阅读说明：本技术 一种血型检测卡及其制备方法 (Blood type detection card and preparation method thereof ) 是由 王小波 毛亚琳 黄谦 于 2019-09-29 设计创作，主要内容包括：本发明公开了一种用于血型检测的凝胶微球,其微球上连接有血型抗体。本发明还公开了一种用于血型检测的凝胶微球的制备方法,包括如下步骤：包括如下步骤：1)活化位于凝胶微球表面的基团,使所述基团能够与血型抗体发生化学反应而相接合；2)将血型抗体与被活化的基团相接合；3)使用封闭剂封闭未接合血型抗体的被活化的基团,洗涤,得到可特异性结合血型抗原的凝胶。本发明的血型检测卡相比现有的血型检测卡,具有更高的稳定性、灵敏度,应用前景良好。(The invention discloses a gel microsphere for blood type detection, which is connected with a blood type antibody. The invention also discloses a preparation method of the gel microsphere for blood type detection, which comprises the following steps: the method comprises the following steps: 1) activating groups on the surface of the gel microsphere to enable the groups to be combined with the blood group antibody through chemical reaction; 2) binding the blood group antibody to the activated group; 3) blocking the activated groups of the unbound blood group antibodies with a blocking agent, and washing to obtain a gel capable of specifically binding blood group antigens. Compared with the existing blood type detection card, the blood type detection card has higher stability and sensitivity and good application prospect.)

1.A gel microsphere for blood group detection is characterized in that: the gel microsphere is connected with blood group antibodies.

2. The microsphere of claim 1, wherein: the blood group antibody and the gel microsphere are connected through the group reaction on the surfaces of the blood group antibody and the gel microsphere;

preferably, the groups on the surface of the gel microsphere are tosyl, epoxy, carboxyl, amino or streptavidin.

3. The microsphere of claim 2, wherein: the surface of the microsphere is also connected with a sealing agent, the sealing agent is connected with a group on the surface of the microsphere through reaction, and the group on the surface of the microsphere reacted by the sealing agent is the same as the group reacted by the antibody;

preferably, the blocking agent is a non-proteinaceous blocking agent.

4. The microsphere of claim 2 or 3, wherein: the blood group antibody and the gel microsphere are connected with the amino group of the blood group antibody through the reaction of the carboxyl on the surface of the microsphere; the blocking agent is a non-protein blocking agent capable of binding carboxyl, preferably a compound with amino, and more preferably polyethylene glycol with amino.

5. The gel microsphere of claim 1, wherein: the gel microspheres are sephadex, agarose gel or polyacrylamide gel microspheres; preferably carboxyl polyacrylamide gel microspheres.

6. A blood type test card characterized in that: the microspheres in the test card are the microspheres of any one of claims 1-5.

7. A preparation method of gel microspheres for blood group detection is characterized by comprising the following steps:

1) activating groups on the surface of the gel microsphere to enable the groups to be combined with the blood group antibody through chemical reaction;

2) binding the blood group antibody to the activated group;

3) using a sealant to seal the activated groups of the blood group antibodies which are not jointed, and washing to obtain gel capable of specifically binding the blood group antigens;

preferably, the group in step 1) is selected from one or more of tosyl, epoxy, carboxyl, amino and streptavidin;

further preferably, the group of step 1) is a carboxyl group; further preferably, the reagent for activating carboxyl in the step 2) is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;

still more preferably, the blocking agent in step 3) is a non-protein blocking agent capable of binding carboxyl, preferably a compound with amino, and still more preferably polyethylene glycol with amino.

8. The method of claim 7, wherein:

the gel microsphere in the step 1) is a carboxyl polyacrylamide gel microsphere;

and/or, step 2) activating the groups with 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride at a final concentration of 0.25-1 mg/ml.

9. The method of claim 7, wherein the microsphere particle size X10 of step 1) is 20.9-85.6 microns;

and/or the particle size X90 of the microsphere is 90.1-180.3 microns;

and/or the microspheres have an average particle size of 53.3-150.9 microns;

x10 denotes the maximum particle size of the microspheres with the smallest particle size of 10%;

the X90 refers to the maximum particle size of the microspheres which is 90% of the smallest particle size.

10. A method for preparing a blood type test card, which is characterized in that the gel microspheres according to any one of claims 1 to 5 or the gel microspheres prepared by the method according to any one of claims 7 to 9 are mixed with a buffer solution and filled in a centrifuge tube of the blood test card;

in the buffer solution, the buffer salt is selected from one or more of morpholine sodium ethanesulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride;

preferably, after mixing the gel microspheres with the buffer, the ratio of the precipitate to the supernatant is 2: (0.5-2); still further preferably, the ratio of precipitate to supernatant is 2: 1.

Technical Field

The invention belongs to the field of blood transfusion detection, and relates to a preparation method of a blood type detection card, wherein the detected blood types include but are not limited to detection of an ABO blood type system and detection of antigens D, C, C, E and E of an Rh blood type system.

Background

As is well known, blood group testing is closely related to blood transfusion, and effective blood group testing must be provided by the transfusions department prior to all procedures that may require blood transfusion. When a recipient inputs blood of an allogeneic donor, alloantibodies can be generated, and transfusion adverse reactions occur, wherein the most serious is acute or delayed hemolytic reaction caused by blood group incompatibility of ABO and RhD. Therefore, it is of great clinical significance to detect blood type.

To date, over 30 human blood types have been discovered. The ABO blood group system is the first to find and the most important blood group system, and blood groups are classified into a type, B type, AB type and O type according to the presence or absence of a antigen and B antigen on erythrocytes. The Rh blood group system is the most complex blood group system and the most important blood group system except the ABO blood group system, and the blood groups are classified into RhD positive and RhD negative according to the existence of D antigen on red blood cells. In the Rh blood group system, D, C, C, E and E are the five most important antigens in the Rh blood group system, and have important clinical significance. When a blood recipient inputs blood of an allogeneic donor, allogenic antibodies can be generated, for example, when individuals negative to D, C, C, E and E antigens input red blood cells positive to corresponding antigens, corresponding immune antibodies can be generated by stimulation of the corresponding antigens, and hemolytic transfusion reaction can occur. Five antigens of D, C, C, E and E of the Rh system are detected to achieve the purpose of homotypic transfusion, and the occurrence of hemolytic transfusion reaction can be effectively reduced.

At present, methods for blood type examination in various domestic hospitals comprise a saline method, a polyamine method, a protease method, a microcolumn gel method and the like. Compared with the traditional saline method and the traditional polybrene method, the microcolumn gel method is simpler, more sensitive and more accurate, and the result can be stored for a long time, so that the microcolumn gel method is the most widely applied method at present and is also the recommended method of FDA. With the advent of the full-automatic blood type analyzer matched with the technology, the card-type microcolumn centrifugation method for blood type analysis is widely applied worldwide and is highly popular with the majority of users.

The principle of the microcolumn gel method is as follows: when the red blood cells are added into an antibody system corresponding to the surface antigens of the red blood cells, the red blood cells can agglutinate, when the red blood cells are added into an antibody system not containing the corresponding antibody, the red blood cells can not agglutinate, the agglutinated red blood cells are distributed on the upper layer of the gel after low-speed centrifugation by utilizing the steric hindrance of the gel, and the unagglutinated red blood cells sink on the bottom of the gel, so the surface antigens of the red blood cells can be judged by detecting whether the red blood cells are positioned on the upper layer or the bottom of the gel.

The sensitivity of the existing micro-column gel method is highly dependent on the particle size of the gel medium (gel microspheres): the smaller the gel microsphere volume, the larger the steric hindrance, and the higher the sensitivity of the detection card prepared by the gel microsphere, but the false positive is easily caused; the larger the gel microsphere is, the smaller the steric hindrance is, and the lower the sensitivity of the prepared detection card is, and the false negative is easily caused. However, the production process of the gel microspheres is complex, and the control of the batch-to-batch difference is difficult, so that the particle size difference of the gel microspheres in different batches is large, and therefore, the sensitivity of the reagent card can be obviously changed along with the batch-to-batch difference of the raw materials, the accuracy of the detection result is influenced, and the standardization of the detection result is not facilitated.

Therefore, there is an urgent need to improve the existing microcolumn gel method to overcome the problems of inaccurate results and difficult standardization.

Disclosure of Invention

The object of the present invention is to provide a new blood group test card which overcomes the aforementioned false negatives and further overcomes the problems of lot-to-lot instability that it brings.

The technical scheme of the invention comprises the following steps:

a gel microsphere for blood type detection is provided, wherein a blood type antibody is connected on the gel microsphere.

Further, the blood group antibodies are selected from one or more of the following antibodies: anti-A antibody, anti-B antibody against ABO blood group, anti-D antibody, anti-C antibody, anti-C antibody, anti-E antibody, anti-E antibody against Rh blood group.

As the microspheres, the blood group antibody and the gel microsphere are connected through the group reaction on the surfaces of the blood group antibody and the gel microsphere;

preferably, the groups on the surface of the gel microsphere are tosyl, epoxy, carboxyl, amino or streptavidin.

As for the microsphere, the surface of the microsphere is also connected with a sealing agent, the sealing agent is connected with a group on the surface of the microsphere through reaction, and the group on the surface of the microsphere reacted by the sealing agent is the same as the group reacted by the antibody;

preferably, the blocking agent is a non-proteinaceous blocking agent.

The blood group antibody and the gel microsphere are connected with the amino group of the blood group antibody through the reaction of the carboxyl on the surface of the microsphere; the blocking agent is a non-protein blocking agent capable of binding carboxyl, preferably a compound with amino, more preferably polyethylene glycol with amino, such as: the Blockmaster CE210 from JSR or the Blockmaster CE510 from JSR.

The microspheres as described above, wherein said gel microspheres are sephadex, sepharose or polyacrylamide gel microspheres; preferably carboxyl polyacrylamide gel microspheres.

The blood type detection card comprises the microspheres.

A preparation method of gel microspheres for blood group detection is characterized by comprising the following steps:

1) activating groups on the surface of the gel microsphere to enable the groups to be combined with the blood group antibody through chemical reaction;

2) binding the blood group antibody to the activated group;

3) using a sealant to seal the activated groups of the blood group antibodies which are not jointed, and washing to obtain gel capable of specifically binding the blood group antigens;

preferably, the group in step 1) is selected from one or more of tosyl, epoxy, carboxyl, amino and streptavidin;

further preferably, the group of step 1) is a carboxyl group; further preferably, the reagent for activating carboxyl in the step 2) is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;

still more preferably, the blocking agent in step 3) is a non-protein blocking agent capable of binding carboxyl, preferably a compound with amino, and still more preferably polyethylene glycol with amino; still more preferably, the blocking agent of step 3) is a Blockmaster CE210 (abbreviated as "CE 210") from JSR or a Blockmaster CE510 (abbreviated as "CE 510") from JSR.

The preparation method of the gel microsphere comprises the following steps of: anti-A antibody, anti-B antibody against ABO blood group, anti-D antibody, anti-C antibody, anti-C antibody, anti-E antibody, anti-E antibody against Rh blood group.

The preparation method of the gel microsphere comprises the following steps:

the gel microsphere in the step 1) is a carboxyl polyacrylamide gel microsphere;

and/or, step 2) activating the groups with 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride at a final concentration of 0.25-1 mg/ml.

The preparation method of the gel microsphere comprises the following steps:

step 1), the particle size X10 of the microsphere is 20.9-85.6 microns;

and/or the particle size X90 of the microsphere is 90.1-180.3 microns;

and/or the microspheres have an average particle size of 53.3-150.9 microns;

x10 denotes the maximum particle size of the microspheres with the smallest particle size of 10%;

the X90 refers to the maximum particle size of the microspheres which is 90% of the smallest particle size.

A preparation method of blood group detection card, it uses the above-mentioned gel microballoon, or uses the gel microballoon that the preparation method of the above-mentioned gel microballoon prepares and gets, mix with buffer solution, pack in the centrifuge tube of the card of testing blood to get final product;

in the buffer solution, the buffer salt is selected from one or more of morpholine sodium ethanesulfonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride;

preferably, after the gel microspheres are mixed with the buffer solution, the proportion of the sediment to the supernatant is 2: 0.5-2; still more preferably, the ratio of precipitate to supernatant is 2: 1.

The invention fixes the antibody on the gel microsphere of the blood type detection card, thereby greatly reducing the influence of the particle size of the gel microsphere on the detection sensitivity and overcoming the problem of poor detection stability caused by the particle size difference of the gel microsphere.

Under the specific preparation method, especially under the specific step parameters of activation (25-100 mu l of 10mg/ml 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is activated) and blocking (CE210 and CE510 are used as blocking agents), not only the accurate presentation of a positive signal is ensured, but also the clear background and no tailing of a negative control are ensured. The detection effect is overall, the identification degree is high, and the stability is good.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a graph showing the effect of detection of 4+ agglutination intensity.

FIG. 2 is a graph showing the effect of detection of 3+ agglutination intensity.

FIG. 3 is a graph showing the effect of detection of 2+ agglutination intensity.

FIG. 4 is a graph showing the effect of detection of 1+ agglutination intensity.

FIG. 5 is a graph showing the effect of detection of the agglutination intensity +/-corresponds.

FIG. 6 is a graph showing the effect of detection of the agglutination strength Dcp.

FIG. 7 is a graph showing the effect of detection on the agglutination strength H.

FIG. 8 is a graph showing the agglutination intensity-corresponding detection effect.

Detailed Description