Test reagent for determining blood group antigens

Abstract

The present application relates to a test reagent for determining blood group antigens. The test reagent comprises: an antibody or antigen-binding fragment thereof against blood group antigens, sodium salt, potassium dihydrogen phosphate, bovine serum albumin, glycine, sugar, optionally a polymerization promoter, a preservative. After the test reagent of the present application is mixed with red blood cells, a positive result can form visible agglutination in an anti-human globulin test card or a blood group reverse typing test card based on micro-column gel method.

Description

Technical Field

[0001] This application relates to the clinical, medical, and biological fields. Specifically, it relates to a detection reagent for identifying blood group antigens on the surface of red blood cells or in blood samples. Background Technology

[0002] The human body has multiple blood group systems. During blood transfusions, incompatible blood group antigens can lead to hemolytic reactions of varying degrees. In addition, some blood group systems are helpful in clinical disease research. Rare blood typing reagents are antigen detection reagents for blood group systems other than the ABO and Rh systems. They are mainly used for the detection and typing of Kidd system Jka / Jkb antigen, Duffy system Fya / Fyb antigen, Kell system K / k antigen, P1 system P1 antigen, MNS system M / N / S / s, and Lewis system Lea / Leb antigen.

[0003] The Miltenberger (MNS) blood group system is the second blood group system discovered after the ABO blood group system. It exhibits complexity in serological, genetic, and biochemical aspects, with over 40 identified antigens. This system contains many variants and satellite antigens. The Miltenberger subtype system shows a higher rate of antigen-antibody reactions in Asian populations than in other groups, making its antigen and antibody research clinically important. In recent years, there have been increasing reports of hemolytic transfusion reactions involving Mur antigens and antibodies.

[0004] The Lewis system refers to antigens in blood plasma. The antigens on red blood cells are adsorbed from blood plasma. The main reason is that antigens are soluble in secretions but not in blood plasma. They are then adsorbed from blood plasma onto red blood cells, where they appear as red blood cell antigens.

[0005] The P system is the only antigen in this blood group system. The P, PK and LKE antigens are closely related to the P antigen in terms of serology and biochemistry, but they are not controlled by its genes. There are many reports in domestic literature about natural anti-P causing blood incompatibility and difficulty in blood typing.

[0006] The Kell system consists of 5 groups of opposing antigens, comprising 12 high-frequency antigens and 5 low-frequency antigens. Among them, the K antigen is more common in Caucasians, while the KK phenotype occurs very rarely in Black and Asians.

[0007] The Kidd system consists of Jka and Jkb antigens. The frequency of the Jka gene is higher in European Caucasians and African Blacks, while the frequency of the Jkb gene is generally higher than that of the Jka gene in East Asian populations.

[0008] The Duffy system, discovered in 1950, mainly consists of Fya, Fyb, and Fy antigens. In East Asian populations such as those from China, Japan, and Korea, the frequency of the Fya gene is higher than that of Caucasians and Black Africans. Caucasians show a higher frequency of the Fyb gene, while Black populations predominantly show the Fy antigen gene. Recent studies by domestic scholars have found that the Fya gene has the highest frequency, suggesting that its gene frequency does not exhibit a clear regularity within China, but varies greatly among different ethnic groups (Xu Ailei, Research Progress on Rare Blood Type Systems, Medical Review, 2011, 2658-2660).

[0009] There is still a need in this field for a tool that can enable rapid identification of rare blood types. Summary of the Invention

[0010] Test reagents

[0011] This application provides a detection reagent for determining Duffy blood type, which contains an anti-Duffy blood type antigen antibody or an antigen-binding fragment thereof.

[0012] This application also provides a detection reagent for determining Kidd blood type, which comprises anti-Kidd blood type antigen antibody or antigen-binding fragment thereof.

[0013] This application also provides a test reagent for determining Kell blood type, which comprises an anti-Kell blood type antigen antibody or an antigen-binding fragment thereof.

[0014] This application also provides a detection reagent for determining P1 blood type, which comprises anti-P1 blood type antigen antibody or antigen-binding fragment thereof.

[0015] This application also provides a detection reagent for determining MNS blood type, which comprises anti-MNS blood type antigen antibody or antigen-binding fragment thereof.

[0016] This application also provides a diagnostic reagent for determining Lewis blood type, which comprises anti-Lewis blood type antigen antibody or antigen-binding fragment thereof.

[0017] Microcolumn gel assays combine gel testing, centrifugation, and antigen-antibody specific reactions. As an example, antigens on the surface of red blood cells react with corresponding antibodies. Upon contact, antigen and antibody bind (cell sensitization). Using a microcolumn gel medium (such as a gel column supplemented with anti-human globulin antibodies), sensitized cells bind to the anti-human globulin antibodies, forming visible agglutinations. After centrifugation, these agglutinations are trapped in the upper layer of the gel column or suspended within the gel, resulting in a positive result. Conversely, if the red blood cells do not have corresponding antigens on their surface, free red blood cells can pass through the gel gaps, centrifuge, and deposit at the bottom of the gel microcolumn, resulting in a negative result.

[0018] In some embodiments, a test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood type is provided, comprising 1 g / L to 10 g / L of antibody or antigen-binding fragment thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g / L, or a range between any two of the aforementioned values, which may be integers or decimals), preferably 1 g / L to 6 g / L of anti-Duffy, anti-Kidd, anti-Kell, anti-P1, anti-MNS, or anti-Lewis blood type antigen antibody or antigen-binding fragment thereof.

[0019] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. Antibodies (immunoglobulins) are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be classified as: IgM, IgD, IgG, IgA, and IgE.

[0020] The term "antigen-binding fragment" refers to a fragment of an antibody and antibody analogue, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains the binding specificity of the parent antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd, dAb; VHH domain; linear antibodies; and single-chain antibody molecules (scFv).

[0021] Determining a Duffy, Kidd, Kell, P1, MNS, or Lewis blood type involves testing a sample and determining its classification within the Duffy, Kidd, Kell, P1, MNS, or Lewis blood group system based on the test results. This determination can be qualitative or quantitative; qualitative is preferred.

[0022] In some embodiments, the test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types further comprises: sodium salt not exceeding 7 g / L, for example, 1, 2, 3, 4, 5, 6, 7 g / L, or a range between any two of the aforementioned values, which can be integers or decimals, preferably 2 g / L to 6 g / L sodium salt, and most preferably 5 g / L sodium salt.

[0023] In some embodiments, the test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types further comprises: 0.1 g / L to 0.5 g / L potassium dihydrogen phosphate (e.g., 0.1, 0.2, 0.3, 0.4, 0.5 g / L, or any range between any two of the foregoing values, which may be integers or decimals), preferably 0.2 g / L to 0.4 g / L potassium dihydrogen phosphate.

[0024] In some embodiments, the test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types further comprises: not less than 20 g / L bovine serum albumin (e.g., 20, 25, 30, 35, 40, 45, 50 g / L, or any range between any two of the foregoing values, which may be integers or decimals), preferably 30 g / L bovine serum albumin.

[0025] In some embodiments, the test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types further comprises: 10 g / L to 15 g / L glycine (e.g., 10, 11, 12, 13, 14, 15 g / L, or any range between any two of the foregoing values, which may be integers or decimals), preferably 11 g / L to 14 g / L glycine.

[0026] In some embodiments, the test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types further comprises: 0.5 g / L to 5 g / L of sugar (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 g / L, or any range between any two of the foregoing values, which may be integers or decimals), preferably 0.5 g / L to 2 g / L of sugar.

[0027] In some embodiments, the sugar is selected from any one or a combination of the following: glucose, trehalose, and sucrose.

[0028] In some embodiments, the testing reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types may optionally contain 1 g / L to 2 g / L of a polymerization promoter (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 g / L, or any range between any two of the foregoing values, which may be integers or decimals), preferably 1 g / L of polymerization promoter.

[0029] In some embodiments, the polymerization promoter is selected from any of the following: PEG6000, PEG8000, PEG20000.

[0030] In some embodiments, the testing reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types further comprises: 0.5 g / L to 5 g / L of preservative (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 g / L, or any range between any two of the foregoing values, which may be integers or decimals), preferably 0.5 g / L to 3 g / L of preservative.

[0031] In some embodiments, the preservative is selected from any of the following: sodium azide, lithium azide, ProClin300.

[0032] In some embodiments, the sodium salt is selected from any one of the following: sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium carbonate.

[0033] In some implementations, the test reagents for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types are prepared as selected from any of the following: microcolumn gel method reagents, colloidal gold reagents, polygluconate method reagents, test tube method reagents, and glass bead method reagents.

[0034] In some implementations, the reagents used to determine Duffy, Kidd, Kell, P1, MNS, or Lewis blood types are microcolumn gel assay reagents.

[0035] In some implementations, the volume of the test reagent used to determine the blood type of Duffy, Kidd, Kell, P1, MNS, or Lewis is 20 μL to 50 μL (e.g., 20, 25, 30, 35, 40, 45, 50 μL or any two of the aforementioned values, which may be integers or decimals), preferably 25 μL.

[0036] In some embodiments, the type of antibody against Duffy, Kidd, Kell, P1, MNS, or Lewis blood group antigen is selected from any of the following: IgG antibody, IgM antibody, IgY antibody, IgA antibody.

[0037] In some embodiments, the antigens of the Duffy blood group system are selected from any of the following antigens or epitopes: Fya(001), Fyb(002), Fy3(003), Fy4(004), Fy5(005), Fy6(006), or combinations thereof.

[0038] In some embodiments, the antigens of the Kidd blood group system are selected from any of the following antigens or epitopes: JKa (JK1), JKb (JK2), JK3 (JK3), or combinations thereof.

[0039] In some implementations, the antigen of the Kell blood group system is XK1 or its epitope.

[0040] In some embodiments, the antigens of the P1 blood group system are selected from any of the following antigens or epitopes: P1PK1 (P1), P1PK3 (PK), P1PK4 (NOR), or combinations thereof.

[0041] In some embodiments, the antigens of the MN blood group system are selected from any of the following antigens or epitopes: M, N, S, s, or combinations thereof.

[0042] In some embodiments, the antigens of the Lewis blood group system are selected from any of the following antigens or epitopes: Lea, Leb, ALeb, Bleb, or combinations thereof.

[0043] The term "epitope" refers to a site on an antigen of the Duffy, Kidd, Kell, P1, MNS, or Lewis blood group system that binds to an immunoglobulin (or antibody). Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids through the tertiary folding of a protein. Epitopes typically consist of at least 3-15 amino acids in a distinctive spatial conformation. Methods for determining which epitope an antibody binds to are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0044] The testing reagents for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types according to this application can be prepared in various forms, such as, but not limited to, kits, test strips, test cards, chips, well plates, tubes, and columns.

[0045] In a representative example, the test reagents used to determine Duffy, Kidd, Kell, P1, MNS, or Lewis blood types are prepared in the form of test cards. A test card is an in vitro diagnostic reagent that is presented as a combination of one or more tubes or columns containing the test reagents for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types, to facilitate the comparison or display of identification results.

[0046] In a representative example, the test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types is prepared in the form of gel microcolumns. When the antibody is IgG type, the microcolumn is loaded with a gel medium (e.g., a gel medium containing anti-human globulin antibodies). When the test cells come into contact with the test reagent of this application, sensitized cells will bind to the anti-human globulin antibodies to form visible agglutinations, which are blocked in the upper layer of the gel column or suspended in the gel; unsensitized cells flow through the gel medium. For the detection principle of IgG type antibodies (involving anti-human globulin antibodies), taking Fya as an example: the red blood cell surface antigen reacts with the corresponding antibody. The reagent containing anti-Fya IgG binds to the Fya antigen on the surface of red blood cells (sensitization), but does not produce a direct agglutination reaction. During centrifugation, red blood cells sensitized by the reagent containing anti-Fya IgG pass through the gel microcolumn containing the anti-human globulin reagent, connecting with each other and causing agglutination.

[0047] In another representative example, the test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types is prepared in the form of gel micropillars. When the antibody is IgM, the micropillars are loaded with a gel medium (a gel medium without anti-human globulin antibodies). When the test cells come into contact with the test reagent of this application, sensitized cells form visible agglutinations, which are blocked on the upper layer of the gel column or suspended in the gel; unsensitized cells flow through the gel medium. Regarding the principle of IgM antibody detection, for example, Fyb: the erythrocyte surface antigen reacts with the corresponding IgM antibody. The reagent containing anti-Fyb IgM can bind to the Fyb antigen on the erythrocyte surface to produce a direct agglutination reaction because the structure of IgM is a pentamer, which readily forms agglutinations.

[0048] In one specific implementation, an anti-human globulin test card is provided for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types, comprising:

[0049] 1 g / L anti-Duffy, Kidd, Kell, P1, MNS, or Lewis blood group antigen antibody (preferably IgG or IgM antibody).

[0050] 5g / L sodium chloride,

[0051] 0.4 g / L potassium dihydrogen phosphate

[0052] 1.5g / L trehalose

[0053] 14g / L glycine

[0054] 30g / L bovine serum albumin

[0055] Choose 1g / L PEG8000

[0056] 2 g / L sodium azide;

[0057] And, a gel medium (preferably a gel medium containing anti-human globulin antibodies).

[0058] use

[0059] This application also provides the use of the aforementioned testing reagents for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types in the preparation of identification devices.

[0060] In some implementations, the identification device is selected from any of the following: reagent kit, test strip, test card, chip, well plate, tube, column; preferably, test card or gel microcolumn.

[0061] In some implementations, the identification device is used to determine the Duffy, Kidd, Kell, P1, MNS, or Lewis blood type of a sample.

[0062] In some implementations, the sample contains red blood cells.

[0063] In other embodiments, the sample is whole blood; more preferably, whole blood containing an anticoagulant (EDTA, citric acid, or heparin, etc.).

[0064] In some implementations, the sample volume is 25 μL to 75 μL (25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 μL or any two of the aforementioned values, which may be integers or decimals), preferably 50 μL.

[0065] In some implementations, the cell concentration in the sample is 0.8% to 1.0% (v / v).

[0066] In some implementations, the identification device may also include instructions for use, auxiliary tools (such as, but not limited to, sampling tools), and packaging.

[0067] In the context of this application, when specific numerical values ​​are mentioned, they should not be construed as precise point values ​​unless otherwise specified, but rather as allowing for appropriate errors (such as errors introduced by the operational process, errors due to measurement accuracy, errors inherent in the methodology, etc.). For example, 0.4 g / L potassium dihydrogen phosphate can include values ​​within the range of 0.4 g / L ± 5%. Detailed Implementation

[0068] Example 1. Anti-human globulin detection card (microcolumn gel method)

[0069] 1. Each card contains multiple (e.g., 6) microtubes, with a reaction chamber at the top and a microcolumn at the bottom; each microtube is filled with a green gel (other colors are acceptable for easy identification) containing anti-human globulin (rabbit IgG polyclonal antibody, mouse IgM monoclonal antibody-C3d, BRIC-8), and stored in a NaN3 solution with a concentration of no more than 0.1%.

[0070] 2. Preparation method of anti-human globulin detection card (microcolumn gel method):

[0071] The gel was immersed in a cleaning solution to remove impurities. Green buffer containing anti-human globulins (rabbit IgG polyclonal antibody, mouse IgM monoclonal antibody-C3d, BRIC-8) was added in the correct proportion, at which point the gel turned green. An empty card membrane was prepared; an appropriate amount of gel was added to the membrane, and the membrane was sealed. Preparation was complete.

[0072] Example 2. Blood type reverse typing test card (microcolumn gel method)

[0073] 1. Each card contains multiple (e.g., six) microtubes, with a reaction chamber at the top and a microcolumn at the bottom; each microcolumn is filled with a white, antibody-free gel. Store in a NaN3 solution with a concentration not exceeding 0.1%.

[0074] 2. Preparation method of blood type reverse typing test card:

[0075] The gel is immersed in a cleaning solution to remove impurities. Buffer solution is then added according to the specified ratio, at which point the gel turns white. An empty card membrane is prepared; an appropriate amount of gel is added to the membrane, and the membrane is sealed. Preparation is now complete.

[0076] Example 3. Test reagents for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types.

[0077] Prepare a test reagent for determining Duffy, Kidd, Kell, P1, MNS, or Lewis blood types by dissolving and mixing the following components:

[0078] 1 g / L anti-Fya antibody (IgG in anti-human globulin detection method, IgM in blood typing reverse typing method) (or anti-Fyb, or Jka, or Jkb, or Lea, or Leb, or M, or N, or S, or s, or K, or k, or P1 antibody);

[0079] 5g / L sodium chloride,

[0080] 0.4 g / L potassium dihydrogen phosphate

[0081] 1.5g / L trehalose

[0082] 14g / L glycine

[0083] 30g / L bovine serum albumin

[0084] Choose 1g / L PEG8000

[0085] 2 g / L sodium azide.

[0086] Optimization Example 1. Reagent Composition Optimization (Sodium Chloride Concentration)

[0087] 1. Component 1 (this application):

[0088] 1 g / L anti-Fya antibody (IgG as an example) (or anti-Fyb, or Jka, or Jkb, or Lea, or Leb, or M, or N, or S, or s, or K, or k, or P1 antibody);

[0089] 5g / L sodium chloride,

[0090] 0.4 g / L potassium dihydrogen phosphate

[0091] 1.5g / L trehalose

[0092] 14g / L glycine

[0093] 30g / L bovine serum albumin

[0094] Choose 1g / L PEG8000

[0095] 2 g / L sodium azide.

[0096] 2. Component 2 (control reagent): The difference from Component 1 is that it contains 7 g / L sodium chloride.

[0097] 3. Experimental Procedure: Detection of Sanquin anti-screening cells I, II, and III. Experimental Data:

[0098] Table 1

[0099]

[0100] Table 2

[0101]

[0102] Table 3

[0103]

[0104] Table 4

[0105]

[0106] Note: w = weak aggregation.

[0107] 4. Experimental conclusion: According to the experimental data, the reaction intensity is better at a sodium chloride concentration of 5 g / L than at 7 g / L. Therefore, the sodium chloride concentration should be 5 g / L.

[0108] When the sodium chloride concentration exceeds 7 g / L (e.g., 7.5, 8, 10 g / L), the reaction strength is significantly and adversely affected (data not shown).

[0109] Optimization Example 2. Adjusting BSA concentration

[0110] 1. Component 1 (this application):

[0111] 1 g / L anti-Fya antibody (IgG as an example) (or anti-Fyb, or Jka, or Jkb, or Lea, or Leb, or M, or N, or S, or s, or K, or k, or P1 antibody);

[0112] 5g / L sodium chloride,

[0113] 0.4 g / L potassium dihydrogen phosphate

[0114] 1.5g / L trehalose

[0115] 14g / L glycine

[0116] 30g / L bovine serum albumin

[0117] Choose 1g / L PEG8000

[0118] 2 g / L sodium azide.

[0119] 2. Component 2 (Control): The only difference from Component 1 is the addition of 20 g / L bovine serum albumin.

[0120] 3. Experimental procedure: Detection of Sanquin anti-screening cells I, II, and III.

[0121] Experimental data:

[0122] Table 5

[0123]

[0124] Table 6

[0125]

[0126] Table 7

[0127]

[0128] Table 8

[0129]

[0130] 4. Experimental conclusion: According to the experimental data, the reaction intensity of 30 g / L BSA concentration is better than that of 20 g / L BSA, so the BSA concentration should be 30 g / L.

[0131] When the concentration of BSA is below 20 g / L (e.g., 10, 15) or above 40 g / L (e.g., 40, 45, 50), the reaction strength is significantly and adversely affected (data not shown).

[0132] Test Example 1. Specificity and Repeatability

[0133] 1. Sample: A sample known to be antigen negative / positive.

[0134] 2. Experimental steps:

[0135] The anti-human globulin detection cards prepared in Example 1 were used to repeatedly detect the Fya+, Fya-, M+, M-, N+, N-, S+, S-, s+, s-, k+, and k- in the samples. The samples were incubated at 37°C for 15 minutes, centrifuged for 10 minutes, and the agglutination intensity was recorded (Table 9).

[0136] In Example 2, the reverse typing card was used to repeatedly test the sample Fyb+, Fyb-, Jka+, Jka-, Jkb+, Jkb-, Lea+, Lea-, Leb+, Leb-, K+, K-, P1+, and P1-. The samples were incubated at room temperature for 15 minutes and centrifuged for 5 minutes. The agglutination intensity was recorded, and the positive and negative results were required to be consistent. The agglutination intensity deviation was allowed to be ≤1+ (Table 10).

[0137] Table 9. Specificity and repeatability

[0138]

[0139]

[0140] Table 10. Specificity and repeatability

[0141] Fyb+ Fyb- Jka+ Jka- Jkb+ Jkb- Lea+ Lea- Leb+ Leb- K+ K- P1+ P1- 1 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 2 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 3 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 4 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 5 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 6 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 7 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 8 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 9 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 10 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 11 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0 12 2+ 0 2+ 0 4+ 0 2+ 0 2+ 0 2+ 0 2+ 0

[0142] Strength determination criteria (semi-quantitative): Agglutination strength is ranked from strongest to weakest as 4+, 3+, 2+, 1+; 0 indicates negative. The stronger the antigenicity, the stronger the agglutination strength; the weaker the antigenicity, the weaker the agglutination strength. If the surface of red blood cells does not have the corresponding antigen, the result is negative.

[0143] As shown in Tables 9 and 10, the results of the 12 tests were consistent, with a deviation of ≤1+, and the positive and negative results were consistent with the results of known cell antigens.

[0144] Test Example 2. Comparison with the test tube method

[0145] 1. Sample: A sample that is negative or positive for an unknown antigen.

[0146] 2. Experimental steps:

[0147] The samples were tested using the anti-human globulin detection card prepared in Example 1. The samples were incubated at 37°C for 15 minutes, centrifuged for 10 minutes, and the agglutination intensity was recorded. The results were compared with those obtained using a commercially available reagent (DIAGAST) via the test tube method.

[0148] The sample was tested using the reverse typing card prepared in Example 2. The sample was incubated at room temperature for 15 minutes, centrifuged for 5 minutes, and the agglutination intensity was recorded. The test tube method was used to compare the agglutination intensity with the reagent.

[0149] Table 11. Comparison between microcolumn gel method (anti-human globulin detection card) and test tube method

[0150]

[0151] Note: w = weak aggregation.

[0152] Table 12. Comparison between the microcolumn gel method (reverse shaping card) and the test tube method

[0153]

[0154] Note: w = weak aggregation.

[0155] As can be seen from the data in Tables 11 and 12 above, the test tube method results of the microcolumn gel method reagent and the comparison reagent (DIAGAST) of this application are consistent in terms of positive and negative results.

[0156] Compared to the test tube method, the reagents in this application are more compatible with various blood group systems, reduce costs, and standardize interpretation.

[0157] Test Example 3. Accelerated Stability

[0158] 1. Sample: A sample known to be antigen negative / positive.

[0159] 2. Experimental Procedure: Sanquin anti-screening cells (I, II, and III) were measured after storage at 37℃ for 3, 5, 7, 10, and 14 days, and the results were compared with the initial results. Agglutination intensity was recorded. Consistency in the interpretation of results was required, with an allowable deviation of ≤1+ in agglutination intensity.

[0160] Table 13. Accelerated stability at 37℃

[0161] Fya 4℃ 37℃ for 5 days 37℃ for 10 days 37℃ for 14 days 37℃ for 16 days I anti-screening cells 0 0 0 0 0 II Anti-screening cells 0 0 0 0 0 III Anti-screening cells 3+ 3+ 3+ 3+ 3+ Sample 1 + + + + + Sample 2 + + + + + Sample 3 + + + + +

[0162] Table 14

[0163] M 4℃ 37℃ for 5 days 37℃ for 10 days 37℃ for 14 days 37℃ for 16 days I anti-screening cells 3+ 3+ 3+ 3+ 3+ II Anti-screening cells 0 0 0 0 0 III Anti-screening cells 3+ 4+ 3+ 3+ 4+ Sample 1 3+w 3+w 3+ 3+w 3+ Sample 2 0 0 0 0 0 Sample 3 3+ 3+ 3+ 3+ 3+

[0164] Table 15

[0165] N 4℃ 37℃ for 5 days 37℃ for 10 days 37℃ for 14 days 37℃ for 16 days I anti-screening cells 3+ 3+ 2+ 2+ 3+w II Anti-screening cells 3+ 3+ 3+ 3+ 3+ III Anti-screening cells 0 0 0 0 0 Sample 1 0 0 0 0 0 Sample 2 3+w 3+w 3+w 2+ 2+ Sample 3 0 0 0 0 0

[0166] Table 16

[0167]

[0168] Table 17

[0169]

[0170] Table 18

[0171]

[0172] Table 19

[0173]

[0174]

[0175] Table 20

[0176]

[0177] Table 21

[0178]

[0179] Table 22

[0180]

[0181] Table 23

[0182] Leb 4℃ 37℃ for 5 days 37℃ for 10 days 37℃ for 14 days 37℃ for 16 days I anti-screening cells 2+ 2+ 4+w 3+ 3+w II Anti-screening cells 2+ 2+ 4+w 2+ 3+w III Anti-screening cells 0 0 0 0 0 Sample 1 2+ 2+ 2+ 2+ 2+ Sample 2 2+ 2+ 2+ 2+ 2+ Sample 3 0 0 0 0 0

[0183] Table 24

[0184] K 4℃ 37℃ for 5 days 37℃ for 10 days 37℃ for 14 days 37℃ for 16 days I anti-screening cells 0 0 0 0 0 II Anti-screening cells 2+ 2+ 2+ 2+ 2+ III Anti-screening cells 0 0 0 0 0 Sample 1 0 0 0 0 0 Sample 2 0 0 0 0 0 Sample 3 0 0 0 0 0

[0185] Table 25

[0186] P1 4℃ 37℃ for 5 days 37℃ for 10 days 37℃ for 14 days 37℃ for 16 days I anti-screening cells ± ± 1+ ± 1+ II Anti-screening cells 2+ 2+ 3+ 2+ 3+ III Anti-screening cells 3+ 3+ 4+ 3+ 4+ Sample 1 0 0 0 0 0 Sample 2 2+ 2+ 2+ 2+ 2+ Sample 3 2+ 2+ 2+ 2+ 2+

[0187] Note: w = weak aggregation.

[0188] 3. Conclusion:

[0189] - The blood group antigen detection reagent based on the microcolumn gel method in this application shows accelerated stability for at least 16 days.

[0190] - The accuracy of the reagents is consistent with that of the test tube method.

[0191] - The reagent reacted with samples known to be positive or negative, and the results were consistent, confirming the specificity of the test reagent.

[0192] - The results were consistent after 10 repeated tests, confirming the repeatability of the test reagent.

Claims

1. A detection reagent for determining blood group antigens, comprising: 1 g / L anti-blood group antigen antibody or its antigen-binding fragment; 5 g / L sodium chloride; 0.4 g / L potassium dihydrogen phosphate; 30 g / L bovine serum albumin; 14 g / L glycine; 1.5 g / L trehalose; 1 g / L PEG8000; 2 g / L preservative; in: The preservative is selected from any one or a combination of the following: sodium azide, lithium azide, PC300; the blood group antigen is selected from any one or a combination of the following: Duffy blood group system antigen, Kidd blood group system antigen, Kell blood group system antigen, P1 blood group system antigen, MNS blood group system antigen, Lewis blood group system antigen. The antigens of the Duffy blood group system are selected from any of the following antigens or epitopes: Fya (001), Fyb (002), Fy3 (003), Fy4 (004), Fy5 (005), Fy6 (006), or combinations thereof; The antigens of the Kidd blood group system are selected from any of the following antigens or epitopes: JKa (JK1), JKb (JK2), JK3 (JK3), or combinations thereof; The antigens of the Kell blood group system are selected from any of the following antigens or epitopes: K, k, or a combination thereof; The antigens of the P1 blood group system are selected from any of the following antigens or epitopes: P1PK1 (P1), P1PK3 (PK), P1PK4 (NOR), or combinations thereof; The antigens of the MNS blood group system are selected from any of the following antigens or epitopes: M, N, S, s, or combinations thereof; The antigens of the Lewis blood group system are selected from any of the following antigens or epitopes: Lea, Leb, ALeb, Bleb, or combinations thereof; The anti-blood type antigen antibody is an IgG or IgM antibody.

2. The detection reagent for determining blood group antigens according to claim 1, wherein the reagent is a microcolumn gel method reagent.

3. The detection reagent for determining blood group antigens according to claim 2, wherein the volume of the detection reagent is 20 μL to 50 μL.

4. The detection reagent for determining blood group antigens according to claim 3, wherein: The volume of the detection reagent is 25 μL.

5. The detection reagent for determining blood group antigens according to any one of claims 1 to 4, further comprising a gel medium; The gel medium may or may not contain anti-human globulin antibodies.

6. The use of the testing reagents in the preparation of blood group antigen identification devices, wherein: The detection reagent is the detection reagent for determining blood group antigens as described in any one of claims 1 to 5; The identification device is a test card or a gel microcolumn.

7. The use according to claim 6, wherein the identification device is used to identify blood group antigens in a sample; The sample was whole blood.

8. The use according to claim 7, wherein the sample is whole blood containing an anticoagulant.

9. The use according to claim 7, wherein the volume of the sample is from 25 μL to 75 μL.

10. The use according to claim 9, wherein the volume of the sample is 50 μL.

11. The use according to claim 7, wherein the red blood cell concentration in the sample is from 0.8% to 1.0% v / v.

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