Species-specificity and reacting site of antiglobulin factors, non-neutralizable by homologous γ-globulin, were studied and the following results were obtained:
1) Many of human sera showing only weak antiglobulin activity of anti-antibody nature against human red cells sensitized with human whole Rh-antibody were found to be capable of agglutinating the red cells sensitized with pepsin fragment —F(ab′)2— of the Rh-antibody.
2) In serum of a rabbit exhibiting antiglobulin activity of anti-antibody nature, there was found, in addition to a factor reacting only with rabbit antibodies, a factor capable of cross-reacting with both rabbit (homologous) and human (heterologous) antibodies.
3) Rabbit anti-antibodies were found to agglutinate sheep red cells sensitized with non-agglutinating pepsin fragment of rabbit anti-sheep erythrocyte antibody. This finding seems to indicate that reacting site against anti-antibody must be located in the Fab portion of rabbit IgG.
4) Cross-reacting antiglobulin factor of anti-antibody nature was also found in a rabbit antiserum against HB antigen, prepared by immunization with specific immune complex.

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During routine post natal screening, two examples of anti-Di^a stimulated by pregnancies were were found. One of the antibodies caused hemolytic disease of the newborn.
Case 1. Mrs. H. K., a Di^a negative, 28-year-old Japanese woman, had no history of blood transfusion and had a healthy child. The routine antibody tests during her first pregnancy revealed no immunization. When her serum was tested again six months after the birth, anti-Di^a with a titre of 1:2-1:4 against Di (a+b+) cells by the anti-globulin technique was detected. A year later the antibody in her serum was not detectable.
Case 2. Mrs. K. O., a 35-year-old Japanese woman, was found to contain anti-Di^a in her serum when a routine direct anti-globulin screening on the cord cells of her third child was found to be positive. She was Di (a-b+), and had never been transfused. Her first child was Di (a-b+), and her husband and second child were Di (a+b+). Primary antibody response may has been due to stimulation by her second pregnancy. The third child became jaundiced within three days of birth; serum bilirubin level at the fifth days was 16.0mg/100ml decreasing to 13.7mg/100ml at the sixth day. The child was placed under phototherapy and survived. The anti-Di^a serum from Mrs. K. O. reacted best by the papain- and ficin-antiglobulin techniques using anti-IgG; the titre of which was 1:64-1:128 against Di (a+b+) cells, it was not denatured by 2-mercaptoethanol. The antibody was also detectable by the papainized or ficinized cells in saline (titre 1:2-1:4), but the antibody dit not combine the complement. In titration studies with five Di (a+b-) and five Di (a+b+) cell samples, the anti-Di^a showed dosage effect; the titration scores were 38.2 (on the average) for Di (a+b-) and 28.2 for Di (a+b+), and the difference was significant (F₀=23.4, P<0.01).
Distribution of the Diego groups. Random 3, 425 blood samples from Japanese in and around Tokyo were tested for Di^a and Di^b antigens by one of the present authors (H. N.). The phenotypes and gene frequencies obtained are shown in Table 1. The agreement with expectation on the assumption of Hardy-Weinberg equilibrium was satisfactory.
The chances of antibody-production by pregnancy and by transfusion are expected to be 0.04-0.08 for anti-Di^a and 0.002 for anti-Di^b. As far as we know, four examples of anti-Di^a, including two examples described in this paper, and three examples of anti-D^b- have been found in Japan, suggesting that the potency of Di^a antigen is lower than that of D^b-.

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