BLOOD TYPING OF DRIED WHALE ERYTHROCYTESWITH 131I LABELLED ANTIBODY*
KAZUO FUJINO AND JOHN E. CUSHING**Blood typing techniques and concepts are now being applied in population studies of several species of marine vertebrates important infisheries (Fujino, 1956 ; Suzuki, Shimizu & Morio, 1958 ; Ridgway, Cushing & Durall, 1958). A major problem of this work is the preservationof samples of blood until they can be brought into a laboratory foranalysis. One method of preservation that has been found to be usefulis that of glycerol-freezing (Cushing, Fujino & Takahashi, 1959). The
present paper is a continuation of research initiated by the senior author(Fujino, 1958) on the possibility of developing another method, using
dried blood and radioactive antibody. The usefulness of 131 I labelled
antibody for this purpose was suggested by a variety of researches(for example, Pressman & Eisen, 1950; Bournsnell, Coombs & Rizk,1953).
MATERIAL AND METHOD

Blood samples from blue-white dolphins (Stenella caeruleo-albus) weretaken from individuals captured at Ito, Shizuoka Prefecture. Dried
samples were prepared by diluting one part of fresh whole blood withnine parts of 1.5% NaCl solution, being careful to equilibrate thecolorimetric concentration of different samples. Single drops of diluted
blood were spotted on pieces of filter paper (Toyo filter paper No. A.5)
and allowed to dry at room temperatures. These samples were thenpreserved in a dessicator until they were used, approximately threemonths after collection. A second set of dolphin bloods was preserved
by glycerol-freezing as described in the paper on this subject cited
above (Cushing, Fujino & Takahashi, 1959).
The antiserum used was prepared by injecting a rabbit with fresh
dolphin cells from a single individual (S. 23). Absorption of the antiserum obtained with heterologous dolphin cells (S. 25) left antibodies
specific to the homologous cells as shown in Table 1. (The antigen* Supported in part by the grants of Japanese Fisheries Agency, of Naval Research,
United States Navy, and of University of California.
** The University of California, Santa Barbara, California, U.S. A. John Simon Guggenheim Memorial Foundation Fellow, Dept. of Fisheries, Faculty of Agriculture, Tokyo
University, 1958-59.
101

K. FUJINO AND J. E. CUSHING

102
TABLE 1.
Treatmentofantibody
Beforesalting-out

Aftersalting-out

Afteriodination

AGGLUTININ TITER OF ANTI-23 DOLPHIN SERUM IN EACH
ST AGE OF TREATMENT

Before or after
absorb. by S. 25.
dolphin cells

lll

beforeDolphin cells
used foragglutination
{S.23
S.25

after{S.23
S.25

before{S.23
S.25

after{S.23
S.25

before{S.23
S.25

after{S.23
S.25

Dilution of antibody
128256512** ** **+ **+ *+ ** *** ** ** **± *+ +* * +** ** ** *+ * +* * + ±+

81632641024

involved has not yet been related to the De system previously described
for this species by Yamaguchi & Fujino, 1952).
Iodination procedures followed those described by Pressman & Eisen(1950). Antibody gamma-globulin was precipitated from the immune
serum following one third saturation with ammonium sulfate at ph 7.0.
The ammonium sulfate was removed from the gamma-globulin fraction
by a series of dialysis against isotonic saline, and against carbowax ofmolecular weight 1,500 (Arai, Sakagishi & Nomiyama, 1956). The solution was restored to its original volume following dialysis. The successof dialysis was checked with barium chloride indicator.
The protein concentration of the final preparation was determinedchemically by the semi-micro Kjeldahl method, and colorimetrically with
the Beckman spectrophotometer at wave length 545 mµ using Sols'
Biuret reagent (Sols, 1947; Matsumoto & Kanamitsu, 1955). A conversion factor was calculated as follows: P=l.16·D, where P is the concentration of protein nitrogen in mg. per ml. and D ( = ln 10/I) is theoptical density per 1 cm. depth of solution at wave length 545 mµ.
Radioactive iodine 1311, manufactured by the Radiochemical Centre,Amersham, England, was used for labelling the antibody. This is prepared as the sodium iodide in 0.01 N sodium thiosulfate. Iodination wasaccomplished by adding the iodinating solution (0.1 ml. of 0.01 M potassium iodide, 1.0 ml. of carrier free 1311 at 0.1 me., 1 drop of 1 M sodium
nitrite and 0.2 ml. of 2.5 N hydrochloric acid) to a solution containing
15.0 mg. of gamma-globulin in 2 ml. of borate buffer at ph 8.0 (0.16 N
sodium hydroxide, 0.2 M boric acid in 0.16 M sodium chloride). After
five minutes the ph of this solution was adjusted to 8.0 with 2.5 N
sodium hydroxide. Twenty minutes later dialysis was started against
saline and carbowax as before.

BLOOD TYPING OF DRIED WHALE ERYTHROCYTES

103

After dialysing, completion of removal of the uncoupled 131 1 waschecked by the radioautography from paperchromatograph of iodinatedantibody as well as by counting of radioactivity of dialysates with G-Mcounter. Fig. 1 shows an example of radioautographs made of an antifinback whale serum that demonstrate the method used. These show

Fig. 1. Radioautographs from paperchromatograms of iodinated antibody,showing the complete removal of uncoupled 1311 from iodinated antifinback antibody, Toyo filter paper 5A, 50 minutes diffusion in solvent
which consists of 1 part of acetic acid, 3 parts of normal butyl alcoholand 4 parts of distilled water, Fuji-X-ray film, 6 hrs. exposed, upper:
just after iodination, lower: after removal of uncoupled 131J by dialysis.
The central spot in each autograph shows the location of iodinated antibody-gamma-globulin. The band in the upper figure shows the locationof migrating uncoupled 1311. This band is absent in the lower figure
after a comparable time of diffusion.

that the serum proteins have been successfully iodinated and that thefree 131 ! was removed successfully by dialysis.

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BLOOD TYPING OF DRIED WHALE ERYTHROCYTES105

BLOOD TYPING PROCEDURE

After titration the labelled antiserum was absorbed three times withthe cells of dolphin S-25 which removed antibodies capable of reactingwith antigens common to both individuals, as well as the remaining
traces of uncoupled 1311 (Table 1).
The reactions of the absorbed antiserum with intact erythrocytes
were observed first. Three drops of a two percent cell suspension fromdolphin S-23 were added to a test tube containing three drops of one
in eight dilution of the absorbed antiserum. A similar mixture wasmade with S-25 cells as a control. After thirty minutes each of thecell suspensions was centrifuged and was washed twelve times withsaline to remove uncombined antibody. The whole washed cells werespotted on filter paper, dried at room temperatures, and their total
radioactivity was determined by G-M counter and radioautograph. The
results of these determinations, Table 2 and Fig. 2, show that the cells ofdolphin S-23 absorbed much more labelled protein than did those of S-25.
Samples of blood dried on filter paper strips were moistened by diffusion in four percent ammonium sulfate and then placed with their ends
in the same solution (Fig. 3). Whendiffusion had started, equal sized
drops of absorbed antiserum (one ineight dilution) were placed so thatthey could diffuse through the blood
spots. After two hours the filter
papers were allowed to dry at room
temperature and the total radioi.a.
activity of the blood spots was Fig. 3. Preparation of antigen-antibody
determined by G-M counter. This
reaction on filter paper, p. b.: spots ofblood preserved by drying, i.a.: spots ofexperiment was repeated five times
iodinated antibody, f.p.: filter paper,with replicated samples from eacha.s.: 4 percent ammonium sulfate.
individual. The results are shownin Table 2. These agree very well with those obtained with intact cells
in the first experiment.
CONCLUSION

These experiments show that it is highly probable that 131 1 labelled antibodies can be used to distinguish individual antigenic differences insamples of dired blood. Such samples have great potential value inblood type studies on marine vertebrates where it may be difficult topreserve intact erythrocytes.

106

K. FUJINO AND J.E. CUSHING

In order to expand these techniques for use in large scale investiga-tions of fisheries problems, further research will be necessary on suchmatters as the extent of the variability introduced during the preparation of dried samples, as well as the general practicability of the method
used.
Authors' acknowledgements are due to Dr. H. Omura, Director ofWhales Research Institute, Prof. Y. Suyehiro, Prof. Y. Hiyama, Dr. M.
Saiki, Mr. K. Hashimoto, Department of Fisheries, Tokyo University
and Mr. J. Matsumoto, Tokai Regional Fisheries Laboratory for theirarrangements and suggestions on this study. The technical assistanceof Mr. K. Takahashi, Department of Fisheries, Tokyo University isgratefully acknowledged.

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