is in cubic function relationship), specifically
Operation and result are as follows:
The combined standard curve of blood group A antigen antibody and blood group A antigen
Firstly, taking 5 small test tubes, physiological saline 0.1ml is added in every pipe, and 0.1ml standard items 1 are added in the 1st pipe, is mixed
0.1ml is drawn afterwards, and the 2nd pipe is added, and so on, until the 5th pipe, finally discards 0.1ml.Then, it is returned to zero with the reagent R1 of 100 μ l
Afterwards, the reagent R1 of 99 μ l is mixed with the sample after 1 μ l dilution, after being incubated at room temperature 5min, in λ340nmDetect its light absorption value.Every group
Data duplication three times after, take its average value to establish the combined standard curve of blood group A antigen antibody and blood group A antigen.
The combined standard curve of blood group B antigen-antibody and blood group B antigen
Firstly, taking 5 small test tubes, physiological saline 0.1ml is added in every pipe, and 0.1ml standard items 2 are added in the 1st pipe, is mixed
0.1ml is drawn afterwards, and the 2nd pipe is added, and so on, until the 5th pipe, finally discards 0.1ml.Then, it is returned to zero with the reagent R2 of 100 μ l
Afterwards, the reagent R2 of 99 μ l is mixed with the sample after 1 μ l dilution, after being incubated at room temperature 5min, in λ340nmDetect its light absorption value.Every group
Data duplication three times after, take its average value to establish the combined standard curve of blood group B antigen-antibody Yu blood group B antigen.
As a result as shown in Figure 4 and Figure 5, Fig. 4 is the anti-human blood group A antigen antibody of mouse and KLH-A combined standard curve, curve
Related coefficient is 0.9957, and real result is credible；Fig. 5 is the anti-human blood group B antigen-antibody of mouse and KLH-B combined standard curve, song
The phase relation coefficient of line is 0.9966, and real result is credible.
5. finally compared with micro-column gel detection card, with blood group antibody quantitative detecting method used in the verifying present invention to just
The accuracy of blood group antibody concentration mensuration in ordinary person's peripheral blood.
Detailed description of the invention
The identification of anti-human blood group A, the B antigen-antibody of Fig. 1 mouse.(A) western- of the anti-human blood group A antigen antibody of mouse
Blotting analysis, Lane 1.Marks, Lane 2.PAGE analysis, Lane 3.western-blotting analysis, secondary antibody are
Sheep anti mouse blood group antigens IgM-HRP (1: 2000)；(B) the western-blotting analysis of the anti-human blood group B antigen-antibody of mouse,
Lane 1.Marks, Lane 2.PAGE analysis, Lane 3.western-blotting analysis, secondary antibody are sheep anti mouse blood group antigens
IgM-HRP(1∶2000)。
Fig. 2 .BSA standard protein concentration-gray value tracing analysis.(A) PAGE electrophoresis, Lane 1.0.5 μ gBSA,
3.2 4.4 5.8 μ g BSA of μ g BSA, Lane of μ g BSA, Lane of Lane2.1 μ g BSA, Lane；(B) BSA protein content-gray scale
Value analysis
Fig. 3 blood group B antigen and the anti-human blood group B antigen-antibody binding time rate-change curve of mouse
The analysis of Fig. 4 human blood type Staphylococal Protein A antibody standard curve
The analysis of Fig. 5 human blood type B antigen-antibody standard curve
Specific embodiment
One, of embodiment is compared with micro-column gel detection card, blood group antibody concentration quantitative used detection side in the verifying present invention
The accuracy of method.
1. micro-column gel detection card separately verify A, in Type B human normal plasma blood group antibody potency.
Firstly, taking 5 small test tubes, physiological saline 0.1ml is added in every pipe.0.1ml A type or Type B blood are added in the 1st pipe
Blood plasma draws 0.1ml and the 2nd pipe is added after mixing, and so on, until the 5th pipe, finally discards 0.1ml.Then, every pipe microtrabeculae is solidifying
Add a certain amount of standard A type blood or Type B erythrocyte physiological saline suspension (3% dilution) and detection B blood group blood in glue detection card
Slurry or A type blood blood plasma, 37 DEG C of incubation 15min.After 1000rpm is centrifuged 10min, detection card, visual results are taken out.
2. the concentration of blood group antibody in immunoturbidimetry measurement A, Type B human normal plasma
Firstly, taking 5 small test tubes, physiological saline 0.1ml is added in every pipe.0.1ml A type or Type B blood are added in the 1st pipe
Blood plasma draws 0.1ml and the 2nd pipe is added after mixing, and so on, until the 5th pipe, finally discards 0.1ml.Then, with the examination of 100 μ l
After agent R2 zeroing, the reagent R1 of 99 μ l is mixed with the sample after 1 μ l dilution, after being incubated at room temperature 5min, in λ340nmDetect its suction
Light value.
As a result as shown in Table 1 and Table 2:
Blood group antibody in 1. micro-column gel of table detection card and immunoturbidimetry analysis A type blood blood plasma
Blood group antibody in 2. micro-column gel of table detection card and immunoturbidimetry analysis Type B blood blood plasma
Data described in Tables 1 and 2 are shown, compared with micro-column gel detection card, with the doubling dilution of blood plasma, ratio is immunized
The data of turbid method measurement are presented to be reduced close to 2 times of ratio of two term, illustrates that immunoturbidimetry used can also react well in the present invention
The variation tendency of blood group antibody, and can be with the concentration of blood group antibody in the sample as the result is shown of quantification, and it can be in certain journey
Reagent stability, human factor etc. is excluded on degree to judge by accident caused by result.
Claims (3)
1. a kind of human blood type antibody quantitative detecting method based on immunoturbidimetry, which is characterized in that include reagent R1, reagent
R2, reagent R3, calibration object 1 and calibration object 2；Wherein reagent R1 group becomes the PBS buffer solution of 0.01M pH 7.4,0.008-
The keyhole limpet hemocyanin of 0.012mg/ml is coupled blood group A antigen；Reagent R2 group as 0.01M pH 7.4 PBS buffer solution,
The keyhole limpet hemocyanin of 0.008-0.012mg/ml is coupled blood group B antigen；Reagent R3 group is buffered as the PBS of 0.01M pH 7.4
Liquid, 0.15M NaCl, 40-60g/L PEG-8000；Calibration object 1 is the anti-human blood group A antigen antibody reagent of mouse；Calibration object 2 is
The anti-human blood group B antigen-antibody reagent of mouse；Reagent R1 and reagent R3, reagent R2 and ratio when reagent R3 use are respectively R1: R3
=5: 94, R2: R3=5: 94；The blood group B antigen in blood group A antigen, reagent R2 in the reagent R1 is respectively to be coupled to
The blood group A antigen specificity trisaccharide of keyhole limpet hemocyanin, the blood group B antigentic specificity trisaccharide for being coupled to keyhole limpet hemocyanin.
2. a kind of human blood type antibody quantitative detecting method based on immunoturbidimetry according to claim 1, feature exist
In the quantitative detection of human blood type antibody A is that general formula formula is calculated and realized, formula are as follows: y=-21819x3+1644x2-
0.9588x+0.0426, y indicate concentration, and x indicates light absorption value.
3. a kind of human blood type antibody quantitative detecting method based on immunoturbidimetry according to claim 1, feature exist
In the quantitative detection of human blood type antibody B is to calculate to realize by formula, formula are as follows: y=-12735x3+1543.2x2-
50.649x+0.5213, y indicate concentration, and x indicates light absorption value.
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| CN111766286B (en) * | 2020-07-09 | 2023-06-16 | 深圳兰度生物材料有限公司 | Method for detecting content of hybrid protein in collagen |
| CN115343485B (en) * | 2022-10-18 | 2023-01-06 | 天津德祥生物技术股份有限公司 | Application of blood group antigen-trisaccharide conjugate in blood group antibody detection |
| CN117269514A (en) * | 2022-10-18 | 2023-12-22 | 天津德祥生物技术股份有限公司 | Blood group antigen trisaccharide A analogue protein conjugate and application thereof |
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| CN105548572A (en) * | 2016-02-02 | 2016-05-04 | 潍坊三维生物工程集团有限公司 | Kit and method for detecting content of immune globulin D and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1584595A (en) * | 2004-05-31 | 2005-02-23 | 广州伟仕科技有限公司 | External blood group and blood serum experimental determining method |
| CN101178410A (en) * | 2006-11-07 | 2008-05-14 | 上海血液生物医药有限责任公司 | Red corpuscle reagent box for reverse typing used for detecting human ABO blood type |
| CN101957366A (en) * | 2009-07-15 | 2011-01-26 | 英科新创(厦门)科技有限公司 | Human erythrocyte membrane antigen coated microsphere and application thereof |
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