CN116593691A - Simple microcolumn detection method for IgG anti-A (B) antibody titer - Google Patents

Simple microcolumn detection method for IgG anti-A (B) antibody titer Download PDF

Info

Publication number
CN116593691A
CN116593691A CN202310490604.8A CN202310490604A CN116593691A CN 116593691 A CN116593691 A CN 116593691A CN 202310490604 A CN202310490604 A CN 202310490604A CN 116593691 A CN116593691 A CN 116593691A
Authority
CN
China
Prior art keywords
blood cells
red blood
microcolumn
reagent
sample
Prior art date
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.)
Pending
Application number
CN202310490604.8A
Other languages
Chinese (zh)
Inventor
李志强
李丽玮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Sixth Peoples Hospital
Original Assignee
Shanghai Sixth Peoples Hospital
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shanghai Sixth Peoples Hospital filed Critical Shanghai Sixth Peoples Hospital
Priority to CN202310490604.8A priority Critical patent/CN116593691A/en
Publication of CN116593691A publication Critical patent/CN116593691A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a simple and convenient microcolumn detection method for the titer of an IgG anti-A (B) antibody, which comprises the following steps: step one, adding type A reagent red blood cells or type B reagent red blood cells corresponding to blood group antibodies into a micro column, wherein a filling material with a molecular sieve function is arranged in the micro column, and only free red blood cells are allowed to pass through so as to separate the free red blood cells and the aggregated red blood cells; diluting the inactivated sample to be tested to a specific final concentration by using a sodium chloride solution, adding the diluted sample into a micro-column, and centrifuging; and thirdly, directly judging the IgG anti-A or anti-B antibody effect value according to the strength of the agglutination intensity in 1 hole of the reaction product. The result interpretation of the microcolumn detection method provided by the invention is simple and visual, the operation is simple and quick, the accuracy of experimental results is obviously improved, and the experimental cost is reduced.

Description

Simple microcolumn detection method for IgG anti-A (B) antibody titer
Technical Field
The invention relates to the technical field of biological detection, in particular to a simple and convenient microcolumn detection method for the titer of an IgG anti-A (B) antibody.
Background
ABO fetal neonatal hemolysis (hemolytic disease of the fetus and newborn, HDFN) refers primarily to fetal or neonatal immune hemolytic disease caused by maternal-fetal ABO blood group incompatibility. Since most of the natural anti-B or anti-a antibodies of type a or B mothers belong to IgM antibodies, they cannot enter the fetus through the placenta, whereas the natural anti-a and anti-B antibodies of type O mothers mainly belong to IgG antibodies, they can enter the fetus through the placenta. Thus, ABO blood group incompatible hemolysis is mainly seen in type O mothers, type a or type B fetuses.
Typically, maternal IgG anti-a or anti-B antibodies enter the fetal blood circulation through the placenta, thereby destroying fetal erythrocytes resulting in intrauterine anemia (dead fetus) or neonatal jaundice (nuclear jaundice), etc. HDFN can occur in the first embryo, with more births, higher morbidity, and more severe than once. In order to prevent HDFN, maternal ABO blood group antibody titer monitoring is necessary.
The titer of the ABO blood group antibody refers to the binding of the ABO blood group antibody with corresponding erythrocyte antigens under certain medium conditions, the occurrence of different degrees of erythrocyte agglutination, and the capacity of the blood group antibody to bind to the antigens is indirectly reflected according to the agglutination intensity. However, because of the high reactivity of the blood group antibodies, the blood group antibodies are subjected to relatively strong reactive agglutination with corresponding red blood cells, and thus the difference of the ability of the ABO blood group antibodies of the specimen of the tested person to bind to the antigen cannot be distinguished. Therefore, the conventional method is that after the anti-A or (and) anti-B antibody with IgM property in serum (or plasma) to be detected is treated by using sulfhydryl reagent, the serum (or plasma) must be diluted continuously and times, and the agglutination reaction strength of the anti-A or (and) anti-B antibody with IgG property in the diluted serum (or plasma) and red blood cells of corresponding blood group antigens is observed, specifically, (1) 10 test tubes are arranged and marked, and the serum (or plasma) to be detected containing the anti-A or anti-B antibody with IgG property after being treated by using sulfhydryl reagent is diluted continuously and times to 1 by using 0.9% sodium chloride solution according to the ratio of 1:1: 512, (2) adding 2-5%A reagent red blood cells or B reagent red blood cells corresponding to blood type antibodies into each test tube one by one, uniformly mixing, incubating each test tube after uniform mixing in a water bath box at 37 ℃ for 30 minutes, washing the red blood cells in the test tube for 3 times, adding 1 drop of anti-human globulin reagent, centrifuging, and immediately judging the experimental result. The reciprocal of the dilution at an agglutination intensity of 1+ is usually determined and can be interpreted as the antibody titer value of the specimen. The method is suitable for observing the difference comparison of the activity of the ABO blood group antibodies between blood samples of different subjects, and is also suitable for monitoring the change of the activity of the ABO blood group antibodies of the same subject in different periods.
However, the above conventional method has the following disadvantages:
(1) In the experimental process, a large amount of test tubes, pipette tips, pipette heads, 0.9% physiological saline and the like are consumed, and the operation is complicated;
(2) In the experimental process, repeated pipetting and dilution are extremely easy to generate sample adding errors, so that the accuracy of experimental results is low; especially, the experimental results of different detection personnel are poor in comparability;
(3) The experimental operation process is quite tedious and takes a very long time (one blood specimen detection time is not less than 60 minutes), etc.
Thus, there is a need for a simpler method of determining the potency of IgG anti-a (B) antibodies.
Disclosure of Invention
In order to change the tedious experimental detection process of the conventional ABO blood group antibody titer, the invention provides a simple microcolumn detection method of the IgG anti-A (B) antibody titer.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a simple and convenient microcolumn detection method for the titer of an IgG anti-A (B) antibody, which comprises the following steps:
step one, adding type A reagent red blood cells or type B reagent red blood cells of corresponding blood group antibodies into a micro column, wherein a filling material with a molecular sieve function is arranged in the micro column, and only free red blood cells are allowed to pass through so as to separate the free red blood cells and the aggregated red blood cells;
diluting the inactivated sample to be tested to a specific final concentration by using a sodium chloride solution, adding the diluted sample to the micro-column, and centrifuging;
and thirdly, directly judging the IgG anti-A or anti-B antibody effect value according to the intensity of the agglutination intensity of the reaction product.
Further, the concentration of the type A reagent red blood cells or the type B reagent red blood cells of the corresponding blood group antibodies in the step one is 2 to 5 percent.
Further, the above-mentioned filling contains an antihuman globulin reagent.
Further, the reagent adopted in the second step is sulfhydryl reagent, and the parameters of the treatment are as follows: 37 ℃ for 15-30 minutes.
Further, the thiol reagent is 2-Me (amphiphobic ethanol).
Further, the volume ratio of the sulfhydryl reagent to the sample to be tested is 1:1.
further, the sample to be tested is serum or plasma.
Further, in the second step, the sample to be measured is diluted with 0.9% sodium chloride solution to a final concentration of 1:256.
Further, the interpretation criteria of step three are as follows:
a second aspect of the invention is to provide a product based on the above-described microcolumn detection method.
Further, the product comprises a micro-column or a micro-column gel card, reagents adopted in the micro-column detection method and a product instruction; the product specification indicates the steps of the microcolumn detection method.
Compared with the prior art, the invention has the following technical effects:
(1) The repeated pipetting and dilution process is not needed in the experimental process, so that the sample adding error is reduced, and the accuracy of the experimental result is remarkably improved; the experimental results of different detection personnel are high in comparability.
(2) The interpretation of the experimental result is simple and visual.
(3) In the experimental process, a large number of test tubes, liquid transfer pipettes, liquid transfer device heads, microcolumn cards, 0.9% physiological saline and the like are saved, and the experimental cost is obviously reduced.
Drawings
FIG. 1 shows the results of anti-human globulin test cards at different dilution ratios for samples of known titer 1:32 according to one embodiment of the present invention;
FIG. 2 shows the results of anti-human globulin test cards at different dilution ratios for samples of known titer 1:64 according to one embodiment of the present invention;
FIG. 3 shows the results of anti-human globulin test cards at various dilution ratios for samples of known titer 1:128 according to one embodiment of the present invention;
FIG. 4 shows the results of anti-human globulin test cards at various dilution ratios for samples of known titer 1:256 in one embodiment of the invention;
FIG. 5 shows the results of anti-human globulin test cards at different dilution ratios for samples of known titer 1:512 in one embodiment of the invention.
Detailed Description
Aiming at the defects of complex operation and time consumption in the existing ABO blood group antibody titer detection technology, the invention creatively provides a simple microcolumn detection method for detecting the IgG anti-A (B) antibody titer, which comprises the following steps:
step one, adding type A reagent red blood cells or type B reagent red blood cells of corresponding blood group antibodies into a micro column, wherein a filler with a molecular sieve function is arranged in the micro column, and only free red blood cells are allowed to pass through;
diluting the inactivated sample to be tested to a specific final concentration by using a sodium chloride solution, and adding the diluted sample to the micro-column;
and thirdly, directly judging the IgG anti-A or anti-B antibody effect value according to the intensity of the agglutination intensity of the reaction product.
In the invention, the design of the micro column is similar to that of a micro column gel tube in a micro column gel card, the bottom of the micro column gel tube is provided with gel filler with a molecular sieve function, and only free red blood cells are ensured to pass through by adjusting the gap or the size of a sieve pore of the filler, so that the effect of separating the free red blood cells and the aggregated red blood cells is achieved. When the specific antibody of blood group serology is combined with the antigen on the red blood cells, the agglutinated red blood cells remain on the upper layer of the micro-column or are dissociated in the micro-column (tube), and positive reaction is presented; when the antigen-antibody of the red blood cells does not react, the non-agglutinated red blood cells can be deposited at the bottom of the micro-column (tube) through gaps or sieve holes of the filling of the micro-column (tube) by the centrifugal technology, and the negative reaction is presented. In a preferred embodiment of the invention, the inclusion of an anti-human globulin reagent in the fill increases the sensitivity of the immune response to sensitized erythrocytes. In a preferred embodiment of the present invention, a currently commercially available microcolumn gel card can be used for batch detection of samples, saving time and operating costs.
In a preferred embodiment of the invention, the concentration of the type A reagent red blood cells or the type B reagent red blood cells of the corresponding blood group antibody in step one is 2 to 5%.
In a preferred embodiment of the present invention, the reagent used for inactivation in the second step is a sulfhydryl reagent, and the parameters of the treatment are: 37 ℃ for 15-30 minutes.
In a preferred embodiment of the present invention, the thiol reagent is 2-Me (amphiphobic ethanol).
In a preferred embodiment of the invention, the volume ratio of sulfhydryl reagent to sample to be tested is 1:1.
in a preferred embodiment of the present invention, the sample to be tested is serum or plasma.
In a preferred embodiment of the present invention, in the second step, the sample to be tested is diluted with 0.9% sodium chloride solution to a final concentration of 1:256.
In a preferred embodiment of the present invention, the interpretation criteria for step three are as follows:
the present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.
The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.
Example 1
The embodiment provides a microcolumn detection method of IgG anti-A (B) antibody titer, comprising the following steps:
(1) 1 test tube and 1 microcolumn well of microcolumn card (anti-human globulin card) were taken.
(2) Adding 15 mu L of 2-5% of A-type reagent red blood cells or B-type reagent red blood cells of corresponding blood group antibodies into 1 hole of the microcolumn gel card according to a sample to be detected;
(3) Respectively adding serum or plasma to be detected containing IgG-property anti-A or anti-B antibody after 30 minutes of treatment at 37 ℃ by using a sulfhydryl reagent (the ratio of serum or plasma to sulfhydryl reagent is 1:1) and 0.9% sodium chloride solution into a test tube by using a pipette, and uniformly mixing the serum or plasma and the 0.9% sodium chloride solution according to a certain ratio; and sucking 40 mu L of liquid after uniformly mixing in the test tube by using a pipette, adding the liquid into the micro-column hole, and putting the micro-column card into a special centrifugal machine for centrifugation.
(3) And directly judging the IgG anti-A or anti-B antibody effect value according to the intensity of the agglutination intensity in the micro-column gel card hole.
Example 2
In this example, on the basis of example 1, a suitable mixing ratio of serum to be tested and 0.9% sodium chloride solution, namely, a dilution ratio of a sample is explored, and specific experimental steps and results are as follows:
plasma samples treated with thiol reagents of known potency were assayed by the method of example 1.
In the method of example 1, the plasma sample treated with thiol reagent 2-Me and 0.9% sodium chloride solution were mixed in a mixing ratio of 1:7 (actual dilution ratio 1:16), 1:15 (actual dilution ratio 1:32), 1:31 (actual dilution ratio 1:64), 1:63 (actual dilution ratio 1:128), 1:127 (actual dilution ratio 1:256), 1:255 (actual dilution ratio 1:572), respectively, to obtain a mixed solution, and 40 μl of the mixed solution was pipetted into 1 microcolumn well of the microcolumn card, and centrifuged. Based on the intensity of the agglutination intensity in the gel card wells of the microcolumns, the IgG anti-A or anti-B antibody efficacy value was directly interpreted, and the specific results are shown in Table 1 below:
table 1 results of the assay of plasma samples of known titer by the method of example 1
Where "w" represents weak (weak) and "s" represents strong (strong).
From the above, it can be seen that the samples of various titers can be well distinguished and the detection results more accurate in the case of the thiol reagent-treated plasma samples and the 0.9% sodium chloride solution according to 1:127 (i.e., the dilution ratio is 1:256), respectively.
Based on the above results, the mixing ratio of the serum to be tested containing the anti-A or anti-B antibody with IgG property and the 0.9% sodium chloride solution in example 1 is preferably 1:127, and the judgment standard of the efficacy value of the IgG anti-A or anti-B antibody is set as follows in Table 2:
TABLE 2 titer interpretation criteria
Cohesive strength Value of effective
Negative of ≤32
± 64
+ 128
2+ 256
3+ ≥512
The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. It will be apparent to those skilled in the art that any equivalent modifications and substitutions of the present invention are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims (10)

1. A simple method for detecting the titer of an IgG anti-a (B) antibody by a microcolumn, comprising the steps of:
step one, adding type A reagent red blood cells or type B reagent red blood cells corresponding to blood type antibodies into a micro column, wherein a filler with a molecular sieve function is arranged in the micro column, and only free red blood cells are allowed to pass through so as to separate the free red blood cells and the aggregated red blood cells;
diluting the inactivated sample to be tested to a specific final concentration by using a sodium chloride solution, adding the diluted sample to the micro-column, and centrifuging;
and thirdly, directly judging the IgG anti-A or anti-B antibody effect value according to the intensity of the agglutination intensity of the reaction product.
2. The method according to claim 1, wherein the concentration of the type a reagent red blood cells or type B reagent red blood cells of the corresponding blood group antibody is 2 to 5%.
3. The method according to claim 1, wherein the reagent used for inactivation in the second step is a sulfhydryl reagent, and the parameters of the treatment are: 37 ℃ for 15-30 minutes.
4. The method for detecting a microcolumn according to claim 3, wherein the thiol reagent is 2-Me.
5. The method according to claim 3, wherein the volume ratio of the thiol reagent to the sample to be measured is 1:1.
6. the method of claim 1, wherein the sample is serum or plasma.
7. The method according to claim 1, wherein in the second step, the sample to be tested is diluted with 0.9% sodium chloride solution to a final concentration of 1:256.
8. the method of claim 7, wherein the interpretation criteria for step three are as follows:
9. a product based on the microcolumn detection method according to any one of claims 1 to 8.
10. The product of claim 9, comprising the microcolumn or microcolumn gel card, reagents used in the microcolumn detection method, and a product instruction; the product specification indicates the steps of the microcolumn detection method.
CN202310490604.8A 2023-05-04 2023-05-04 Simple microcolumn detection method for IgG anti-A (B) antibody titer Pending CN116593691A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310490604.8A CN116593691A (en) 2023-05-04 2023-05-04 Simple microcolumn detection method for IgG anti-A (B) antibody titer

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310490604.8A CN116593691A (en) 2023-05-04 2023-05-04 Simple microcolumn detection method for IgG anti-A (B) antibody titer

Publications (1)

Publication Number Publication Date
CN116593691A true CN116593691A (en) 2023-08-15

Family

ID=87594765

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310490604.8A Pending CN116593691A (en) 2023-05-04 2023-05-04 Simple microcolumn detection method for IgG anti-A (B) antibody titer

Country Status (1)

Country Link
CN (1) CN116593691A (en)

Similar Documents

Publication Publication Date Title
US6114179A (en) Method and test kit for detecting antigens and/or antibodies
US5460940A (en) Method for detecting antigens and/or antibodies
US5236826A (en) Immunoassay for the detection or quantitation of an analyte
Perrault et al. Automated Red Cell Antibody Analysis. A Parallel Study: I. Detection and Quantitation
EP0746767B1 (en) Analyzer cuvette, method and diagnostic test kit for determination of analytes in whole blood samples
JPH034170A (en) Method of forming aggulutinated lump in blood sample
CN110174519B (en) Confluent-detection type erythrocyte blood type irregular antibody detection kit based on solid-phase agglutination technology and preparation method thereof
CN113252914B (en) Antibody diluent for Rh system parting detection card and detection card
CN113156143A (en) Blood group irregular antibody specificity identification method and reagent thereof
CN116493061B (en) Blood detection micro-fluidic chip and detection method thereof
CN110308288B (en) Novel blood platelet cross matching kit
CN110824157B (en) Method for quickly separating red blood cells for immunochromatography detection kit
US6461825B1 (en) Immunometric assay kit and method applicable to whole cells
Spada et al. Comparison of conventional tube and gel-based agglutination tests for AB system blood typing in cat
CN115754311A (en) Method for quantitatively detecting ABO blood group antigen by using erythrocyte endogenous peroxidase
CN103706340B (en) A kind of water-base cement chromatography media and the method for detecting
CN116593691A (en) Simple microcolumn detection method for IgG anti-A (B) antibody titer
CN103599817A (en) Immunological assay system and method
CN111398578A (en) Brucella antibody gel detection kit
TATSUMI et al. Trial of ABO and Rh blood typing with an automated blood cell counter
Zhou et al. The progress in pre-transfusion test technique research
CN113009144B (en) Antibody detection kit and detection method based on microfluidic technology
EP0163631A1 (en) Method for the determination of the results of agglutination reactions
CN115753652A (en) Method for quantitatively detecting RhD antigen by using erythrocyte endogenous peroxidase
US20220252622A1 (en) Single tube preparation comprising a panel of differently labeled cells for serology

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination