JPS6234106B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to methods and reagents for use in blood classification, and more particularly for detecting Rh antigens, such as Rh ₀ (D) antigens, as well as other antigens.

Detection of blood type and in particular the presence or absence of the Rh ₀ (D) antigen is a routine task in modern medicine. 1930
Since the discovery of the relationship between the Rh factor and disease in the 1990s, there has been research into the relationship between Rh incompatibility between mother and fetus so that it can be treated or avoided in the future child.
There has been increasing interest in detecting Rh incompatibility. Currently, many U.S. state laws protect pregnant women and infants born to Rh(-) women.
Requires Rh test. In 1968, the introduction of Rh immune globulin made it possible for the first time to treat Rh(-) women who gave birth to Rh(+) children and to prevent diseases in the Rh(+) children of these women. .

Two types of diagnostic tests are currently used to detect the presence of D antigen. It must be understood that IgG antibodies directed against the D antigen are so-called "incomplete" antibodies. That is, IgG antibodies having specificity for the D antigen often fail to agglutinate Rh(+) red blood cells suspended in saline. In contrast, for D antigen
IgM antibodies cause Rh(+) red blood cell agglutination in saline.

The first of these tests uses an IgG anti-D antibody as a reagent.
Anti-D IgG itself is Rh(+) in saline.
Since it will not cause red blood cell agglutination, some driving means must be found to cause agglutination. This drive is achieved by relatively high concentrations of biological polymers (eg non-specific proteins such as albumin) or synthetic polymers (eg polyvinylpyrrolidone or dextran sulfate). Typically, the concentration of albumin in the final reagent composition is about 20-25%. While this added protein successfully drives the agglutination reaction of red blood cells with specific IgG anti-D antibodies and D antigen, it also causes non-specific agglutination of red blood cells with IgG coated on the surface. cause. Such coatings occur, for example, in individuals with diseases such as neonatal autoimmune hemolytic anemia or hemolytic diseases in which antibodies are produced against and bind to the patient's own red blood cells. Therefore, using the above test in these individuals would give a false positive result for the presence of Rh antigen. However, one important advantage of this test is its rapidity, requiring little (if any) incubation before reading the results. This test can be performed either in test tubes or on glass slides.
A second advantage is the ability to detect certain weak antigens D ^u as discussed further below.

A second test method currently in use for detection of D antigen uses IgM antibodies instead of IgG antibodies. Since IgM is a complete antibody against the D antigen, it does not require the addition of a potentiator. Typically, the reagent composition contains a total protein concentration of about 8% by weight or less, achieved by (eg) adding bovine albumin to the IgM antiserum. This test can only be performed in vitro. This test was driven
Although it eliminates the nonspecific agglutination of IgG-coated cells that occurs with IgG testing, it also has some drawbacks. The main drawback is that this test requires an incubation step of 15 to 60 minutes at 37°C, which is not required in the case of a driven IgG test, and furthermore, it is rarely obtained and This means that it requires IgM antibodies, which are difficult to obtain. Furthermore, since the so-called D ^u test uses an antibody against IgG as its reagent, it is not possible to perform this D ^u test in combination with this second test method. This reagent antibody clearly does not react with IgM.

The same two clinical tests have shown that IgG antibodies are also deficient for antigens of other blood groups (e.g. C, E, e).
etc.) can also be used in general.

Thus, a long felt need would be satisfied if a rapid test method for blood group antigens could be developed by using IgG antibodies in low protein formulations.

The conversion of incomplete anti-D IgG to agglutinin directly in saline by reductive cleavage of disulfide bonds has been described in several publications. The first report was in 1960 by Chan and Deutsch (J.
Immunol. 85 , 37-45), and the following
Pirofsky and Cordova, 1963 (Nature, 197 , 392-393). However, these discoveries were made in 1965 by Mandy et al. (J. Clin. Invest., 44 , 1352-1363).
The question is posed by. Further research confirming Pirofsky and Cordova's conclusions
In 1977 Romans et al. (Proc. Natl.
Acad.Sci.USA, 74 (6), 2531-2535). These latter authors are anti-D
We report the direct conversion of anti-c, anti-E, and anti-K IgG, as well as IgG, to agglutinin in saline. However, in this document, the agglutination reaction is achieved only in vitro after incubation of red blood cells and reduced IgG for 2 hours. Furthermore, although the Romance et al. document teaches that incomplete IgG antibodies can be converted to complete antibodies by reduction, there are no special steps or reagents (not to mention special steps or reagents) for use in performing blood classification tests. does not teach anything about compliance with FDA standards). In fact, the article notes in its last sentence that there remains some doubt as to whether reduced antibodies are practical for use as blood grouping reagents, given the unpublished research underway at the time. It shows.

International Society July 24-28, 1978
of Hematology 1st Conference and International
In a report at the joint meeting of the Society of Blood Transfusion, an excerpt was published that appeared to disclose some of the work that referenced the Romance et al. literature. This excerpt from Laschinger et al. states that ``a reliable monospecific anti-D agglutinin with a titer of 16
Production and testing of "incomplete" anti-D serum pools. Pooled “incomplete” antis
Production of a direct reagent with a titer of 8 by improvement of c. ”
is reported. These reported potencies provide no guarantee that the manufactured material will meet FDA standards. Also, this publication does not provide any information regarding the method of manufacturing these agglutinins, the testing methods used, or the extent of incubation required to achieve the agglutination reaction. Furthermore, the excerpt shows that the anti-K or anti-F _y ^a that can be used "... still has to be obtained by this method."

The present invention provides Rh ₀ (D), c, C, e and E antigens (Rh 0 (D), c, C, e and E antigens (Rh
Provides a rapid method for detecting Kell (K) (present in the Rh system) and Kell (K) (not present in the Rh system), and reagents for carrying out the method that eliminates the drawbacks associated with conventional methods. do. The subject antibody reagents firstly allow the use of IgG antibodies in low protein slides or rapid in vitro tests. This test does not show non-specific reactions with IgG-coated cells due to the low protein content. More books
Rh antibody reagents meet and exceed FDA standards for efficacy and specificity for slide or rapid test tube tests, and for anti-D, c , an average titer of at least 32 on e cells and, in the case of anti-C, an average titer of at least 5 on d, C, c, e or D, C, c, E, e cells. with an average titer of at least 16 in d, C, c, e cells for anti-c and at least 8 in d, c, E, e cells for anti-E. have an average titer, and in the case of anti-e, d, c, E,
have an average titer of at least 2 in e cells and anti-
In the case of K, it comprises reduced and S-alkylated IgG antibodies having an average titer of at least 8 against cells heterozygous for the K antigen. These FDA standards apply to Federal Food, Drug
and Cosmetic Act, Part 660, Subpart C, Subchapter F (see, October 7, 1977,
42Federal Regulations 54542ff, No. 195). The Anti-K antibody reagent meets and exceeds corresponding FDA standards for in vitro testing of uncontrolled materials in saline.

Anti-D reagents are generally produced by the following method. This method is exemplary of methods used to produce other reagents. Serum is obtained from individuals who have high amounts of anti-D IgG in their bloodstream and is processed to remove certain contaminants and purify the IgG. Although the specific method of generating anti-D antiserum for use in making the present reagents is not part of this invention, one suitable method is as follows. Other methods of generating suitable anti-D antisera are known.

If necessary, the plasma may be defibrinated with an appropriate amount of bovine thrombin; decalcified with sodium oxalate; clarified by centrifugation; and addition of blood group specific substances A and/or AB. neutralize it.

Lipids are substantially removed from serum by extraction with trichlorotrifluoroethane. best serum
heated to 56.6°C, then cooled to 2-8°C,
The trichlorotrifluoroethane is then mixed at high speed until the serum foams. The serum is stored at 2-9°C until the foam has dissipated, after which it is centrifuged, heated again and stored at 2-8°C. PH
7.3-8.3 and store serum at -20°C or below.

This substantially lipid-free serum is processed in the following manner to produce the reagent of the present invention.

The PH of substantially lipid-free serum is approximately 8.6 using an effective amount of a compatible buffer such as 2M Tris-(hydroxymethyl)aminomethane (TRIS).
adjusted to. One volume of 0.04M dithiothreitol in saline is added to about 4 volumes of buffered serum and the mixture is allowed to stand at about 20-30°C for a minimum of about 30 minutes.

Following this treatment, 1 volume of 0.4M iodoacetamide in distilled water is added to approximately 4 volumes of previously treated serum. Heat this mixture at about 20-30℃ for a minimum of about 60 minutes.
Leave it there. The obtained serum was then diluted with 0.13M
Dialyze twice at 2-8°C by using 10 volumes of freshly prepared 0.02M TRIS buffer in NaCl at PH 7.5-8.3, preferably 7.7-8.1. After the second dialysis, sodium azide was added to the treated serum as a preservative to a final concentration of 0.1%, and the total protein concentration was diluted with polymerized bovine serum a albumin from ca. Adjust to 10% by weight. The polymerized bovine serum albumin used in the present invention and its production method are known, for example, the polymerized bovine serum albumin described in U.S. Pat. No. 4,046,871 is preferably used. The concentration of antibody in the reagent can be adjusted by diluting with 0.02M TRIS buffer in 0.13M NaCl containing 6-8% by weight of polymerized bovine serum albumin.

US Pat. No. 4,046,871 describes polymerized bovine serum albumin useful for use in the antibody reagents of the present invention and methods for making the same. Namely, polymerized bovine serum albumin is defined as "bovine serum albumin containing at least one polymer of bovine serum albumin in an aqueous solution and useful for the detection of specific antigens and the detection of antibody-coated red blood cells. a composition wherein the polymer or polymers have a
It is a homopolymer of up to 15 monomeric albumin units linked together through covalent peptide bonds.”
(See U.S. Pat. No. 4,046,871, column 15, lines 12-19). and ``the albumin component of the aqueous composition comprises (i) bringing bovine serum albumin into intimate contact with a peptide bond-forming agent selected from the group consisting of 3-H-isoxazolium salts and carbodiimide reagents to form an albumin polymer; (ii) adding a basic nitrogen compound to the polymerization mixture containing the polymer to terminate the reaction and subsequently decompose excess peptide former;
(iii) acidifying the mixture to a pH of about 4.5-7; and (iv)
"Albumin monomers and polymers are then recovered" (U.S. Pat. No. 4,046,871).
(See column 15, lines 23-36).

Although specific methods for making the reagents of the invention have been described above, it is contemplated that other suitable reducing agents can be used in place of the preferred dithiothreitol described above. Such a reducing agent may be, for example, 2
-Contains mercaptoethanol and dithioerythritol. Iodoacetamide is S as mentioned above.
- used as alkylating agent, but obviously other alkylating agents such as iodoacetic acid can also be used.
Other equivalent methods of preventing regeneration of disulfide bonds may also be used.

A PH range of 7.5 to 8.3, preferably 7.7 to 8.1 is important for the proper action of the reagent. 7.5~8.3
Outside the PH range of , the reaction of this antibody reagent tends to be somewhat weaker. This PH range can be maintained with an effective amount of a compatible buffer (ie, one that does not adversely affect the antibodies or red blood cells), with TRIS buffers being preferred.

The present invention provides D, C, c, E,
The present invention provides an improved method for rapidly determining the presence or absence of E, or Kel (K) antigens. According to the invention:
Except for the use of the reagents of the present invention in place of the use of conventional IgG antisera, conventional rapid blood classification methods, such as slide tests, modified ("rapid") tube tests or stick test methods, can be used. It is expected that both can be used. Anti-K of the present invention
With reagents, only test tube tests in saline are performed. The method of using the present antibody reagent in a conventional test is as follows, but the use of the present antibody reagent is not limited to that test method, and generally involves the following steps: A. Collecting a sample of red blood cells to be tested; B. C. adding an antibody reagent to and mixing to form a mixture; and C. observing the mixture without substantial incubation to determine whether an agglutination reaction occurs or not; It is intended that it can also be used in the method of

As used herein, "without substantial incubation" means that only a 2-15 minute delay occurs prior to the observation step. This contrasts with the statement in the Romance et al paper that a substantial incubation step is required. In many cases in the present invention, the observation step can occur "without incubation." No incubation here means that there is no intentional delay between the mixing and observation steps. The observation step can occur without incubation for the present Rh reagent in the slide test and for the present Anti-D reagent in the modified test tube method. For other methods and antibody reagents, about 2 minutes of incubation is desirable to obtain a high degree of agglutination.

The modified test tube test can be performed as follows. First, a suspension of red blood cells to be tested is prepared. Conveniently, this may be a 5% suspension in compatible serum of the normal group, in the patient's own serum or plasma, or in blood isotonic saline.

Second, add a small amount (one drop for convenience) of the antibody reagent to
Mix with an equal volume of red blood cell suspension in a small test tube and then centrifuge the tube without prior incubation to remove red blood cell "butlons" and clear cells as known in the art. Generate a supernatant liquid.

Finally, the presence or absence of agglutination is determined, for example, by resuspending the red blood cells by gentle agitation and inspecting them visually. If desired, the use of a control of saline or a non-specific protein in saline (eg, 6-8% bovine albumin) facilitates this determination.

The slide test is conducted as follows. Primarily,
A suspension of red blood cells to be tested is prepared in compatible serum of a normal population or in the patient's own serum or plasma. The concentration of red blood cells in the test sample is conveniently approximately 40 ~
It has to be 50%, so it's best to use whole blood undiluted.

Second, a small amount (e.g. about 1 drop) of the antibody reagent
is mixed with approximately twice the volume of the red blood cell suspension on a pre-warmed glass slide. Preferably the temperature of the slide is about 40-45°C, but at room temperature (e.g. 20°C).
~30℃) can also be used.

Finally, after the red blood cell suspension and antibody reagent are thoroughly mixed without further incubation, the presence or absence of agglutination is assessed by tilting the slide back and forth for several minutes and observing the agglutination reaction. A control of saline or a non-specific protein in saline (eg, 6-8% bovine albumin) can also be subjected to the same process in place of the antibody reagent for use as a negative control.

Negative results for D antigen in any of these tests can be confirmed by the ^Du test as follows. Add an equal volume of the anti-D antibody reagent to a small volume of the 5% suspension of red blood cells described above with mixing. After incubating this mixture at about 37°C for about 15 minutes, the red blood cells are preferably incubated with blood isotonic saline for 30 minutes.
Wash twice and add human antiserum to the washed red blood cells. The red blood cells are then centrifuged as described above and the presence or absence of agglutination determined by observation. A positive result in this test (but a negative result in the D test and control) indicates Rh(+) cells of the ^Du species. A suspension of the same red blood cells in saline (without antibody reagent) is used as a control.

Although the usage of this antibody reagent has been exemplified above, this Rh reagent can be used in agglutination tests to detect any of the above antigens, and especially in such tests that do not require incubation. is intended to be suitable for The Anti-K reagent is suitable for use in test tube tests in saline.

The invention will be further explained with reference to the drawings.

Figures 1 to 5 are comparative examples that do not contain polymerized bovine serum albumin, and Figures 6 to 14 are comparative examples that do not contain polymerized bovine serum albumin at a total protein concentration of 6 to 10% by weight. This is an example.

Figure 1 shows the direct antiglobulin test and Rh
Figure 2 shows a comparison of the present anti-D reagent lot and commercially available anti-D serum in a saline test tube test using -hr control positive cells.

Figure 2 shows a comparison in a modified test tube test using cells suspended in saline, i.e., a slide and modified test tube test between the present anti-D reagent lot and a commercially available anti-D serum; It was used to conduct a modified test tube test.

FIG. 3 shows a modified in vitro test using serum-suspended cells, the same comparison as FIG. 2, but in which the reagents are reacted with test red blood cells in serum suspension.

FIG. 4 shows the same comparison as FIGS. 2 and 3, but for the stake test.

Figure 5 shows the same comparison for the slide test.

Figure 6 shows the reaction with saline suspended cells, ie, a comparison of the present Anti-D reagent with polymerized bovine albumin and normal bovine albumin.

Figure 7 shows a comparison of this anti-C reagent lot with a commercially available anti-C serum in slide and modified test tube tests, where both reagents were used to perform the modified test tube test. .

FIG. 8 shows a comparison between this lot of Anti-C reagent and a commercially available Anti-C serum in a test tube test in saline.

Figure 9 shows a comparison of this anti-c reagent lot with commercially available anti-c serum in slide and modified test tube tests, both reagents used to perform the modified test tube test. .

Figure 10 shows a comparison of this anti-E reagent lot with a commercially available anti-E serum in slide and modified test tube tests, where both reagents were used to perform the modified test tube test. .

FIG. 11 shows a comparison of the present Anti-E reagent lot with a commercially available Anti-E serum in a test tube test in saline.

Figure 12 shows a comparison of this anti-e reagent lot with commercially available anti-e serum in slide and modified test tube tests, where both reagents were used to perform modified test tube tests. .

FIG. 13 shows a comparison between this lot of anti-e reagent and a commercially available anti-e serum in a test tube test in saline. and Figure 14 shows a comparison of the present Anti-K reagent lot with a commercially available Anti-K serum, both reagents used to perform a modified test tube test.

The performance of this antibody reagent was determined by slide and modified test tube tests and by test tube tests in saline.
This is shown by comparison with that of currently used reagents. The antibody reagents of the invention exhibit at least the performance of current reagents as shown in these tests, and offer significant advantages due to their unique properties.

This Anti-D antibody reagent lot number 78-AS-
The specificity of No. 074 was demonstrated in comparison to Ortho-Anti-D Serum Lot No. R1497 for in vitro testing in saline. This test selected 284 positive red blood cells (as indicated by the polyspecific human antiserum) in a direct antiglobulin test. Cells positive for these direct antiglobulin tests were chosen because of their natural tendency to aggregate in high protein media such as those used in conventional IgG antibody reagents. They therefore provide good cells to demonstrate the benefits of the present anti-D antibody reagents with relatively low protein concentrations.
Fifty-five of these cells reacted with the orthoRh-hr control (reagent solution without antibody). This would invalidate test results obtained with high protein, high viscosity reagents such as anti-D serum for slide and modified test tube tests. Therefore, these 55 cells could be successfully tested using the present anti-D antibody reagent, which is not possible with conventional high-protein IgG anti-D antibody reagents. . Three of the 55 cells aggregated with saline and could not be tested in routine work. The remaining 52 cells were tested according to the in vitro saline test recommended by the labeling method. The results are shown graphically in FIG. 10 cells reacted negatively with both reagents. 42 cells showed comparable strong positive responses with both reagents. These results demonstrate that the present anti-D antibody reagent exhibits similar specificity to ortho-anti-D serum for in vitro tests in saline, and that it exhibits similar specificity to ortho-anti-D serum for slide and modified in vitro tests. It was concluded that it can be used in tests that react nonspecifically.

This Anti-D antibody reagent lot number 78-AS-
No. 074, by conducting the improved test tube test, stake test and slide test recommended by the labeling method, the Ortho-Anti-D Serum Lot No. R5736 and No.
Compared with R5773. This modified test tube test was conducted using saline suspension test cells and serum suspension test cells, respectively. The results obtained with the saline suspension test cells are shown in Figure 2.
This shows that the reaction with the species antiserum can be compared. The results obtained with serum suspension test cells are shown in FIG. From these results, it appears that the reaction with the present anti-D antibody reagent is slightly stronger than the reaction with anti-D serum on slides and modified test tube tests. The Stake test reaction shown in Figure 4 shows that the reaction with the present anti-D antibody reagent is more potent. The results of the slide test are shown in Figure 5. In these tests, the present anti-D antibody reagent showed a slightly weaker response than the anti-D serum to the slide and modified test tube tests. The use of polymerized bovine serum albumin (Figure 6) improves the strength of the agglutination reaction. The figure shows the strength of the agglutination reaction for each composition containing 2-3 week old red blood cells (3-5%) suspended in saline.

This Anti-C antibody reagent lot number 80-AS-
The specificity of No. 023 is that the Ortho-Anti-C Serum Lot No.
Comparison with RCST-175 and Anti-C Serum Lot No. R3225 for test tube test in saline. The present anti-C antibody reagents are as specific as commercially available anti-C sera as are those for the present anti-D antibody reagents, while ortho-anti-C for slide and modified test tube tests.
It was found that this method can also be used in tests where the compound reacts nonspecifically. The data are as shown in FIGS. 7 and 8.

The specificity of the present anti-c antibody reagent lot number NC106 is demonstrated in comparison with ortho-anti-c serum lot number R0384 for slide and modified test tube tests. The data shown in Figure 9 show that the anti-c antibody reagent is weaker than commercially available slide and modified test tube antisera, indicating that the reagent is acceptable. Furthermore, for the preparation of test tube test reagents in saline for normal anti-c
Since IgM is not commonly available, the present anti-c antibody reagent is the only reagent that can be used routinely without the possibility of non-specific reactions.

The specificity of this Anti-E Reagent Lot No. 79-AS-169 is as follows:
Comparison with REST No. 178 and Anti-E Serum Lot No. R4196 for test tube test in saline. This anti-e reagent lot number
The specificity of DEV 79025 is demonstrated by comparison with Ortho-Anti-e Serum Lot No. RSE 136 for Slide and Modified Test Tube Tests and Anti-e Serum Lot No. RSET 156 for Test Tube Tests in Saline. The present anti-E and anti-e reagents were found to be as specific as the commercially available anti-E and anti-e sera, respectively, as were the present anti-D antibody reagents. The antibody reagents also have the advantage that they can be used in tests where commercially available antisera show non-specific reactions to slides and modified test tube tests. Book anti-
The data for the E reagent are shown in Figures 10 and 11 and for the present anti-e reagent in Figures 12 and 1.
Shown in Figure 3.

The results of a comparative test of the present anti-K reagent lot number 79-AS-176 and ortho-anti-K serum lot number RKST141 for the slide test and indirect Coombs test are shown in FIG. These results indicate that this anti-K
Reagents can be successfully used to perform modified test tube tests, whereas commercially available reagents have been shown to be unacceptable for this test.

In Figures 1 to 14, the degree of agglutination reaction is 0,
Displayed as 1 ⁺ , 2 ⁺ , 3 ⁺ , 4 ⁺ and S. Here, 0 indicates that no aggregation reaction was observed, 1 ⁺ to 4 ⁺ indicates a progressively increasing degree of agglutination reaction, and S indicates "solid" or highest agglutination reaction.

[Brief explanation of the drawing]

Figure 1 shows a comparison between a lot of the present Anti-D reagent and a commercially available anti-D serum in a test tube test in saline; Figure 2 shows a comparison between a lot of the present Anti-D reagent and a commercially available anti-D serum; Figure 3 shows the same comparison as Figure 2, but in a slide and modified test tube test, with both reagents used to perform the modified test tube test; Figure 4 shows the same comparison as Figures 2 and 3, but for the STATE test;
Figure 5 shows the same comparison for the slide test; Figure 6 shows the comparison of the present Anti-D reagent with polymerized bovine albumin and normal bovine albumin; Figure 7 shows the comparison of the present Anti-D reagent with polymerized bovine albumin and normal bovine albumin; Comparison of anti-C reagent lot and commercially available anti-C serum in slide and modified test tube tests, where both reagents were used to perform modified test tube tests;
Figure 8 shows the present anti-C reagent and commercially available anti-C.
Figure 9 shows a comparison between the present anti-c reagent lot and commercially available anti-c serum in slide and modified test tube tests; Both reagents were used to perform modified test tube tests; Figure 10 shows a comparison of this anti-E reagent lot and commercially available anti-E serum in slide and modified test tube tests. , both reagents were used to perform a modified test tube test; Figure 11 shows a lot of this Anti-E reagent and a commercially available Anti-E reagent.
Figure 12 shows a comparison between this anti-e reagent lot and a commercially available anti-e serum in slide and modified test tube tests. , both reagents were used to conduct a modified test tube test; Figure 13 shows a comparison between this anti-e reagent lot and a commercially available anti-e serum in a test tube test in saline. Figure 14 shows a comparison of this anti-K reagent lot with a commercially available anti-K serum, both reagents being used to perform a modified in vitro test.

Claims (1)

Claims: 1. A reduced and S-alkylated IgG antibody against an antigen selected from the group consisting of D, C, c, E, e and K and a total protein concentration of about 6-10% by weight. D, C, c, E, e on red blood cells containing polymerized bovine albumin in the medium to give
an antibody reagent useful for rapidly determining the presence or absence of an antigen selected from the group consisting of , an average titer of at least 32 on e cells and, in the case of anti-C, an average titer of at least 5 on d, C, c, e or D, C, c, E, e cells. with an average titer of at least 16 in d, C, c, e cells for anti-c and at least 8 in d, c, E, e cells for anti-E. with an average titer of anti-e
an average titer of at least 2 on d, c, E, e cells in the case of anti-K, and an average titer of at least 8 on cells heterozygous for the K antigen in the case of anti-K. An antibody reagent with 2 Depending on the effective buffer amount of compatible buffers, approximately
The antibody reagent according to claim 1, which is maintained at a pH of 7.5 to 8.3. 3. The antibody reagent according to claim 2, wherein the selected antigen is D and the IgG antibody is anti-D. 4. a. taking a sample of red blood cells to be tested; b. adding an antibody reagent to the sample and mixing to form a mixture; and c. observing the mixture substantially without incubation to determine if an agglutination reaction occurs. determining whether the antibody reagent occurs or not; containing albumin of D, C,
A method for rapidly determining the presence or absence in a given red blood cell of an antigen selected from c, E, e, and K, wherein the antibody reagent is anti-D in the slide or rapid test tube test. have an average titer of at least 32 on D, c, e cells and for anti-C at least 5 on d, C, c, e or D, C, c, E, e cells. have an average titer of at least 16 in d, C, c, e cells for anti-c and d, c, E, e cells for anti-E. with an average titer of at least 8, and in the case of anti-e, d, c, E,
have an average titer of at least 2 in e cells and anti-
In the case of K, a method having an average titer of at least 8 against cells heterozygous for the K antigen. 5. The method of claim 4, wherein the antibody reagent is maintained at a pH of about 7.5-8.3. 6. The method of claim 5, wherein the antibody reagent further comprises a total protein concentration of about 6-10% by weight. 7. The method according to claim 4, 5 or 6, wherein the antigen is D antigen and the IgG antibody is anti-D. 8 a. taking a sample of red blood cells to be tested; b. adding an antibody reagent to the sample and mixing on a test slide to form a mixture; and c. observing the mixture substantially without incubation. determining whether an agglutination reaction occurs or not; the antibody reagent provides a reduced and S-alkylated IgG antibody against the selected antigen and a total protein concentration of 6-10% by weight. D, C, comprising polymerized bovine albumin.
A slide test method for rapidly determining the presence or absence in red blood cells of a given antigen selected from c, E and e, wherein the antibody reagent is applied to the slide or in a rapid test tube test. In case of D, at least for D, c, and e cells.
d for anti-C, with an average titer of 32;
have an average titer of at least 5 in C, c, e or D, C, c, E, e cells, and in the case of anti-c an average titer of at least 16 in d, C, c, e cells. and has an average titer of at least 8 in d, c, E, e cells in the case of anti-E, and at least an average titer of 8 in d, c, E, e cells in the case of anti-e. A slide assay having an average titer of 2 and, in the case of anti-K, an average titer of at least 8 for cells heterozygous for the K antigen. 9. Test slides are mixed for approximately 40 to 40 minutes prior to mixing step b.
9. The method according to claim 8, wherein the method is heated to 45°C. 10. The method according to claim 8 or 9, wherein the selected antigen is D and the IgG antibody is anti-D. 11 a. Harvesting a substantially lipid-free human antiserum containing IgG antibodies against a selected antigen; b. Bringing the pH of the antiserum to about 8.6; c. reducing the buffered antiserum with a reducing agent; d. reducing the reduced and buffered antiserum with S-
S-alkylating with an alkylating agent; e dialyzing the S-alkylated and reduced antiserum at pH 7.5-8.3; and f adding polymerized bovine albumin to reduce the total protein concentration to approximately of the antibody reagent useful for rapidly determining the presence or absence in red blood cells of an antigen selected from D, C, c, E, e and K; A manufacturing method, in which the antibody reagent is anti-D in a slide or rapid test tube test, D, c,
have an average titer of at least 32 against e cells and d,C,c,e or D,C, in the case of anti-C;
have an average titer of at least 5 in c, E, e cells;
For anti-c, have an average titer of at least 16 in d, C, c, e cells; for anti-E, have an average titer of at least 8 in d, c, E, e cells. and in the case of anti-e, an average titer of at least 2 in d, c, E, e cells, and in the case of anti-K, against cells that are heterozygous for the K antigen. A method of production having an average titer of at least 8. 12. The method according to claim 11, wherein the buffer used in step b is 2M tris-(hydroxymethyl)aminomethane. 13. Mix 1 volume of 0.04 M dithiothreitol in saline with about 4 volumes of buffered antiserum from step b and stir this mixture for at least about 30 minutes for about 20 minutes.
13. The method according to claim 11 or 12, wherein the reduction step c is carried out by standing at ~30°C. 14 Mix 1 volume of 0.4 M iodoacetamide in distilled water with about 4 volumes of reduced antiserum from step c and stir this mixture for at least about 60 minutes for about 20 to 30 minutes.
S-alkylation step d by standing at ℃
A method according to any one of claims 11 to 13 for carrying out the following. 15 About 10 volumes of 0.02M tris-(hydroxymethyl)aminomethane in 0.13M NaCl, pH about 7.5-8.3
15. The method of any of claims 11 to 14, wherein the S-alkylated antiserum from step d is dialyzed twice at about 2 to 8° C. to carry out dialysis step e. . 16 Claim 1 in which the PH is about 7.7 to 8.1
The method described in Section 5. 17. The method according to any of claims 11 to 16, wherein the antigen selected is D and the IgG antibody is anti-D.