CN111638373A - Kit for ABO blood group positive typing and Rh blood group detection and preparation method and detection method thereof - Google Patents
Kit for ABO blood group positive typing and Rh blood group detection and preparation method and detection method thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kit for ABO blood group positive typing and Rh blood group detection, and a preparation method and a detection method thereof. Comprises an external clamping shell, and a detection film, a water absorption pad and a back lining which are sequentially arranged in the clamping shell from top to bottom; the detection membrane is composed of a character area and a non-character area, wherein the character area is composed of letters and symbols, the letters comprise A, B, O letters and letters of Rh positions, and the letters of the Rh positions are one or more of C, C, D, E and E; the symbols include the minus sign in the middle of the letter O and the plus sign above the right of the letter in Rh position; each character includes a hydrophilic region and a hydrophobic region, the hydrophilic region being a reaction region. According to the principle of immunodiafiltration, the invention utilizes the specific reaction of erythrocyte antigen and antibody to produce erythrocyte agglutination, observes whether there is erythrocyte agglutination color, and interprets the text composed of colors to realize the detection of blood type. The invention has simple operation, can realize the visual interpretation of the detection result and avoid blood transfusion accidents caused by misjudgment.
Description
Technical Field
The invention relates to the technical field of in-vitro detection, in particular to a kit for ABO blood group positive typing and Rh blood group detection, and a preparation method and a detection method thereof.
Background
At present, ABO blood typing and RhD blood type detection are required before clinical blood transfusion, and the detection of other antigens of the Rh system is particularly necessary on the basis. ABO typing involves two aspects, positive and negative typing. The positive typing is erythrocyte typing, which means that anti-A/anti-B blood typing reagent with known specificity is used for detecting whether corresponding antigen exists on erythrocytes; the retrotyping is serotyping, which means that whether corresponding antibodies exist in the serum or plasma to be detected is detected by using A type/B type red blood cells of known antigens. The blood type of the specimen to be detected can be correctly judged by integrating the positive and negative shaping results. The RH blood group system contains 5 antigens, namely C, C, D, E and E, and the D antigen is a necessary item before clinical blood transfusion at present. Rh blood typing also uses anti-C/C/D/E/E antibodies of known specificity to detect the presence of C/C/D/E/E antigens on erythrocytes.
Both ABO blood group orthotyping and Rh blood group detection are based on the agglutination reaction principle of erythrocyte antigen antibodies. The detection methods currently used include: the assay method includes a test tube method, a microplate method, a microcolumn gel method and a solid phase agglutination method, and the detection methods require special containers and/or equipment. And judging and reading the detection result through the negative and positive reaction patterns, wherein the positive reaction is the occurrence of erythrocyte agglutination, and the negative reaction is the absence of erythrocyte agglutination. The detection method needs to be analyzed by a professional trained technician to make a blood type judgment, but the method still has the risk of wrong interpretation even though the technician is the professional trained technician, and transfusion accidents caused by wrong interpretation are easy to occur.
Therefore, with the needs of developing population census and emergency examination, a blood type detection method and/or reagent which is convenient to carry, simple to operate and easy to interpret results, especially for operators in remote areas, and can interpret results without special training is needed.
Disclosure of Invention
The invention aims at the technical problems and provides a kit for ABO blood group positive typing and Rh blood group detection and a preparation method and a detection method thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention firstly provides a kit for ABO blood group positive typing and Rh blood group detection, which comprises a reagent card, wherein the reagent card comprises a card shell wrapped outside, and a detection film, a water absorption pad and a back lining which are sequentially arranged inside the card shell from top to bottom;
the detection membrane is composed of a character area and a non-character area, wherein the character area is composed of letters and symbols, the letters comprise A, B, O letters and letters at Rh positions, and the letters at the Rh positions are one or more of C, C, D, E and E in combination; the symbols include the minus sign in the middle of the letter O and the plus sign above the right of each letter of the Rh position;
each character comprises a hydrophilic area and a hydrophobic area, wherein, the horizontal lines in the first oblique vertical stroke of the A, B letters, the O letters, the letters at Rh positions and the plus signs above the right of the letters at Rh positions are coated with non-bioactive colored hydrophobic ink which is the hydrophobic area;
the rest areas of the characters are hydrophilic areas, wherein the A letter hydrophilic area is coated with an anti-A antibody, the B letter hydrophilic area is coated with an anti-B antibody, the vertical line of the right upper plus sign of the letter at the Rh position is coated with an anti-C antibody or an anti-C antibody or an anti-D antibody or an anti-E antibody or an anti-E antibody, and the minus sign in the middle of the O letter is coated with an anti-A and anti-B mixed antibody;
the area of the detection film except the character is a non-character area, coated with hydrophobic solution and is a hydrophobic area.
Specifically, the hydrophobic solution is 0.5-1% AKD-ethanol solution.
The non-bioactive, colored hydrophobic ink is a commercially available red hydrophobic ink.
The detection membrane is made of porous polymer material with the pore diameter not larger than 15 mu m.
The second purpose of the invention is to provide a preparation method of the kit for ABO blood group orthotyping and Rh blood group detection, which comprises the following steps:
s1, preparing A, B, O, C/c/D/E/E in advance+A pattern design of character shape, wherein a hydrophobic solution is sprayed on a non-character area on the detection membrane by an ink-jet printer, a hydrophobic area is formed by a non-bioactive colored hydrophobic ink sprayed on the first oblique vertical line of the letters A and B, the letter at the position of the letter O, Rh and the horizontal line of the plus sign above and to the right of the letter at the position of Rh, and the rest area of the character is a hydrophilic area;
s2, coating an anti-A antibody, an anti-B antibody, an anti-C/C/D/E/E antibody and an anti-A and anti-B mixed antibody on a hydrophilic area of an A letter, a hydrophilic area of a B letter, a vertical line of a plus sign on the right side of a letter of Rh position and a minus sign in the middle of an O letter of the detection membrane respectively by a physical adsorption or chemical crosslinking method;
s3, drying the detection membrane coated with the antibody at 37 ℃ for 30-60 minutes or overnight at room temperature to form the detection membrane coated with the antibody and provided with the permanent mark.
Still another object of the present invention is to provide the above detection method for ABO blood group orthotyping and Rh blood group detection, wherein the detection method comprises:
dripping a sample to be detected into a reaction area which is coated with an antibody in advance, adding 150-250 mu l of flushing liquid into the reaction area after the reaction time is not more than 60 seconds, and if the antigen corresponding to the coated antibody exists on the erythrocyte sample to be detected, carrying out antigen-antibody specific reaction to form erythrocyte agglutination, wherein the erythrocyte is intercepted at the antibody and forms a positive text form result with a permanent mark; on the other hand, if the antigen corresponding to the coated antibody is not present on the red blood cells, the unreacted red blood cells are eluted by the washing solution, and a result in the form of a negative text is formed in the reaction region.
Specifically, the sample to be detected is whole blood or packed red blood cells.
The sample to be detected comprises a hemolytic blood sample, a jaundice blood sample, a high fat blood sample and a normal blood sample.
The dropping amount of the sample to be detected is 5-10 mul.
The washing solution is physiological saline or phosphate buffer solution.
Compared with the prior art, the invention has the beneficial effects that:
the kit and the method for ABO blood group positive typing and Rh blood group detection utilize the specific reaction of erythrocyte antigen and antibody to generate erythrocyte agglutination according to the principle of immune infiltration, observe whether the agglutination color of the erythrocyte exists, and judge the text consisting of the colors to realize the detection of the blood group. The invention has simple operation, can realize the visual interpretation of the detection result and avoid blood transfusion accidents caused by misjudgment. The blood sample to be detected is not more than 10ul in use amount, the whole detection process is not more than 2 minutes, and the method is simple and rapid to operate and convenient to carry.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
Fig. 1 is a schematic diagram of the graphical design of ABO and D in the same row of reaction regions, which is provided in embodiment 2 of the present invention, and which exemplifies ABO blood type positive typing and RhD blood type detection.
FIG. 2 is a schematic diagram of the graphical design of the reaction areas in the same row of ABO and D, which is provided by example 2 of the present invention, and is exemplified by ABO blood type positive typing and RhD blood type detection.
Fig. 3 is a schematic plan view of a detection membrane provided in embodiment 2 of the present invention.
Fig. 4 is a schematic side view of a detection membrane provided in embodiment 2 of the present invention.
Fig. 5 is a schematic diagram of a graphical design of an example ABO blood group typing test.
Fig. 6 is a schematic diagram of a diagram design of an example of the individual detection of a certain antigen in the Rh blood group system.
Fig. 7 is a schematic diagram of a Rh blood group system antigen detection.
FIG. 8 is a schematic diagram of a graphical design illustrating ABO blood typing and Rh blood typing.
Fig. 9 shows eight forms of the text reaction in ABO blood group orthotyping and RhD blood group detection as examples provided in example 3 of the present invention.
Detailed Description
The invention provides a kit for ABO blood group positive typing and Rh blood group detection, which comprises a reagent card, wherein the reagent card comprises a card shell wrapped outside, and a detection film, a water absorption pad and a back lining which are sequentially arranged inside the card shell from top to bottom;
the detection membrane is composed of a character area and a non-character area, wherein the character area is composed of letters and symbols, the letters comprise A, B, O letters and letters at Rh positions, and the letters at the Rh positions are one or more of C, C, D, E and E in combination; the symbols include the minus sign in the middle of the letter O and the plus sign above the right of each letter of the Rh position;
each character comprises a hydrophilic area and a hydrophobic area, wherein, the horizontal lines in the first oblique vertical stroke of the A, B letters, the O letters, the letters at Rh positions and the plus signs above the right of the letters at Rh positions are coated with non-bioactive colored hydrophobic ink which is the hydrophobic area;
the rest areas of the characters are hydrophilic areas, wherein the A letter hydrophilic area is coated with an anti-A antibody, the B letter hydrophilic area is coated with an anti-B antibody, the vertical line of the right upper plus sign of the letter at the Rh position is coated with an anti-C antibody or an anti-C antibody or an anti-D antibody or an anti-E antibody or an anti-E antibody, and the minus sign in the middle of the O letter is coated with an anti-A and anti-B mixed antibody;
the area of the detection film except the character is a non-character area, coated with hydrophobic solution and is a hydrophobic area.
Specifically, the hydrophobic solution is 0.5-1% AKD-ethanol solution.
The non-bioactive, colored hydrophobic ink is a commercially available red hydrophobic ink.
The detection membrane is made of porous polymer material with the pore diameter not larger than 15 mu m. The pore size of the membrane is at least capable of allowing free single red blood cells to pass through and can retain weak agglutination of the red blood cells. For example, the film may be a glass fiber film or a paper towel. In the embodiment of the present invention, a commercially available film made of glass fiber is used, for example: the membrane of GE's Accuflow G, Rapid27, gold labeled RB45, RB65, etc. has a pore size that allows free red blood cells to pass through, but does not allow weakly agglutinated red blood cell antigen-antibody complexes to pass through.
The water absorption pad is made of cotton fiber with strong water absorption capacity, can also be made of glass fiber, is compact in manufacturing, is not prone to fiber falling, and is suitable for production and use.
One side of the back lining close to the water absorption pad is provided with glue, and the material of the back lining is not limited to a polyester plate.
The preparation of the symbols on the detection film can adopt a mode of printing by an ink-jet printer, and also can adopt a mode of manual drawing or type matrix printing.
The washing solution is normal saline or phosphate buffer solution with isotonic property.
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and examples.
Example 1 determination of hydrophobic solution
Preparing 0.3%, 0.5%, 0.7%, 0.9% and 1% AKD solution by absolute ethyl alcohol, dripping a drop of hydrophobic solution on a glass fiber membrane, drying at room temperature or 37 ℃ after the hydrophobic solution is completely diffused, dripping a colored liquid solution on the membrane after the membrane is completely dried, and observing whether the solution is absorbed by the membrane or not to determine that the AKD concentration of the solution is 0.5-1%.
EXAMPLE 2 preparation of a kit for ABO blood group typing and Rh blood group detection
The kit contains a reagent card, a blood sampling tool and flushing liquid, wherein the reagent card comprises an external card shell, a detection membrane 1, a water absorption pad 2 and a back lining 3 which are sequentially arranged in the card shell from top to bottom;
taking a kit for ABO blood type positive typing and RhD blood type detection as an example, the letter of Rh position is D, the vertical line of the upper right plus sign of the D letter is coated with an anti-D antibody, and the preparation method of the detection membrane comprises the following steps:
s1, preparing A, B, O, D on computer in advance+The graphic design of the character shapes, ABO and D, can be in the same row of reaction areas or can be designed into different rows of reaction areas, as shown in FIGS. 1 and 2. All letters in the figures are italicized. Spraying a hydrophobic solution on a non-character area on the detection membrane by adopting an ink-jet printer, and forming a hydrophobic area with non-biological activity colored hydrophobic ink sprayed on the first oblique vertical letters of the letters A and B, the letters O and D and the horizontal line of the plus sign above the right side of the letter D; and other areas of the characters are hydrophilic areas (reaction areas), wherein the hydrophilic area of the letter A is blue, the hydrophilic area of the letter B is yellow, the minus sign in the letter O is green, and the vertical line of the plus sign at the upper right of the letter D is light yellow.
S2, respectively coating an anti-A antibody, an anti-B antibody, an anti-D antibody and an anti-A and anti-B mixed antibody on a hydrophilic region of an A letter, a hydrophilic region of a B letter, a vertical line of a plus sign at the upper right of the D letter and a minus sign region in the middle of the O letter of the detection membrane by a physical adsorption or chemical crosslinking method; specifically, high titer blood group antibody reagents of anti-A, anti-B and anti-D are taken and diluted by 0.01M phosphate buffer solution containing 2% of bovine serum albumin and 2% of sucrose, so that the titer is not lower than 1: 128. adding the anti-A, anti-B, anti-D and anti-A and anti-B mixed reagents with equal volume to the hydrophilic area by a dropper, wherein the total amount of the added reagents is 5-10 ul.
S3, drying the detection membrane coated with the antibody at 37 ℃ for 30-60 minutes or overnight at room temperature to form the detection membrane coated with the antibody and provided with the permanent mark. As shown in fig. 3.
Assembling the reagent card:
a water absorption pad is adhered to a polyester plate with glue on one surface, and positions for adhering detection films are left on the left side and the right side of the polyester plate. And covering the cut detection film above the water absorption pad, and fixing the detection film left and right by glue on the polyester plate. The detection membrane side is shown in fig. 4.
In addition, except that the reagent card that above-mentioned ABO blood group and Rh blood group detected together, according to the demand of testing project, this application can also design the combination form of several kinds of detections, prepares out the kit of multiple blood group system. For example, the graphic in the shape of a character is designed to be tested for ABO blood group positive typing alone as shown in FIG. 5, for Rh system antigen alone as shown in FIG. 6, for Rh system antigen as shown in FIG. 7, or for both ABO blood group positive typing and Rh system together as shown in FIG. 8. The preparation method is similar to that of example 2 and is not repeated herein.
Example 3 detection for ABO blood group orthotyping and Rh blood group detection
The sample to be detected is whole blood or packed red blood cells.
The sample to be detected comprises a hemolytic blood sample, a jaundice blood sample, a high fat blood sample and a normal blood sample.
The detection method comprises the following steps:
5-10 mul (10 mul of whole blood and 5 mul of packed red blood cells) of the sample to be detected is dripped into the reaction area which is coated with the antibody in advance, and after the reaction time of no more than 60 seconds, 150-250 mul (3-5 mul and 50 ul/drop) of washing liquid is added into the reaction area until clear text is displayed. The blood type of the sample to be detected is judged through the text display on the detection membrane.
If the antigen corresponding to the coated antibody exists on the erythrocyte sample to be detected, the antigen-antibody specific reaction can occur, the erythrocyte agglutination is formed, the erythrocyte is intercepted at the antibody, and the result in the form of positive text is formed with the permanent mark; on the other hand, if the antigen corresponding to the coated antibody is not present on the red blood cells, the unreacted red blood cells are eluted by the washing solution, and a result in the form of a negative text is formed in the reaction region.
Taking the kit for ABO blood group positive typing and RhD blood group detection of example 2 as an example, the following eight forms exist as text for reaction, as shown in FIG. 9.
When the specimen to be detected is added into the reaction area and washed by the reaction washing liquid, if the letter AOD shown as the first letter in the table is formed+And then, the surface of the red blood cell of the sample to be detected contains the A antigen and the D antigen, and the A antigen and the D antigen respectively react with the anti-A antibody and the anti-D antibody coated on the membrane, and the positive stereotype of the sample to be detected is positive for the A-type D antigen. Similarly, the ABO blood type and RhD blood type detection results of the sample to be detected can be obtained through interpretation in a text mode.
Other ABO blood group is just stereotyped and Rh blood group detects kit preparation method and result show and are similar with this embodiment, and this application is no longer repeated.
The invention has the advantages of convenient carrying, simple and convenient operation, rapidness and easy result interpretation when the blood type detection is carried out, and is suitable for the blood type detection under the conditions of bedside blood type reinspection, field operation detection, emergencies, a large amount of population blood type general survey and the like.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: it is to be understood that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof, but such modifications or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The kit for ABO blood group orthotyping and Rh blood group detection is characterized by comprising a reagent card, wherein the reagent card comprises a card shell wrapped outside, and a detection film, a water absorption pad and a back lining which are sequentially arranged inside the card shell from top to bottom;
the detection membrane is composed of a character area and a non-character area, wherein the character area is composed of letters and symbols, the letters comprise A, B, O letters and letters at Rh positions, and the letters at the Rh positions are one or more of C, C, D, E and E in combination; the symbols include the minus sign in the middle of the letter O and the plus sign above the right of each letter of the Rh position;
each character comprises a hydrophilic area and a hydrophobic area, wherein, the horizontal lines in the first oblique vertical stroke of the A, B letters, the O letters, the letters at Rh positions and the plus signs above the right of the letters at Rh positions are coated with non-bioactive colored hydrophobic ink which is the hydrophobic area;
the rest areas of the characters are hydrophilic areas, wherein the A letter hydrophilic area is coated with an anti-A antibody, the B letter hydrophilic area is coated with an anti-B antibody, the vertical line of the right upper plus sign of the letter at the Rh position is coated with an anti-C antibody or an anti-C antibody or an anti-D antibody or an anti-E antibody or an anti-E antibody, and the minus sign in the middle of the O letter is coated with an anti-A and anti-B mixed antibody;
the area of the detection film except the character is a non-character area, coated with hydrophobic solution and is a hydrophobic area.
2. The kit for ABO blood group orthogonalization and Rh blood group detection according to claim 1, wherein the hydrophobic solution is 0.5-1% AKD-ethanol solution.
3. The kit of claim 1, wherein said non-bioactive colored hydrophobic ink is a commercially available red hydrophobic ink.
4. The kit for ABO blood group orthotyping and Rh blood group detection according to claim 1, wherein the detection membrane is made of a porous polymer material with a pore size of not more than 15 μm.
5. The method for preparing a kit for ABO blood group orthogonalization and Rh blood group detection according to any one of claims 1 to 4, comprising the steps of:
s1, preparing A, B, O, C/c/D/E/E in advance+A pattern design of character shape, wherein a hydrophobic solution is sprayed on a non-character area on the detection membrane by an ink-jet printer, a hydrophobic area is formed by a non-bioactive colored hydrophobic ink sprayed on the first oblique vertical line of the letters A and B, the letter at the position of the letter O, Rh and the horizontal line of the plus sign above and to the right of the letter at the position of Rh, and the rest area of the character is a hydrophilic area;
s2, coating an anti-A antibody, an anti-B antibody, an anti-C/C/D/E/E antibody and an anti-A and anti-B mixed antibody on a hydrophilic area of an A letter, a hydrophilic area of a B letter, a vertical line of a plus sign on the right side of a letter of Rh position and a minus sign area in the middle of an O letter of the detection membrane respectively by a physical adsorption or chemical crosslinking method;
s3, drying the detection membrane coated with the antibody at 37 ℃ for 30-60 minutes or overnight at room temperature to form the detection membrane coated with the antibody and provided with the permanent mark.
6. The test method for ABO blood group orthogonalization and Rh blood group test according to any one of claims 1 to 4, wherein the test method is as follows: dripping a sample to be detected into a reaction area which is coated with an antibody in advance, adding 150-250 mu l of flushing liquid into the reaction area after the reaction time is not more than 60 seconds, and if the antigen corresponding to the coated antibody exists on the erythrocyte sample to be detected, carrying out antigen-antibody specific reaction to form erythrocyte agglutination, wherein the erythrocyte is intercepted at the antibody and forms a positive text form result with a permanent mark; on the other hand, if the antigen corresponding to the coated antibody is not present on the red blood cells, the unreacted red blood cells are eluted by the washing solution, and a result in the form of a negative text is formed in the reaction region.
7. The method for detecting ABO blood group orthogonalization and Rh blood group detection according to claim 6, wherein the sample to be detected is whole blood or packed red blood cells.
8. The method of claim 6, wherein the sample to be tested comprises hemolyzed blood, icteric blood, hyperlipidemia blood and normal blood.
9. The method for detecting ABO blood group positive typing and Rh blood group detection according to claim 6, wherein the amount of the sample to be tested is 5 μ l to 10 μ l.
10. The method of claim 6, wherein the washing solution is physiological saline or phosphate buffer.
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| CN112345773A (en) * | 2020-09-24 | 2021-02-09 | 北京健乃喜生物技术有限公司 | Immunoassay device for in vitro instant determination of ABO and/or Rh blood type of human |
| CN112710859A (en) * | 2020-12-07 | 2021-04-27 | 珠海贝索生物技术有限公司 | Rh blood group antigen detection method convenient to observe |
| CN113092793A (en) * | 2021-04-16 | 2021-07-09 | 江苏力博医药生物技术股份有限公司 | Method and system for detecting Rh blood group antigen and application thereof |
| CN114236140A (en) * | 2021-12-27 | 2022-03-25 | 江苏贝索生物工程有限公司 | Blood type intelligent interpretation method based on test tube method |
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| CN114236140B (en) * | 2021-12-27 | 2024-06-11 | 江苏贝索生物工程有限公司 | Blood type intelligent interpretation method based on test tube method |
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