A Novel Method for Screening Monoclonal Antibodies Reacting with Antigenic Determinants on Soluble Antigens; A Reversed Indirect-Enzyme Linked Immunosorbent Assay(RI-ELISA)

A novel screening method was established to select new monoclonal antibodies which react with unknown antigenic determinants on molecules bearing antigen determinants reactive with established monoclonal antibodies. This new method is a sandwich assay termed "reversed indirect-enzyme linked immunosorbent assay" (RI-ELISA). Goat antimouse immunoglobulin antibodies are used as the primary immobilized antibody in this assay. They allow the non-purified monoclonal antibodies contained in hybridoma culture supernatants to bind to the microtest plate for enzyme immunoassay (EIA plate) much more efficiently than in the usual sandwich assay where the non-purified monoclonal antibodies are adsorbed directly to the polystyrene surface. The antigen solution is then reacted with the monoclonal antibodies and thereafter enzyme labeled monoclonal antibody with known specificity is added. Therefore, if the hybridoma culture supernatant contains monoclonal antibodies which were bound to the EIA plate and react with antigenic determinants on the soluble molecules which have antigen determinants recognized by the enzyme labeled antibody, the enzyme labeled antibodies will bind to induce an enzymatic reaction. The most important technical consideration in the RI-ELISA is the inhibition of direct binding of the enzyme labeled monoclonal antibodies to free sites remaining in the immobilized goat anti-mouse immunoglobulins antibodies. This problem could be effectively overcome by using normal mouse serum as blocking substance. These studies indicate that the RI-ELISA may be a useful screening method for selecting new monoclonal antibodies which react with unknown antigenic determinants on soluble molecules

Detection of Lymphocyte Subsets by Monoclonal Antibodies in Aged and Young Humans

With the use of Leu-series monoclonal antibodies, peripheral blood lymphocyte subsets in aged and young humans were determined. 1) In comparison with young individuals, Leu-1+ cells and Leu-2a+ cells were decreased, whereas Leu-7+ cells and Leu-3a/Leu-2a were increased in aged individuals. 2) No sex difference could be observed in lymphocyte subsets. 3) PHA response of lymphocytes showed a negative correlation with Leu-2a+ cells and a positive correlation with Leu-3a/Leu-2a in aged individuals

Production of Interleukin 2 in Human Peripheral Blood Lymphocytes: Optimal Condition for its Culture

For the assay of the production of Interleukin 2 (IL-2) in human peripheral blood lymphocytes (human PBL), a study was made on the optimal condition for its culture. 1) The optimal condition for the production of IL-2 was considered to be incubation time of 24 hr, number of PBL of 1 x 106 cells/ml, and phytohemagglutinin-M (PHA-M) concentration of 1 — 5% 2) By one way mixed lymphocyte reaction (one way MLR), IL-2 activity similar in level to that by PHA-M stimulation could also be obtained. It was maximal at the 4th day of culture. 3) By PHA-P (0.06%) stimulation, IL-2 could also be produced similar to that by PHAM stimulation but it decreased in the order of Con A, PWM and PPD and hardly any production of IL-2 could be observed by LPS

Interleukin-2 Production and Lymphocyte Proliferation in Aged and Young Humans

A study was made on the effect of aging on the production of Interleukin-2 (IL-2) and lymphocyte proliferation of human peripheral blood lymphocytes. 1) In comparison with young individuals, IL-2 production tended to decrease in aged individuals, while lymphocyte proliferation showed a significant decrease. 2) A significant correlation was observed between IL-2 production and lymphocyte proliferation in both the aged and young human populations. 3) IL-2 production showed a negative correlation with Leu-2a positive rate and a positive correlation with Leu-3a/Leu-2a ratio in aged individuals

Immune Functions of Former Poison Gas Workers I. Mitogenic response of lymphocytes and serum factors

The relation of depressed immune function to carcinogenesis has been estimated in the living body. The authors have measured the immunological parameters in former poison gas workers, a group having a high risk of carcinogenesis, for comparison with age matched normal controls and the following results were obtained. 1) With regard to serum factors, no significant difference could be demonstrated between normal controls and poison gas workers in such immunoglobulins as IgG, IgA, and IgM, in acute phase reactants such as α1-AT, α1-AG, α2-HS and C3 and in such tumor markers as CEA, ferritin, and β2-microglobulin. Furthermore, no difference could be observed in the positive rate of immune complex and in complement activity. 2) No difference could be observed between the two groups with regard to tuberculin skin reaction and number of lymphocytes, but the longer the duration of work at the poison gas factory, the more significant was the increase in those who showed negative tuberculin skin reaction. 3) In comparison with normal controls, mitogenic response to PHA showed a significant decrease in poison gas workers, but no significant difference could be seen in mitogenic response to Con A and PPD and in mixed lymphocyte reaction. 4) No significant difference could be demonstrated between the two groups in the inhibitory effects of serum on mitogenic response to PHA and Con A and on mixed lymphocyte reaction

Cellular Immunity of Patients with Lung Cancer and Other Lung Diseases II. Analysis of interleukin-2 production

Interleukin-2 (IL-2) production by stimulated peripheral blood lymphocytes (PBL) from patients with lung cancer and noncancerous respiratory diseases was determined. The results are as follows: 1) Neither sex nor age difference was observed for IL-2 production among healthy people. 2) IL-2 production showed a positive correlation with the Leu-3a/Leu-2a ratio and a negative correlation with the percentage of HLA-DR+ cells. 3) IL-2 production of patients with lung cancer and noncancerous respiratory diseases did not differ from that of healthy persons. 4) No difference in IL-2 production was found in relation to the clinical stage of lung cancer, but subjects with low IL-2 production were mostly observed in the advanced stage group (Stage IV)

Reactivity of the Serum from A-Bomb Survivors with the Tissues of Stomach, Liver and Kidney of Normal Rats

In order to evaluate delayed effects of radiation on pathological immune response an attempt was made to detect antibodies in the serum of atomic bomb survivors against kidney, liver, and parietal cells from rats. The following results were observed. Analysis of changes in antibody detection frequencies by age and exposure dose without considering sex showed that the rates for those exposed to 100 + rad showed a trend to increase with age for all three organs (P<0.01). However, in the 0 rad group, a significant trend to increase with age was noted for anti-kidney and antiliver antibodies only (P<0.01 for both). Analysis of changes in antibody detection frequencies by sex, age, and exposure dose showed that the detection frequencies increased significantly with age for all three organs in males exposed to 100 + rad (P < 0.05), but only the anti-liver antibody frequency increased significantly with age in males in the O rad exposure group. Females failed to shown any statistical changes in any exposure group

加令によるヒトリンパ球の mitogen による lgG 産生の増強 : 特に T 細胞の効果について

When in vitro IgG production by mitogen-stimulated peripheral lymphocytes from healthy aged (70-93 year old) and young (20-29 year old) subjects was compared, it was found that the level of IgG production was elevated in the aged subjects. Co-culture studies were performed to determine whether the increase was clue to changes in T or non-T cells. IgG production was significantly higher when reference non-T cells from normal young adults were mixed with T cells from aged subjects than with T cells from young adults. In contrast, no significant difference in IgG production was observed when reference T cells from normal young adults were mixed with non-T cells from either young or aged subjects. The suppressor activity of Tγ cells and helper activity of non-Tγ T cells of young and aged subjects were then determined. The results revealed that the suppressor activity of Tγ cells of aged individuals was significantly lower than that of young adults, but the helper activity of non-Tγ T cells of young and aged subjects was comparable. These results indicate that the increase in production of IgG by the peripheral lymphocytes of aged individuals is due in part to changes in the T cells which are related to a decrease in suppressor activity of Tγ cells

Analysis of Cellular Immunity in Patients with Graves' Disease

Graves' disease has attracted considerable attention as an autoimmune disease. In this study, cellular immunity in patients with this disease was assessed. Specifically examined was the lymphocyte response to mitogens, Interleukin-2 (IL-2) production from peripheral blood mononuclear cells (PBMCs) and the percentage of lymphocyte subsets. No significant difference was observed in the lymphocyte response to phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) between untreated patients with Graves' disease and healthy people. IL-2 production in untreated patients, however, was significantly greater than that of healthy people. While a significant decrease was observed in the percentage of CD8+ cells in untreated patients, no difference was found in the percentage of CD5+, CD4+ and HLA-DR+ cells between them and healthy people. It is thought that the enhancement of IL-2 production by PBMCs and the decrease in the percentage of CD8+ cells (cytotoxic/suppressor cell) are associated with abnormalities in the immune system