Tohoku J. exp. Med., 1967, 91, 331-348

Agglutinin-adsorbing Latex Method for Blood-grouping of Blood-stains and Other Specimens

Tsuyoshi Tajima

Department of Forensic Medicine(Prof. S. Murakami), Hirosaki UniversitySchool of Medicine,Hirosaki

A new serological technique was introduced into blood grouping of blood stains and other test specimens. In this method, latex-adsorbed agglutinin was prepared by mixing polyvinyl-toluene latex particles of 2.015ƒÊ in diameter with antiserum. Accurate blood grouping was possible with the agglutinin-adsorbing latex particles directly even on a small piece of blood or saliva-stained fibrils in only about ten minutes. The described technique was recommended for wide use not only in blood group determinations, but also in general serological tests.

For blood-grouping of blood-stains, secretions or tissues, the agglutinin inhibition test has been widely used. This test, however, is nowadays regarded as

an obsolete one, because not only it requires a large quantity of material and troublesome techniques, but also it is of low sensitivity. Recently, the adsorption-elution method by Kind,1 the so-called mixed

method by Coombs and Dodd2 and its radical modification, the

double combination method by Akaishi, 3 have been worked out and have con

tributed much to meeting the requirement of practical blood-grouping tests.

Even by these methods, however, the - reaction can be ascertained only indirectly. That is, Kind's adsorption-elution method utilizes

antigen-antibody complex and is able to detect with indicator cells free antibody

eluted from the complex at high temperature. Coombs and Dodd's mixed agglu

tination method and Akaishi's double combination method prepare an antigen antibody complex at the first step and agglutinate indicator cells on the com

bined antibody at the second step.

On the other hand, the fluorescent antibody method by Coons et al.4 and the radio-isotope labeled antibody method by Pressman et al.5 are based on direct

reactions. However, these methods require not only special apparatuses and

materials, but also complicated operation and high-level technique. There

fore, many points must still be improved to establish a method for routine use

in the study of blood groups. The author has devised a new method, 'agglutinin-adsorbing latex method',

for blood-grouping. This method consists in the use of agglutinin adsorbed to

Received for publication, December 23, 1966. 331 332 T. Tajima

latex particles of 2.051,u in diameter which agglutinate directly on blood-stains

and other test specimens. This method has such an advantage as to make possible a direct observation of antigen-antibody reaction and furthermore to admit of an

accurate blood grouping in a short time through a simple operation.

MATERIALS AND METHODS

Materials

Latex particles. Ten per cent suspension of polyvinyltoluene latex particles

of 2.051,a with 0.081,u standard deviation in diameter, which was supplied from

Dow Chemical Company, U.S.A., was used.

Antisera. Rabbit anti-A serum immunized with human group A cells

and chicken anti-B serum immunized with human group B cells were used. Non specific were eliminated by adsorption with human group B and A cells

respectively. And each agglutinin preparation had a titer higher than 210 to the so-called standard cells by Furusawa.6 Normal human sera of a higher titer can be

also used.

Saturated ammonium sulfate solution. This was used for salting out antisera and obtaining ƒÁ-globulin.

Buffered saline solution. A small quantity of 0.5 M, pH 8.5 and 0.1 M, pH 8.5

veronal buffered solutions of 0.85% sodium chloride was used.

Preparation of agglutinin-adsorbing latex suspension

Preparation of y-globulin solution. Anti-sera were salted out three times with 1/3 saturated ammonium sulfate, and then dialyzed through cellophane at 4°C

against 0.85% buffered saline solution, which was changed several times until no more NH4+ could be detected with Nessler's reagent. In addition, it was

ascertained by electrophoresis with cellulose-acetate membrane that albumin was

completely eliminated. Natural sediments produced in the process of dialysis

were eliminated by centrifugation at 10,000 rpm for 10 minutes at 4°C, and

the supernatant was used as a y-globulin solution. This solution was kept in a small test tube in 0.8 ml and stored in a deep freezer.

Adsorption of ƒÁ-globulin onto latex. A volume of 0.2 ml of the 0.5 M, pH

8.5 veronal buffered saline was added to 0.8 ml of 1.0% ƒÁ-globulin solution, and

then 5 drops of 10% suspension of polyvinyl toluene latex particles of 2.051,u in size were added. The mixture was agitated at room temperature for 60

minutes with a magnetic stirrer. The mixture was diluted tenfold with 0.1 M, pH

8.5 veronal buffered saline and centrifuged twice for 5 minutes at 10,000 rpm to eliminate the remaining antibody. Then the sediment was strongly agitated to resuspend homogeneously, and centrifuged for 5 minutes at 2 ,000 rpm to eliminate natural agglomerates. After that, the supernatant was used as an agglutinin-adsorbing latex suspension. Agglutinin-adsorbing Latex for Blood-grouping 333

Blood-grouping of blood-stains and other specimens

In the case of stains of blood or secretions on fibers, the latter were cut into

segments of several millimeters in length. In the case of stains on other materials, stains were transferred onto cotton fibers and dried. After fixation of the

stains with pure methanol for five minutes, the fiber was teased apart into fibrils.

Several fibrils were put into small test tubes, and 0.2-0.5 ml of the anti-A and anti

- B agglutinin-adsorbing latex suspension was added to the tubes, respectively. After centrifugation for 10 minutes at 1,000 rpm, the fibrils were taken out and

washed gently in the 0.1 M, pH 8.5 buffered saline to eliminate free latex parti

cles as much as possible. Then, the agglutination of latex on the fibrils was

examined under a microscope (X 200-400). A-group was demonstrated by the agglutination of anti-A agglutinin-adsorbing latex, B-group by anti-B, AB-group

by both anti-A and anti-B, and 0-group by absent agglutination by both.

RESULTS

1) The adsorption of agglutinin onto latex

It is most important to ascertain that agglutinin is adsorbed onto latex.

For demonstration of protein on such fine grains as blood cells, Moreschi's or

Coombs' method and its modifications are generally used. In the present experi ment, the guinea pig anti-rabbit globulin serum was used for the latex adsorbed

rabbit anti-A serum, and the guinea pig anti-chicken globulin serum for the

latex adsorbing chicken anti-B serum. An obvious agglutination was observed

with the agglutinin-adsorbing latex (Fig. 1), but not with the non-agglutinin-adsorb ing latex (Fig. 2). The agglutination was also observed when the agglutinin

adsorbing latex was added to the corresponding blood cell suspension (Fig. 3),

while no agglutination was observed in the case of the non-corresponding one

(Fig. 4). Accordingly it was ascertained that the agglutinin was adsorbed onto the latex.

2) Various preparations of latex, pH and ionic strength

By comparative experiments with 0.81,a polystyrene latex, 2.051,a polyvinyl

toluene latex and 6-14ƒÊ styrene-divinylbenzene copolymer latex supplied by Dow

Chemical Company (U.S.A.), it was found that 2.501ƒÊ polyvinyl toluene latex was most satisfactory, as shown in Table 1. Polystyrene latex of 0.81ƒÊ in diameter

has been used in the serological field since Singer and Plotz.7 However, this

particle is too small in size for the direct observation on fibrils and other materials, and free latex particles remain on the fibrils even after careful washing. Styrene

- divinylbenzene copolymer latex of 6-14,a in diameter was satisfactory in size for

microscopical observation, but blood-grouping was impossible as shown in Table 1.

Experiments were repeated under various conditions, and consistent results were

always obtained. The weight of spherical bodies increases in proportion to the 334 T. Tajima

TABLE 1. Results of blood-grouping with various latexes

Blood-stains: AB group, within one week. Anti-serum: Animal immune serum, titer 1,024.

third power of their diameter, while the surface area increases proportionally to the second power of their diameter. When the size of the particles increases until the ratio of weight to surface area exceeds a certain limit, the latex particles lose the ability of agglutination. After a series of experiments, it was found that polyvinyl-toluene latex of 2.051ƒÊ in diameter was almost satisfactory for blood-grouping except for a little inconvenience in microscopical observation. However, better results will be obtained in future if latex particles with larger diameter and lower specific weight become available. Oreskes and Singer8 reported that the stability of the polystyrene latex suspension was influenced by ionic strength and pH; namely, polystyrene latex particles coated with protein were most unstable at a pH near the LE.P. of the protein, and protein-free latex particles were influenced only by ionic strength, agglutinating spontaneously in the range of pH 4-9 when the ionic strength was over ƒÊ=0.30 and in the range of pH 1-3 when it was over ƒÊ=0.01. With regard to these problems on the stability, several experiments were carried out. Five milliliters of the buffer solutions of

0.1 M, 0.5 M and 1.0 M in the range of pH 4-11 were mixed with 5 drops of 10% suspension of 2.051ƒÊ polyvinyl toluene latex in a small test tube. The mixture was then strongly agitated and left at room temperature for 5 hours, and agglutination was observed. As shown in Table 2, the buffer solution of 0.1 M in the range of pH 7-9 was most stabilizing, whereas in the other solutions sponta Agglutinin-adsorbing Latex for Blood-grouping 335

TABLE2. Spontaneous agglutination of polyvinyl-toluene latex suspension in various pHs and ionic strengths

•}: Agglutination observed only by microscopy. +: Agglutination observed by agglutinoscopy. ??: Definite agglutination. ?? Strong agglomeration.

neous agglutination was strong. Then, the three kinds of agglutinin-adsorbing latex, which were suspended in 0.1 M pH 8.5 buffer solutions such as veronal, phosphate and borate, were compared. The result revealed that the latex suspension in the veronal buffer was most stable. As mentioned above, 0.1 M pH 8.5 veronal buffer solution was most satis factory.

3) Antisera

In antigen-antibody reaction, the optimum ratio between antigen and antibody is important. However, when a small amount of antiserum protein adsorbed onto latex reacts with antigen, it is assumed that the higher the titer is, the better is the result. As shown in Table 3, the antisera with titers over 1,024 showed better results, and the results were obviously influenced also by the antigen titer. In the case of immune sera from animals, non-specific agglutinin should be completely eliminated.

Singer et al.9 showed that protein with lower molecular weight such as albumin was more strongly adsorbed onto the latex particles than y-globulin. Donaldson and Pennington10 reported that the adsorption of human albumin onto latex particles was inhibited by normal rabbit whole sera. Therefore, the antisera used in the author's method must have higher titer and contain possibly pure agglutinin.

4) The concentration of y-globulin and the time of adsorption onto latex

Examining the protein concentration of the solution before and after adsorp tion onto polystyrene latex, Oreskes and Singers reported that the amount of protein adsorbed onto latex particles was expressed as follows: r/A=K(N-r),

r: amount of protein adsorbed onto latex,

A : amount of protein at the equilibrium concentration, 336 T. Tajima

TABLE 3. Results of agglutinin-adsorbing latex method with various antisera

a) ƒÁ-Globulin fraction of antiserum

b) Whole antiserum

Blood-stains: AB group, within one week.

K: the binding constant, and N: the maximum amount of protein that can be adsorbed onto latex . They explained that the protein formed multilayers with different constants on the surface of latex particles, and in the case of the latex particles of 2,200 A, i n diameter, the mean value of N was 0.170 mg protein/mg latex, and in the case of those of 8,020 A in diameter it was 0.045 mg protein/mg latex. Therefore, for an increase of r, A should be increased , but the amount of protein over a certain extent is useless because N is limited. Agglutinin-adsorbing Latex for Blood-grouping 337

TABLE5. Results of agglutinin-adsorbing latex method in reference to time of adsorption onto latex

Blood-stains: AB group, within 10 days. Antiserum: Animal immune serum, titer 1,024.

TABLE6. Results of agglutinin-adsorbing latex method in reference to centrifugation for blood-grouping

Blood-stains: A group, within one week. Antiserum: Animal serum, titer 1,024.

Centrifugation: 1,000 rpm.

Adjusting ƒÁ-globulin of the same antiserum at 4.0 g/100 ml, 1.0 g/100 ml,

0.5 g/100 ml and 0.1 g/100 ml, the author prepared the agglutinin-adsorbing latex suspension respectively, and made blood-grouping of the same blood-stains. As shown in Table 4, a satisfactory result was obtained when ƒÁ-globulin concentra tion was over 1.0g/100 ml. Therefore, the 1.0g/100 ml ƒÁ-globulin solution was used for the agglutinin-adsorbing latex suspension. 338 T. Tajima

Then, the time for adsorption of ƒÁ-globulin onto latex was examined. As shown in Table 5, a satisfactory result was obtained when ƒÁ-globulin was adsorbed for 1 hour or longer. As for the temperature, no particular difference was observed between 37•Ž and room temperature.

5) Washing and natural agglomerates

The elimination of surplus antibody is essential in the author's method. The mixture of antiserum and latex was filtrated through a millipore filter with pores of 1.2,u in diameter under a negative pressure. But, only a small amount of filtrate was obtained. So, the filtration is not recommended. On the other hand, mixing ten volumes of the 0.1 M, pH 8.5 buffered saline solution with one volume of antiserum and latex, the mixture was centrifuged twice at 10,000 rpm for

5 minutes at 4•Ž. In this case, some natural agglomerates were seen in the sediment. These agglomerates were eliminated by centrifugation at 2,000 rpm for 5 minutes. The supernatant contained a few agglomerates, but they did not lead to misgrouping.

6) The time for blood-grouping

The agglutinin-adsorbing latex particles are easily agglutinated by centrifuga tion. So, the mixture of blood stained fibrils and the agglutinin-adsorbing latex suspension was centrifuged at 1,000 rpm for various lengths of time. As shown in Table 6, centrifugation for 10 minutes gave a satisfactory result without non specific reaction. Centrifugation for 20 minutes or more caused an obvious agglutination, but at the same time the result was obscured by non-specific reaction.

TABLE 4. Results of agglutinin-adsorbing latex method in reference to various concentrations of antiserum protein

Blood-stains: AB group, within one week. Anti-serum: Animal serum, titer 1,024. Agglutinin-adsorbing Latex for Blood-grouping 339

TABLE7. Results of agglutinin-adsorbing latex method in reference to lapse of time after blood-stain formation

Antiserum: Animal immune serum, titer 1,024. 340 T. Tajima

TABLE 8. Results of agglutinin-adsorbing latex method with saliva stains from weak secretors

Antiserum: Animal immune serum, titer 2,048.

7) Preservation The stability of the agglutinin-adsorbing latex suspension deteriorates in a few days. This may be a weak point to be improved in future. But, the ƒÁ-globulin solution is preservable for a long time by refrigeration, and the agglutinin Agglutinin-adsorbingLatex for Blood-grouping 341:

adsorbing latex suspension, which is mixed with a preservative, is durable at least for a month. Accordingly, there is little inconvenience for its practical use.

8) Result of blood-grouping of various blood-stains and saliva-stains When the agglutinin-adsorbing latex suspensions reacted directly with the fibrils with group-compatible blood-stains or other test specimens, numerous latex particles agglutinated on the fibrils as shown in Figs. 5 and 6. In strong reactions, agglomerates like bunches of grape were observed. On the other hand, there was no agglutination in cases of group-incompatibility as shown in Fig. 7. Sometimes free latex particles remain in the folds of fibrils even after gentle washing, because latex particles are much smaller than fibrils as shown in Fig. 8. Even in this case, the particles are attached plainly on the fibril and they are easily differentiated from ones involved in true agglutination. The author examined 80 cases of fresh blood stains consisting of each 20 cases of 0, A, B and AB groups, and obtained satisfactory results in all cases. As for blood-stains, a satisfactory result was obtained when the stains were within 1 month old,as shown in Table 7. This was the same in 40 cases of saliva stains from strong secretor, whereas those from `weak secretor' gave a positive reaction within 20 days as shown in Table 8.

DISCUSSION Many attempts have been made to observe the antigen-antibody reaction in the form of agglutination or flocculation by means of fine grains. Loeb11 reported that the electric property of surface of protein-adsorbing collodion particles was equal to that of protein itself, and Jones12 succeeded in observing antigen-antibody reaction between bovine serum and anti-bovine serum by means of collodion particles. Since then, this collodion method has been widely used in the fields of bacteriology and .13-18 On the other hand, since Bozicevich et al.19 reported the reaction between the antigen of trichinella adsorbed onto bentonite particles and the serum of patient with trichinellosis, the bentonite method has been used too.20-22 But these methods have a great disadvantage in that the particles are variable in size and form. Pressman et al.23 devised a method of agglutination between the anti-protein serum and the red cells which were treated with bisdiazotized benzidine and adsorbed protein. Since then, the treated red cells were used as a medium of serological reaction.24 Stavitsky25-27 made a comparative study of these methods and concluded that the tannic acid method was more sensitive than the bisdiazotized benzidine method. Because of the sensibility and simplicity, the tannic acid method has been used in the serological field. The author tried blood-grouping by means of human red cells of O-group, which were treated with tannic acid, and observed a strong non-specific adsorption. In the case of the bisdiazotized benzidine method, non-specific reaction was 342 T. Tajima weaker, but the specific reaction was too weak for practical use.

Moskowitz and Carb28 reported that human red cells treated with formalin could not be agglutinated by antiserum, but could adsorb agglutinin which was eluted at 56°C. Gold et al.29 used this formalin method to preserve blood cells of the rare blood group. The author examined formalin-treated red cells, but no

satisfactory result was obtained. The treated red cell method has a weak point in preservation. However, this method will be more widely applied after technical improvement in future.

In fact, frozen dry red cells adsorbing antigen are now commercially available.

Such red cells will be used in the study of blood groups. In any way, serological application of the fine grains mentioned above has

weak points in preservation, uniformity and others.

Owing to the recent advance in synthetic chemistry, many kinds of synthetic latex are produced. Latex is uniformly spherical in shape, and it has a colloidal

nature like gum-latex and adsorbs protein onto the surface. Bradford and

Vanderhoff30 classified synthetic latex into soft latex and hard latex according to the ability to make film at room temperature, and reported that a kind of

hard latex, polystyrene latex, was agglutinated with 10% potassium persulfate.

Singer and Plotz7 made use of 0.81,a polystyrene latex in the serological reac

tion of rheumatoid arthritis and named this reaction `latex fixation test'. Since then, latex has been widely used for rheumatism reaction, 31-39 detection of anti

nuclear antibody of lupus erythematosus,40-43 pregnancy reaction,44 detection of anti-histamin antibody and so on.45,46 Further, it is used in the field of bac

teriology for the detection of anti-histoplasma capsulatum antibody,41-50 flagellum

agglutination, 51 and recently for the syphilis reaction using Reiter antigen.52, 53

On the other hand, Hagiwara54 made use of 0.81,a polystyrene latex for blood-grouping of FL cells. Thereafter he made a series of serological studies

using latex55-60 and named this method 'antigen-coated latex particles adsorp

tion method'. In this method, the antigen-adsorbing latex is used in place of

indicator cells of the so-called mixed agglutination method by Coombs and Dodd.2 Milgrom and Goldstein61 described that 0.81,u polystyrene particles easily

adsorbed ƒÁ-globulin, and used these particles in place of Coomb's anti-globulin

serum. However, this is not used in practice because of its strong non-specific

reaction. The author's experiment arrived at the same conclusion.

As mentioned above, various kinds of fine grains have been introduced into serological reactions. However, all of them are devised for a detection of

antibody by means antigen-adsorbing fine grains. For the direct detection of

antigen, there are such methods as the fluorescent antibody method by Coons et al.4

and the radio-isotope labeled antibody method by Pressman et al.5 Alexander62

introduced the fluorescent antibody method into the study of blood groups, but

the rate of success of the direct method is rather low, and the two or three step

methods are mainly used. In any case, these methods require special apparatuses Agglutinin-adsorbing Latex for Blood-grouping 343 and highly specific technique. On the other hand, Singer et al.63 succeeded in directly detecting 'antigen' by means of the antibody-adsorbing latex. In this case, however, the antigen was also water-soluble protein. In the field of blood groups, there has been no report on the direct detection of antigen by means of the antibody adsorbing latex or other fine grains. So the author's method is the first one, in which the direct detection of antigen adhering to materials can be made by means of latex. This method can be used not only for blood-grouping of blood-stains and other test specimens, but also for the detection of antigen in tissue cells.

Most of the latex particles used in the serological field are 0.81,u polystyrene latex. This latex is satisfactory in size for the tube or slide method, but too small to admit of direct observation of agglutination on blood cell stroma. On the other hand, 2.051,u polyvinyl toluene latex is most satisfactory in size and weight among various kinds of latex. The author's method may be the first one which uses the latex of this size in the serological field.

SUMMARY AND CONCLUSION

The author devised a new method, 'agglutinin-adsorbing latex method', for the blood grouping of blood-stains and other materials. In this method, the agglutinin-adsorbing latex was prepared by mixing 5 drops of 10% suspension of polyvinyl toluene latex of 2.051,u in diameter with 1 ml of 1 g/100 ml ƒÁ-globulin solution, which was adjusted to 0.1 mol and pH 8.5 with veronal buffer. The mixture was agitated at room temperature for an hour. The remaining free antibody and natural agglomerates were eliminated by centrifugation.

The main results and conclusions are as follows: 1) The adsorption of agglutinin onto latex was demonstrated by both anti globulin test and the direct reaction to the group-compatible blood cells. 2) The titer of antibody to be adsorbed onto latex must be high, and all the protein components except y-globulin should be excluded.

3) By this method, accurate blood-grouping can be made even with a piece of blood or salvia-stained fibril in only about ten minutes.

4) For this method 2.051,u polyvinyl-toluene latex is more suitable than

0.81,u latex. This method may be the first one in the field of serology, in which 2.051,u polyvinyl toluene latex is used.

5) This method promises a wide application not only to blood groups, but also to general serological reactions.

Acknowledgments

The author wishes to express his thanks to Prof. S. Akaishi and Prof. S. Murakami for their valuable advice and encouragement, and to Assistant Prof. Y. Yoshida, the First Department of Internal Medicine, for his adequate advice, and to Dow Chemical Company (U.S.A.) for generous supply of various kinds of latex suspension. 344 T. Tajima

References 1) Kind, S.S. Adsorption-elution grouping of dried blood-stains on fabrics. Nature, 1960, 187, 789-790. 2) Coombs,R.R.A. & Dodd, B. Possible application of the principle of mixed agglutina tion in the identification of blood stains. Med. Science Law., 1961, 1, 359-377. 3) Akaishi, S. Studies on the group-specific double combination method. Nippon Hoigaku Zasshi (Jap), 1965, 19, 177-187. 4) Coons, A.H., Greech, H.J. & Jones, R.N. Immunological properties of an antibody containing a fluorescent group. Proc. Soc. exp. Biol. Med., 1941, 47, 200-202. 5) Pressman, D. & Keighley, G. The zone of activity of antibodies as determined by the use of radioactive tracers; the zone of activity of nephrotoxic antikidney serum. J. Immunol., 1948, 59, 141-146. 6) Furusawa, M. A proposal for the standard serum with standardized titer. Nippon Hoigaku Zasshi (Jap.), 1955, 9, 245. 7) Singer, J.M. & Plotz, C.M. The latex fixation test. I. Application to the sero logical diagnosis of rheumatoid arthritis. Amer. J. Med., 1956, 21, 888-892. 8) Oreskes, I. & Singer, J.M. Stability and gamma globulin binding studies on poly styrene latex particles. Arthr. Rheum., 1960, 3, 273-274. 9) Singer, J.M., Altman, G., Oreskes, I. & Poltz, C.M. The mechanism of particles carrier reactions. III. The stabilizing effect of serum proteins. Amer. J. Med., 1961, 30, 772-778. 10) Donaldson, P. & Pennington, K. Agglutination of latex particles without antigen adsorption. J. Immunol., 1965, 94, 710-714. 11) Loeb, J. The influence of electrolytes on the cataphoretic charge colloidal particles and the stability of their suspensions. I. Experiments with collodion particles. J. Gen. Physiol., 1922-1923, 5, 109-126. 12) Jones, F.S. Agglutination by precipitin. J. exp. Med., 1927, 46, 303-314. 13) Cannon, P.R. & Marshall, C.E. An improved serologic method for the determina tion of the precipitative titers of antisera. J. Immunol., 1940, 38, 365-376. 14) Goonder, K. Collodion fixation: A new immunological reaction. Science, 1941, 94, 241-242. 15) Eisler, D.M. Influence of collodion particles on the visible endpoint in antibody titrations. J. Immunol., 1941, 42, 405-418. 16) Burger, M. Microscopic observation of collodion particles as indicators of type specific pneumococcic immune reactions. J. Lab. clin. Med., 1943, 28, 1138-1139. 17) Lowell, F.C. A comparison of the collodion-particle technic with other methods of measuring antibody. J. Immunol., 1943, 46, 177-182. 18) Cavelti, P.A. Studies on the technic of collodion agglutination. J. Immunol., 1945, 49, 365-373. 19) Bozicevich, J., Tobie, J.E., Thomas, E.H., Hoyen, H.M. & Ward, S.B. A rapid flocculation test for the diagnosis of trichinosis. Public Hlth Rep. (Wash.), 1951, 66, 806-814. 20) Bozicevich, J., Bunim, J.J., Freund, J. & Ward, S.B. Bentonite flocculation test for rheumatoid arthritis. Proc. Soc. exp. Biol. Med., 1958, 97, 180-183. 21) Bloch, K.J. & Bunim, J.J. Simple, rapid diagnostic test for rheumatoid arthritis. Bentonite flocculation test. J. Amer. med. Ass., 1959, 169, 307-314. 22) Lerner, E.M., Bloch, K.J. & Williams, R.R. "Rheumatoid" serological reactions in experimental animals. II. Bentonite flocculation test in rats with experimental arthritis. Arthr. Rheum., 1960, 3, 26-40. 23) Pressman, D., Campbell, D.H. & Pauling, L. The aggultination of intact azo erythrocytes by antisera homologous to the attached groups. J. Immunol., 1942, 44, 101-105. 24) Boyden, S.V. The adsorption of proteins on erythrocytes treated with tannic acid Agglutinin-adsorbing Latex for Blood-grouping 345

and subsequent by antiprotein sera. J. exp. Med., 1951, 93,107 120. 25) Stavitsky, A.B. Micromethods for the study of proteins and antibodies. I. Procedure and general applications of hemagglutination and hemagglutination-inhibition reactions with tannic acid and protein-treated red blood cells. J. Immunol., 1954, 72, 360-367. 26) Stavitsky, A.B. Micromethods for the study of proteins and antibodies. II. Specific applications of hemagglutination and hemagglutination-inhibition reactions with tannic acid and protein-treated red blood cells. J. Immunol., 1954, 72, 368-376. 27) Stavitsky, A.B. Micromethods for the study of proteins and antibodies. III. Procedure and applications of hemagglutination and hemagglutination-inhibition reactions with bis-diazotized benzidine and protein-conjugated red blood cells. J. Immunol., 1955, 74, 306-312. 28) Moskowitz, M. & Carb, S. Surface alteration and the agglutinability of red cells. Nature, 1957, 180, 1049-1050. 29) Gold, E.R., Lockyer, W.J. & Tovey, G.H. Use of lyophilized formol-treated red cells in bloodgroup serology. Nature, 1958, 182, 951. 30) Bradford, E.B. & Vanderhoff, J.W. The morphology of synthetic latexes. J. Polymer Sci., Part C, 1963, 1, 41-64. 31) Plotz, C.M. & Singer, J.M. The latex fixation test. II. Results in rheumatoid arthritis. Amer. J. Med., 1956, 21, 893-896. 32) Singer, J.M. & Plotz, C.M. Slide latex fixation test. A simple screening method for the diagnosis of rheumatoid arthritis. J. Amer. med. Ass., 1958, 168,180-181. 33) Singer, J.M. & Plotz, C.M. The latex fixation test for rheumatoid arthritis using patient's own gamma globulin. Arthr. Rheum., 1958, 1, 142-146. 34) Morgan, G. Comparative study of the hemagglutination test and a simple latex fixation test for the detection on the rheumatoid factor. Ann. rheum. Die., 1959, 18, 322-324. 35) Coke, H. Experience with British latex suspension for agglutination tests in rheuma toid arthritis. Ann. rheum. Die., 1959, 18, 301-304. 36) Singer, J.M., Altmann, G., Goldenberg, A. & Plotz, C.M. The mechanism of particles carrier reactions with rheumatoid sera. II. Sensitizing capacity of various human gamma globulins for latex particles. Arthr. Rheum., 1960, 3, 515-521. 37) Heimer, R. & Freyberg, R.H. Examination of the latex fixation test using sulfated mucopolysaccharides. Arthr. Rheum., 1960, 3, 158-163. 38) Bernhard, G.C., Cheng, W. & Talmage, D.W. The reaction of rheumatoid factor and complement with y-globulin coated latex. J. Immunol., 1960, 88, 750-762. 39) Koch, D. & Odenthal, II. Der Nachweis des Rheumafaktors durch den Latex-Schnell test and durch Ultrazentrifugenuntersuchung bei der primarchronischen Polyar thritis. Dtsch. med. Wschr., 1961, 86, 1767-1768. 40) Christian, C.L., Mendez-Bryan, R. & Larson, D.L. Latex agglutination test for disseminated lupus erythematosus. Proc. Soc. exp. Biol. Med., 1958, 98, 820-823. 41) Fessel, W.J. Nucleoprotein-latex agglutination test in connective tissue diseases. Ann. rheum. Die., 1959, 18, 255-258. 42) Dubois, E.L., Drexler, E. & Arterberry, J.D. A latex nucleoprotein test for diagnosis of systemic lupus erythematosus. J. Amer. med. Ass., 1961, 177, 131-133. 43) Barnes, R., Carmichael, D. & Johnson, G.D. Comparison between the latex nucleoprotein test and the fluorescent method for the demonstration of antinuclear factor. Ann. rheum. Die., 1962, 21, 287-291. 44) Robbins, J.L., Hill, G.A., Carle, B.N., Carlquist, J.H. & Marcus, S. Latex agglutina tion reactions between chorionic gonadotropin and rabbit antibody. Proc. Soc. exp. Biol. Med., 1961, 109, 321-325. 45) Mikol, C., Renoux, M. & Merlien, F.P. Sero-agglutination a l'histmaine sur latex et allergie. Bull. Soc. Med. Paris., 1961, 77, 441-446. 346 T. Tajima

46) Hamelin, A. & Burdet, M. Recherche du facteur serique antihistaminique par agglu tination de particules de latex. Proph. sanit. morale, 1963, 35, 83-85. 47) Carlisle, H.N. & Saslaw, S. A histoplasmin-latex agglutination test. I. Results with animal sera. J. Lab. clin. Med., 1958, 51, 793-801. 48) Wiedermann, G. Anwendung des Latextestes zur serologischen Diagnostik bakteriel ler Infektionen. Zbl. Bakt., I. Abt. Orig., 1961, 81, 106-112. 49) Murasehi, T.F. Latex-leptospiral agglutination test. Proc. Soc. exp. Biol. Med., 1958, 99, 235-238. 50) Fleck, L. & Evenchik, Z. Latex agglutination test with brucella antigen and anti serum. Nature, 1962, 194, 548-550. 51) Bader, R.E. Die Latex-Geissel-Agglutination. Z. Hyg. Infekt.-Kr., 1964, 150, 199-210. 52) Fischman, A. Reiter protein latex test; a preliminary report. Brit. J. vener. Die., 1964, 40, 225-227. 53) Stevens, R.W. Agglutination of Reiter protein-coated latex particles . Amer. J. clin. Path., 1965, 43, 490-493. 54) Hagiwara, A. A new method for the detection of blood group antigen and its application to FL human amnion cell. Exp. Cell Res., 1962, 28, 615-617. 55) Hagiwara, A. & Mura, S. Latex absorption method for the detection of celullar antibody production. Exp. Cell Res., 1963, 30, 618-620. 56) Yoshikawa, H. & Hagiwara, A. Genetical biochemistry of immunity. Igaku no Ayurmi (Jap.), 1963, 44, 600-604. 57) Hagiwara, A. Separation of an antibody molecule by a specific reaction with an antigen latex particle. Nature, 1964, 202, 1019-1020. 58) Hagiwara, A. On the antigen-coated latex particles adsorption method . Seibut8u Kagaku (Jap.), 1964, 16, 49-53. 59) Hagiwara, A. Studies on the antibody formation in cultured mouse spleen cells. Symposia for the Society Cellular Chemistry, 1964, 14, 101-108. 60) Hagiwara, A. The practice of the antigen-coated latex particles adsorption method. Sogorinsho (Jap.), 1964, 14, 445-450. 61) Milgrom, F. & Goldstein, R. Agglutination of sensitized red cells by latex particles . Vox Sang (Basel), 1962, 7, 86-88. 62) Alexander, W.R.M. The Application of the fluorescent-antibody technique to haemagglutinating systems. , 1958, 1, 217-223. 63) Singer, J.M., Plotz, C.M., Pader, E. & Elster, S.K. The latex-fixation test. III. Agglutination test for C-reactive protein and comparison with the capillary precipitin method. Amer. J. clin. Path., 1957, 28, 611-617.

Legends Fig. 1. Agglutination of rabbit anti-A agglutinin-adsorbing latex by guinea pig anti rabbit globulin serum. Fig. 2. Negative reaction of non-agglutinin-adsorbing latex by guinea pig anti -rabbit globulin serum. Fig. 3. Agglutination of group-A blood cells by anti -A agglutinin-adsorbing latex .Fi g. 4. Negative reaction of group-B blood cells by anti-A agglutinin-adsorbing latex .Fi g. 5. Strong agglutination of agglutinin-adsorbing latex on the fibrils with group compatible blood-stains. Fig. 6. Strong magnification of a part of Fig. 5. Fig. 7. Negative reaction of agglutinin-adsorbing latex on the fibrils with incompatible blood-stains. group Fig. 8. Free latex particles remaining in the fold of fibril. Agglutinin-adsorbing Latex for Blood-grouping 347 348 T. Tajima