Journal of Immunological Methods, 147 (1992) 21-25 © 1992 Elsevier Science Publishers B.Y. All rights reselVed 0022-1759/92/S05.00
21
JIM 06183
A simple and sensitive method for the determination of blood group antigens in secretions Shin Yazawa a and Hitoshi Ohkawara b II
Department of Legal Medicine, School of Medicine, GU1I1TIIJ University, 3-39-22 Showa-mtlch~ Maebashi 371, Japan, and b Scientific Crime Laboratory, Gunma Prefecture Police Headquarters, 523 Sohja-mach~ Maebashi 371, Japan (Received 3 June 1991, revised received 30 August 1991, accepted 4 September 1991)
A simple method for the determination of ABH blood group antigens in secretions has been developed. Blood group ABH specific monoclonal antibodies were covalently bound to blue dyed microspheres of acryl polymer with a diameter of 2.2 ILm. The dyed microspheres coated with anti-A, -B and -H antibodies were found to be agglutinated specifically on a plastic microtiter plate by the corresponding blood group antigens secreted in saliva. The agglutination reactions with saliva samples were also observed rapidly and conveniently in a glass capillary tube which contained the same antibody immobilized dyed microspheres. The procedure provides a simple and sensitive method for the determination of blood group antigens through the visible agglutination reaction of dyed microspheres despite the invisibility of the antigens. Key words: Microsphere; Blood group antigen; Agglutination; Blood typing; Saliva, human; Capillary tube
introduction Human blood groups are commonly determined by the hemagglutination reaction of red blood cells with blood group specific antibodies and some of them can also be determined by demonstrating the blood group substances in secretions and body fluids using the same antibodies (Race and Sanger, 1975; Salmon et aI., 1984). However, blood group antigens from the latter sources are unable to be detected directly by the visible agglutination reactions. Although immunological methods for detecting such antigens have
Correspondence to: S. Yazawa. Department of Lela! Medicine, School of Medicine, Gunma University, 3·39-22 Showa-machi, Maebashi 371, Japan (Tel.: 0272-31-7221 (ext.) 2597; Fax: 0272-32-3379).
previously been reported (Springer, 1977; AABB, 1980), a simple and sensitive procedure has not yet been demonstrated. Recently, a number of chemically synthesized microspheres have been prepared for immunological agglutination reactions, and various antigens or antibodies have been detected through the agglutination reactions with antibody- or antigen-coated microspheres, respectively (Hosaka et aI., 1983; Uchida et aI., 1984; Ikeda et aI., 1984; Koga et aI., 1990; Kondo et aI., 1990). In this report we describe a novel agglutination reaction for detecting the ABH blood group antigens in saliva that is both rapid, sensitive and suitable for routinely processing large numbers of samples. The-proCedure employs dyed microspheres to which anti-A, anti-B and anti-H monoclonal antibodies were immobilized, respectively, and the agglutination reactions with such micro'-
22
spheres were observed in a microtiter plate well and a glass capillary tube. Materials and methods Materials Blue-dyed microspheres of polystyrene (Toresphere, GN-type) with a diameter of 2.2 ,urn (Hosaka et aI., 1983) were obtained from Toray, Kanagawa, Japan. Plastic round-bottomed microtiter plates were obtained from Nunc, Roskilde, Denmark. Glass microcapillary tubes (75 mm long and 1.5 mm in diameter) were from Hirschmann Laborgerate, Germany. Saliva samples were collected from healthy volunteers without any artificial stimulus or preservative and stored at -20 0 C until use. The ABO blood group types of the samples were determined by the hemagglutination test and the hemagglutination inhibition test as described previously (Yazawa et aI., 1984). Purified anti-H (Synaff H, lot no. 2PHR-OO(3) was obtained from Chembiomed, Canada. Anti-A Oot no. BBA112D-l) and anti-B Oot no. 07911) were from Ortho, USA and Knickerbocker, Spain, respectively. Glutaraldehyde, bovine serum albumin (BSA) and tannic acid were from Sigma. All other materials used were of the highest quality commercially available. Methods Immobilization of monoclonal antibodies. Each monoclonal antibody was immobilized to blue-dyed microspheres according to the method reported previously (Hosaka et aI., 1983) with minor modifications. To 1 ml of 1% Toresphere microspheres in distilled water, 15 ,ul of 25% glutaraldehyde and 50 ,ul of 0.02 N NaOH were added and the mixture was incubated at 30 0 C for 30 min. 25 ,ul of 10% arabic gum solution was added and the mixture was incubated at 30 0 C for 15 min, and then washed three times with 0.01 M phosphate buffered saline, pH 7.0 (PBS). The microspheres suspended in 200 ,ul of PBS were mixed with 200 ,ul of PBS solution containing 0.1 % of tannic acid and the mixture was incubated at 30 0 C for 15 min. After washing three times with PBS, the beads were resus-
pended in 200 ,ul of PBS. To these activated microspheres, 200 ,ul of antibody diluted with PBS containing 0.1 % of BSA was added and the mixture was incubated at 30 0 C for 60 min. After washing two times with PBS, the beads were suspended in 0.15 M lactate-borate buffer (pH 7.0) and then suspended in 800 ,ul of the same buffer containing 0.1 M triethanolamine and the mixture was incubated at 30 0 C for 60 min. The microspheres were washed again two times with PBS and suspended and stored in 1 ml of PBS. The suspension of the microspheres coated with three different antibodies was used for the agglutination reaction both in a plastic microtiter plate well and in a glass microcapillary tube as described below. Agglutination reaction with antibody-immobilized microspheres. The agglutinability of the antibody-immobilized microspheres was examined in a plastic microtiter plate well by hemagglutination reaction with corresponding types of red blood cells: one drop of the microspheres prepared as described above was placed in each well of the plate and mixed with one drop of 2% suspension of respective red blood cells. The hemagglutination pattern was determined after standing for 15 min at room temperature with gentle shaking. 25 ,ul of saliva samples to be tested was diluted serially in a two-fold dilution with PBS containing 0.1 % BSA using a microtiter plate. Then an equal volume of the suspension of the microspheres was added to each well and the plate was placed on a shaker for 15 min. The agglutination pattern of microspheres was observed and the reciprocal of the highest dilution that produced visible agglutination reaction was determined (maximum agglutination titer). 20 ,ul of the suspension of the microspheres was also added to a glass microcapillary tube (approximately 20 mm long), then lyophilized and stored at - 20 0 C until use. The capillary was placed into the sample solution until it was allowed to flow into the capillary tube up to about 20 mm. After the lyophilized microspheres in the tube were dissolved completely with the sample solution, the tube was placed on the horizontal plate and then the agglutination pattern was observed.
23
Results and discussion
TABLE I
The microspheres coated with anti-A and antiB antibody were found to react specifically with A and B type red blood cells, respectively, and large solid agglutinates were produced in both reaction s. The microspheres coated with anti-H antibody were observed to react not only with 0 type red blood cells but also with A and B type ones, although the agglutinates with A and B type red blood cells were smaller than those with 0 type ones (Table I). The same agglutination patterns were also observed in the direct hemagglutination reactions of A, Band 0 red blood cells with anti-H antibody (data not shown). Fig. 1 shows the agglutination pattern of serially diluted saliva samples from blood type A with the microspheres coated with anti-A antibody on a microtiter plate. Clear agglutinates were observed up to 1/ 65,536 dilution of the sample. The agglutination reaction of saliva samples from various blood types and secretor status was examined with thc microspheres coated with anti-A, -B and -H antibodies and the maximum agglutination
n
HEMAGGLUTINA nON REACTIONS OF RED BLOOD CELLS WITH ANTI-A, ANTI-B AND ANTI-H ANTIBODY IMMOBILIZED MICROSPHERES Antibody immobilized on microspheres Anti-A Anti-B Anti-H
7
8
9
10
11
12
A
B
0
++
0
0 0
0
+
++ +
++
Key: + + ,one solid agglutinate; + , several large agglutinates.
titers of each sample were also determined at the same time (Table 11). The microspheres with antiA antibody demonstrated agglutination with saliva samples from blood type A and AB specifically, irrespective of their secretor status. No agglutination reaction was observed in saliva samples from blood type 0 and B. The microspheres with anti-B antibody agglutinated only with samples from type Band AB and no agglutination reaction was found with the others. On the other hand, the microspheres with anti-H antibody agglutinated with saliva samples not only from type 0 but
Di lution, 1 : 2
=
Red blood cells
n
13
14
15
16
17
18
Fig. I. Agglutination patterns of serially diluted saliva samples from blood A type with the mierospheres coated with anti-A antibody in plastic microliter wells.
TABLE II AGGLUTINATION REACTIONS OF SALIVA SAMPLES WITH ANTI-A, ANTI-B AND ANTI-H IMMOBILIZED MICROSPHERES Maximum agglutination titer with
Saliva Blood type
Secretor status
n
Anti-A
Anli-B
Anli-H
A A B
Sec. Non-sec. Sec. Non-sec. Sec. Non-sec. Sec. Non-sec.
6 6 6 6 6 3
16.384-262,144 64- 16.384 0 0 4.096- 65.538 64- 4,096 0 0
0 0 65,536-262.144 256- 65,536 4.096-262,144 256- 4,096 0 0
1,024- 16,384 4 01,024-262,144 256 01,024- 65,538 4 04,096- 262, 144 256 16-
B
AB AB
0 0
6
6
ant i -A
-
ont i -B
ont l- H
A type
-
B type
o type
Fig. 2. Agglutination patterns of saliva samples from A, Band 0 types with the microspheres coated with anti-A, -B and -H antihodies in glass microcapiliary tubes.
from the other types, as demonstrated in the hemagglutination reactions with A, Band 0 red blood cells. These results show that ABH antigens secreted in saliva can be detected through the agglutination reaction with corresponding antibody immobilized microspheres, and that the specificity of reaction coincides with that of the antibody immobilized to the microspheres. It was of particular interest that saliva samples from non-secretor individuals of A, B, 0 and AB types tested in this study showed distinct agglutination reactions with respective antibody immobilized microspheres. Saliva samples of non-secretor individuals often contain a small amount of ABH antigens, but it is hardly possible to determine their blood types using the conventional methods such as hemagglutination inhibition test and absorption test (Springer, 1977; AABB, 1980). In fact, some saliva samples from non-secretor individuals tested in this study showed weak hemagglutination inhibition titers (data not shown). ABH
non-secretors are assumed to be lack for Se gene which controls the synthesis of ABH antigens in the secretor system. The presence of ABH antigens in saliva from non-secretor individuals has not yet been interpreted genetically, but it has been proposed that the presence of different genes from Se which produce enzymes related to the synthesis of ABH antigens in secretor systems (Salmon et aI., 1984). The agglutinability depending on the secretor status was observed in all the samples tested. The maximum agglutination titers of the samples from secretors were found to be higher than those from non-secretors. Most of the samples from non-secretors showed strong agglutination reactions in this study, but two of the ten (one type A and one type AB) did not show agglutination with the anti-H immobilized microspheres. It might depend on the amount of H substance in their saliva samples which could react with the anti-H antibody immobilized to the microspheres. When the agglutination reactions were per-
25
formed with an excess of antigen, a prozone effect occurred and in some cases false negative agglutination reactions were observed (data not shown). Therefore, the dilution of samples may be helpful to prevent the false negative reaction and to get beyond the prozone effect. The agglutination reaction using the microtiter plate is applicable in particular to determine the maximum agglutination titer of the sample. Although the reactions were demonstrated to be sensitive and specific, the agglutinates with the microspheres tended to adhere to well walls of the plastic microtiter plate and thus in some cases it was difficult to read the weak agglutination reaction. Furthermore, in many cases, it must be required to determine the presence of a specified antigen as soon as possible. Fig. 2 shows the agglutination pattern of diluted saliva samples from A, Band 0 types with the microspheres coated with anti-A, -B and -H antibodies, respectively in glass microcapillary tubes. It involves the formation of visible agglutinates inside capillary tubes, and in almost all the cases, the agglutination reactions occurred quickly (within 0.5 min). The superiority of this capillary method over the microtiter plate method described above was its convenience and rapidity for the determination of agglutination. This capillary method seems to have a greater potential versatility than do the other agglutination procedures. Therefore, the use of glass microcapillary tubes is advantageous for the examination of a large number of samples at a time. In conclusion, the advantage of the method described in this study is that the presence of antigen can be determined easily and rapidly through the visible agglutination reaction with antibodies bound to dyed microspheres, even though the antigen to be tested is invisible and in trace amount. Furthermore, the use of a capillary tube for the agglutination reaction makes it possible to produce agglutinates more quickly and clearly. In addition to the work described here, we have successfully extended the use of the dyed microspheres to determine the presence of some
other antigens for diagnostic or forensic purposes (results to be published elsewhere). Acknowledgements
We thank Mr. I. Terasawa and Dr. S. Hosaka, Toray Research Center, and Toray Basic Research Laboratories, Kamakura, Japan, for the supply of Toresphere beads and valuable discussions. Supported in part by Grants-in-Aid for Scientific Research *6257061 and *01010001 from the Ministry of Education, Science and Culture, Japan. References American Association of Blood Banks (1981) In: T.K Widsmann et al. (Eds.), Technical Manual of the American Association of Blood Banks. J.B. Lippincott, Philadelphia, PA, p. 392. Hosaka, S., Murao, Y., Masuko, S. and Miura, K (1983) Preparation of microspheres of poly(glycocidylmethacrylate) and its derivatives as carriers for immobilized proteins. Immunol. Commun., 12, 509. Ikeda, M., Fujino, R., Matui, T., Yoshida, T., Kodama, H. and Iwai, J. (1984) A new agglutination test for serum antibodies to adult T-cell leukemia virus. Gann 75, 845. Koga, T., Senpaku, H., Nakashima, K, Ishihara, Y. and Nishihara, T. (1990) Monoclonal antibody-coated latex agglutination assay for identification of Actinobacillus actinomycetem comitans. Int. J. Med. Microbiol. 274,91. Kondo, A., Komano, T., Itoh, F. and Higashitani, K (1990) Immunological agglutination kinetics of latex particles with physically adsorbed antigens. J. Immunol. Methods 135, 111.
Race, R.R. and Sanger, R. (1975) Blood Groups in Man, 6th edn. Backwell, Oxford, p. 229. Salmon, C., Cartron, J.-P. and Rouger, P. (1984) The Human Blood Groups. Masson Publishing, p. 94, p. 158. Springer, G.F. (1977) In: C.A. Williams and M.W. Chase (Eds.), Methods in Immunology and Immunochemistry, Vol. 4, Inhibition of Hemagglutination. Academic Press, New York, p. 67. Uchida, T., Hosaka, S. and Murao, Y. (1984) Complement activity by polymer binding IgG. Biomaterials 5, 281. Yazawa, S., Furukawa, K and Kochibe, N. (1984) Isolation of fucosyl glycoproteins from human erythrocyte membranes by affinity chromatography using Aleuria aurantia lectin. J. Biochem. 96, 1737.
21
JIM 06183
A simple and sensitive method for the determination of blood group antigens in secretions Shin Yazawa a and Hitoshi Ohkawara b II
Department of Legal Medicine, School of Medicine, GU1I1TIIJ University, 3-39-22 Showa-mtlch~ Maebashi 371, Japan, and b Scientific Crime Laboratory, Gunma Prefecture Police Headquarters, 523 Sohja-mach~ Maebashi 371, Japan (Received 3 June 1991, revised received 30 August 1991, accepted 4 September 1991)
A simple method for the determination of ABH blood group antigens in secretions has been developed. Blood group ABH specific monoclonal antibodies were covalently bound to blue dyed microspheres of acryl polymer with a diameter of 2.2 ILm. The dyed microspheres coated with anti-A, -B and -H antibodies were found to be agglutinated specifically on a plastic microtiter plate by the corresponding blood group antigens secreted in saliva. The agglutination reactions with saliva samples were also observed rapidly and conveniently in a glass capillary tube which contained the same antibody immobilized dyed microspheres. The procedure provides a simple and sensitive method for the determination of blood group antigens through the visible agglutination reaction of dyed microspheres despite the invisibility of the antigens. Key words: Microsphere; Blood group antigen; Agglutination; Blood typing; Saliva, human; Capillary tube
introduction Human blood groups are commonly determined by the hemagglutination reaction of red blood cells with blood group specific antibodies and some of them can also be determined by demonstrating the blood group substances in secretions and body fluids using the same antibodies (Race and Sanger, 1975; Salmon et aI., 1984). However, blood group antigens from the latter sources are unable to be detected directly by the visible agglutination reactions. Although immunological methods for detecting such antigens have
Correspondence to: S. Yazawa. Department of Lela! Medicine, School of Medicine, Gunma University, 3·39-22 Showa-machi, Maebashi 371, Japan (Tel.: 0272-31-7221 (ext.) 2597; Fax: 0272-32-3379).
previously been reported (Springer, 1977; AABB, 1980), a simple and sensitive procedure has not yet been demonstrated. Recently, a number of chemically synthesized microspheres have been prepared for immunological agglutination reactions, and various antigens or antibodies have been detected through the agglutination reactions with antibody- or antigen-coated microspheres, respectively (Hosaka et aI., 1983; Uchida et aI., 1984; Ikeda et aI., 1984; Koga et aI., 1990; Kondo et aI., 1990). In this report we describe a novel agglutination reaction for detecting the ABH blood group antigens in saliva that is both rapid, sensitive and suitable for routinely processing large numbers of samples. The-proCedure employs dyed microspheres to which anti-A, anti-B and anti-H monoclonal antibodies were immobilized, respectively, and the agglutination reactions with such micro'-
22
spheres were observed in a microtiter plate well and a glass capillary tube. Materials and methods Materials Blue-dyed microspheres of polystyrene (Toresphere, GN-type) with a diameter of 2.2 ,urn (Hosaka et aI., 1983) were obtained from Toray, Kanagawa, Japan. Plastic round-bottomed microtiter plates were obtained from Nunc, Roskilde, Denmark. Glass microcapillary tubes (75 mm long and 1.5 mm in diameter) were from Hirschmann Laborgerate, Germany. Saliva samples were collected from healthy volunteers without any artificial stimulus or preservative and stored at -20 0 C until use. The ABO blood group types of the samples were determined by the hemagglutination test and the hemagglutination inhibition test as described previously (Yazawa et aI., 1984). Purified anti-H (Synaff H, lot no. 2PHR-OO(3) was obtained from Chembiomed, Canada. Anti-A Oot no. BBA112D-l) and anti-B Oot no. 07911) were from Ortho, USA and Knickerbocker, Spain, respectively. Glutaraldehyde, bovine serum albumin (BSA) and tannic acid were from Sigma. All other materials used were of the highest quality commercially available. Methods Immobilization of monoclonal antibodies. Each monoclonal antibody was immobilized to blue-dyed microspheres according to the method reported previously (Hosaka et aI., 1983) with minor modifications. To 1 ml of 1% Toresphere microspheres in distilled water, 15 ,ul of 25% glutaraldehyde and 50 ,ul of 0.02 N NaOH were added and the mixture was incubated at 30 0 C for 30 min. 25 ,ul of 10% arabic gum solution was added and the mixture was incubated at 30 0 C for 15 min, and then washed three times with 0.01 M phosphate buffered saline, pH 7.0 (PBS). The microspheres suspended in 200 ,ul of PBS were mixed with 200 ,ul of PBS solution containing 0.1 % of tannic acid and the mixture was incubated at 30 0 C for 15 min. After washing three times with PBS, the beads were resus-
pended in 200 ,ul of PBS. To these activated microspheres, 200 ,ul of antibody diluted with PBS containing 0.1 % of BSA was added and the mixture was incubated at 30 0 C for 60 min. After washing two times with PBS, the beads were suspended in 0.15 M lactate-borate buffer (pH 7.0) and then suspended in 800 ,ul of the same buffer containing 0.1 M triethanolamine and the mixture was incubated at 30 0 C for 60 min. The microspheres were washed again two times with PBS and suspended and stored in 1 ml of PBS. The suspension of the microspheres coated with three different antibodies was used for the agglutination reaction both in a plastic microtiter plate well and in a glass microcapillary tube as described below. Agglutination reaction with antibody-immobilized microspheres. The agglutinability of the antibody-immobilized microspheres was examined in a plastic microtiter plate well by hemagglutination reaction with corresponding types of red blood cells: one drop of the microspheres prepared as described above was placed in each well of the plate and mixed with one drop of 2% suspension of respective red blood cells. The hemagglutination pattern was determined after standing for 15 min at room temperature with gentle shaking. 25 ,ul of saliva samples to be tested was diluted serially in a two-fold dilution with PBS containing 0.1 % BSA using a microtiter plate. Then an equal volume of the suspension of the microspheres was added to each well and the plate was placed on a shaker for 15 min. The agglutination pattern of microspheres was observed and the reciprocal of the highest dilution that produced visible agglutination reaction was determined (maximum agglutination titer). 20 ,ul of the suspension of the microspheres was also added to a glass microcapillary tube (approximately 20 mm long), then lyophilized and stored at - 20 0 C until use. The capillary was placed into the sample solution until it was allowed to flow into the capillary tube up to about 20 mm. After the lyophilized microspheres in the tube were dissolved completely with the sample solution, the tube was placed on the horizontal plate and then the agglutination pattern was observed.
23
Results and discussion
TABLE I
The microspheres coated with anti-A and antiB antibody were found to react specifically with A and B type red blood cells, respectively, and large solid agglutinates were produced in both reaction s. The microspheres coated with anti-H antibody were observed to react not only with 0 type red blood cells but also with A and B type ones, although the agglutinates with A and B type red blood cells were smaller than those with 0 type ones (Table I). The same agglutination patterns were also observed in the direct hemagglutination reactions of A, Band 0 red blood cells with anti-H antibody (data not shown). Fig. 1 shows the agglutination pattern of serially diluted saliva samples from blood type A with the microspheres coated with anti-A antibody on a microtiter plate. Clear agglutinates were observed up to 1/ 65,536 dilution of the sample. The agglutination reaction of saliva samples from various blood types and secretor status was examined with thc microspheres coated with anti-A, -B and -H antibodies and the maximum agglutination
n
HEMAGGLUTINA nON REACTIONS OF RED BLOOD CELLS WITH ANTI-A, ANTI-B AND ANTI-H ANTIBODY IMMOBILIZED MICROSPHERES Antibody immobilized on microspheres Anti-A Anti-B Anti-H
7
8
9
10
11
12
A
B
0
++
0
0 0
0
+
++ +
++
Key: + + ,one solid agglutinate; + , several large agglutinates.
titers of each sample were also determined at the same time (Table 11). The microspheres with antiA antibody demonstrated agglutination with saliva samples from blood type A and AB specifically, irrespective of their secretor status. No agglutination reaction was observed in saliva samples from blood type 0 and B. The microspheres with anti-B antibody agglutinated only with samples from type Band AB and no agglutination reaction was found with the others. On the other hand, the microspheres with anti-H antibody agglutinated with saliva samples not only from type 0 but
Di lution, 1 : 2
=
Red blood cells
n
13
14
15
16
17
18
Fig. I. Agglutination patterns of serially diluted saliva samples from blood A type with the mierospheres coated with anti-A antibody in plastic microliter wells.
TABLE II AGGLUTINATION REACTIONS OF SALIVA SAMPLES WITH ANTI-A, ANTI-B AND ANTI-H IMMOBILIZED MICROSPHERES Maximum agglutination titer with
Saliva Blood type
Secretor status
n
Anti-A
Anli-B
Anli-H
A A B
Sec. Non-sec. Sec. Non-sec. Sec. Non-sec. Sec. Non-sec.
6 6 6 6 6 3
16.384-262,144 64- 16.384 0 0 4.096- 65.538 64- 4,096 0 0
0 0 65,536-262.144 256- 65,536 4.096-262,144 256- 4,096 0 0
1,024- 16,384 4 01,024-262,144 256 01,024- 65,538 4 04,096- 262, 144 256 16-
B
AB AB
0 0
6
6
ant i -A
-
ont i -B
ont l- H
A type
-
B type
o type
Fig. 2. Agglutination patterns of saliva samples from A, Band 0 types with the microspheres coated with anti-A, -B and -H antihodies in glass microcapiliary tubes.
from the other types, as demonstrated in the hemagglutination reactions with A, Band 0 red blood cells. These results show that ABH antigens secreted in saliva can be detected through the agglutination reaction with corresponding antibody immobilized microspheres, and that the specificity of reaction coincides with that of the antibody immobilized to the microspheres. It was of particular interest that saliva samples from non-secretor individuals of A, B, 0 and AB types tested in this study showed distinct agglutination reactions with respective antibody immobilized microspheres. Saliva samples of non-secretor individuals often contain a small amount of ABH antigens, but it is hardly possible to determine their blood types using the conventional methods such as hemagglutination inhibition test and absorption test (Springer, 1977; AABB, 1980). In fact, some saliva samples from non-secretor individuals tested in this study showed weak hemagglutination inhibition titers (data not shown). ABH
non-secretors are assumed to be lack for Se gene which controls the synthesis of ABH antigens in the secretor system. The presence of ABH antigens in saliva from non-secretor individuals has not yet been interpreted genetically, but it has been proposed that the presence of different genes from Se which produce enzymes related to the synthesis of ABH antigens in secretor systems (Salmon et aI., 1984). The agglutinability depending on the secretor status was observed in all the samples tested. The maximum agglutination titers of the samples from secretors were found to be higher than those from non-secretors. Most of the samples from non-secretors showed strong agglutination reactions in this study, but two of the ten (one type A and one type AB) did not show agglutination with the anti-H immobilized microspheres. It might depend on the amount of H substance in their saliva samples which could react with the anti-H antibody immobilized to the microspheres. When the agglutination reactions were per-
25
formed with an excess of antigen, a prozone effect occurred and in some cases false negative agglutination reactions were observed (data not shown). Therefore, the dilution of samples may be helpful to prevent the false negative reaction and to get beyond the prozone effect. The agglutination reaction using the microtiter plate is applicable in particular to determine the maximum agglutination titer of the sample. Although the reactions were demonstrated to be sensitive and specific, the agglutinates with the microspheres tended to adhere to well walls of the plastic microtiter plate and thus in some cases it was difficult to read the weak agglutination reaction. Furthermore, in many cases, it must be required to determine the presence of a specified antigen as soon as possible. Fig. 2 shows the agglutination pattern of diluted saliva samples from A, Band 0 types with the microspheres coated with anti-A, -B and -H antibodies, respectively in glass microcapillary tubes. It involves the formation of visible agglutinates inside capillary tubes, and in almost all the cases, the agglutination reactions occurred quickly (within 0.5 min). The superiority of this capillary method over the microtiter plate method described above was its convenience and rapidity for the determination of agglutination. This capillary method seems to have a greater potential versatility than do the other agglutination procedures. Therefore, the use of glass microcapillary tubes is advantageous for the examination of a large number of samples at a time. In conclusion, the advantage of the method described in this study is that the presence of antigen can be determined easily and rapidly through the visible agglutination reaction with antibodies bound to dyed microspheres, even though the antigen to be tested is invisible and in trace amount. Furthermore, the use of a capillary tube for the agglutination reaction makes it possible to produce agglutinates more quickly and clearly. In addition to the work described here, we have successfully extended the use of the dyed microspheres to determine the presence of some
other antigens for diagnostic or forensic purposes (results to be published elsewhere). Acknowledgements
We thank Mr. I. Terasawa and Dr. S. Hosaka, Toray Research Center, and Toray Basic Research Laboratories, Kamakura, Japan, for the supply of Toresphere beads and valuable discussions. Supported in part by Grants-in-Aid for Scientific Research *6257061 and *01010001 from the Ministry of Education, Science and Culture, Japan. References American Association of Blood Banks (1981) In: T.K Widsmann et al. (Eds.), Technical Manual of the American Association of Blood Banks. J.B. Lippincott, Philadelphia, PA, p. 392. Hosaka, S., Murao, Y., Masuko, S. and Miura, K (1983) Preparation of microspheres of poly(glycocidylmethacrylate) and its derivatives as carriers for immobilized proteins. Immunol. Commun., 12, 509. Ikeda, M., Fujino, R., Matui, T., Yoshida, T., Kodama, H. and Iwai, J. (1984) A new agglutination test for serum antibodies to adult T-cell leukemia virus. Gann 75, 845. Koga, T., Senpaku, H., Nakashima, K, Ishihara, Y. and Nishihara, T. (1990) Monoclonal antibody-coated latex agglutination assay for identification of Actinobacillus actinomycetem comitans. Int. J. Med. Microbiol. 274,91. Kondo, A., Komano, T., Itoh, F. and Higashitani, K (1990) Immunological agglutination kinetics of latex particles with physically adsorbed antigens. J. Immunol. Methods 135, 111.
Race, R.R. and Sanger, R. (1975) Blood Groups in Man, 6th edn. Backwell, Oxford, p. 229. Salmon, C., Cartron, J.-P. and Rouger, P. (1984) The Human Blood Groups. Masson Publishing, p. 94, p. 158. Springer, G.F. (1977) In: C.A. Williams and M.W. Chase (Eds.), Methods in Immunology and Immunochemistry, Vol. 4, Inhibition of Hemagglutination. Academic Press, New York, p. 67. Uchida, T., Hosaka, S. and Murao, Y. (1984) Complement activity by polymer binding IgG. Biomaterials 5, 281. Yazawa, S., Furukawa, K and Kochibe, N. (1984) Isolation of fucosyl glycoproteins from human erythrocyte membranes by affinity chromatography using Aleuria aurantia lectin. J. Biochem. 96, 1737.
