Dinitrophenylated Bovine Gamma Globulin Research Articles

Competitive Homogeneous Chemiluminescent Immunoassay Using Antigen-modified Sheep Red Blood Cells

Abstract A novel method for immunoassay is reported. A competitive assay for dinitrophenylated bovine gamma globulin (DNP-GGB) was performed using anti-DNP serum and DNP-sheep red blood cells (DNP-SRBC). After complement-mediated hemolytic reaction, the chemiluminescent measurements were performed without any separation procedure. The sensitivity was 0.1 μg/ml of DNP-BGG.

Antibody affinity maturation in selectively bred high and low-affinity mice.

The affinity of serum antibodies produced by selectively bred lines of mice [high affinity, low affinity, low nonmaturing (N/M)] injected with T-dependent [human serum albumin (HSA), dinitrophenylated bovine gamma-globulin (DNP-BGG)] and T-independent (DNP-Ficoll) antigens in saline and adjuvant has been determined. The lines of mice differ significantly in the affinity of antibody produced to T-dependent antigens injected in saline but not to the T-independent antigen. Unlike mice of the high and low-affinity lines, low-affinity N/M mice failed to show affinity maturation to HSA and DNP-BGG injected in Freund's incomplete adjuvant. However, low-N/M mice responded to DNP-Ficoll injected in adjuvant by the production of antibody of affinity comparable to that produced in the other lines and with a similar maturation in affinity. Carrier priming resulted in the suppression of anti-hapten antibody affinity in all lines but low-N/M mice showed significantly greater suppression late in the response to challenge. Low doses of cyclophosphamide produced a significant increase in affinity in low-N/M mice. These results suggest that the failure of low-N/M mice to show affinity maturation results from increased suppressor T cell activity. The availability of the selectively bred mice provides a useful model for the detailed study of the cellular basis of the control of antibody affinity maturation.

Oral induction of the secretory antibody response by soluble and particulate antigens.

The ability of gastric intubation of a soluble or a particulate form of dinitrophenylated bovine gamma-globulin (DNP-BGG) to induce salivary IgA antibody was examined. Gastric intubation of the particulate form of the antigen tended to induce a greater response than did equivalent doses of the soluble form. However, systemic immunization with the soluble form of the antigen prior to gastric intubation of antigen increased the salivary IgA response. Prior systemic injection of the particulate form did not increase the salivary IgA response. Immunoreactivity of the particulate moiety of the particulate antigen did not seem to be a factor in the observed differences, since three different synthetic particulate antigens with identical hapten-protein moieties produced the same results (i.e. prior injection with the synthetic particulate antigens did not increase the salivary IgA response).

Inhibition of secondary in vitro antibody responses by the third component of complement.

Purified human C3 was found to inhibit rat in vitro secondary antibody responses. Fifty percent inhibition of antibody-forming cell development occurred with C3 concentrations of 26 micrograms/ml. This decrease was not the result of a general toxicity or a shift in the antibody response kinetics. Using cell mixing experiments, we could not detect a C3-induced suppressor lymphocyte or macrophage. C3 was active when added to culture early (day 0 or 1 or during a 24-hr antigen prepulse) or late (day 3, 5, or 7)--the early addition being more suppressive. Regardless of the addition time, there was a characteristic 48- to 72-hr lag before the inhibitory effect was manifested. C3 could inhibit antibody-forming cell development after stimulation with the thymus-independent antigens, trinitrophenyl-Brucella abortus and dinitrophenyl-Ficoll, as well as the thymus-dependent antigens, dinitrophenyl-bovine gamma-globulin and chicken gamma-globulin suggesting that C3 was not selective for B memory cell subpopulations. Further characterization of our C3 preparation indicated that the majority of the suppressive activity resided in a small m.w. protein resembling the C3a fragment of C3. Human C3a preparations generated either by trypsin cleavage or zymosan activation of C3 were also tested in our antibody response system and were able to inhibit antibody-forming cell development. These data implicate C3 cleavage products as negative regulators of antibody formation.

The Immunosuppressive Effect of Mycoplasma meleagridis on Nonreplicating Antigens

The humoral responses of Mycoplasma meleagridis-free and -infected turkeys were compared following primary and secondary antigenic stimulation with inactivated Salmonella pullorum or dinitrophenyl-bovine gamma globulin. The mycoplasma-infected groups had significantly lower antibody responses to both antigens. The suppression was more evident in the secondary response. The results suggest that the immune response in turkeys infected with M. meleagridis is similar to that of bursectomized chickens: the ability of chickens to synthesize immunoglobulin G is affected more readily than the ability to synthesize IgM.

Influence of PUVA and UVB Radiation on Delayed Hypersensitivity in the Guinea Pig

Exposure of guinea pigs to UVA (320--400 nm) radiation following administration of 8-methoxypsoralen by gavage (referred to by the acronym, PUVA) or exposure to UVB (290--320 nm) radiation, produced suppression of the cutaneous delayed hypersensitivity reaction at the site of exposure to radiation and at distant nonexposed sites. In these experiments, the animals were immunized by injection of dinitrophenyl-bovine gamma-globulin (DNP-BGG) in complete Freund's adjuvant and delayed hypersensitivity responses were provoked by intradermal injections of DNP-BGG, DNP and BGG on the flanks. Exposure to erythemogenic doses of either PUVA or UVB radiation for 7 days prior to immunization and for the 7 days between immunization and challenge (total period of radiation: 14 days) produced inhibiton of responses to each of the test substances. In addition, treatment with erythemogenic doses of PUVA either for 7 days prior to immunization or during the interval between immunization and challenge with DNP-BGG, inhibited the delayed hypersensitivity responses at the site of irradiation and at a nonexposed site. These findings suggest that in vivo exposure to nonionizing radiation leads to both local and systemic alteration of certain immune responses.

Suppression of Contact Sensitivity by IgG1 Antihapten Antibody in Contact-Sensitized Guinea Pigs

The guinea pig sera obtained 2 weeks after 1-fluoro-2,4-dinitrobenzene sensitization had ability to suppress elicitation of contact sensitivity when transferred intravenously into contact sensitized guinea pigs. The activity was found only in the sera obtained 2 weeks after the sensitization, whereas the sera obtained 1 and 3 weeks after the sensitization had no effect on contact sensitivity. Pretreatment of cyclophosphamide abolished the generation of the suppressive factor. The suppressive factor was hapten-specific. It was eluted from a Sephadex G-150 column into 7S gamma-globulin fraction and from DEAE ion exchange cellulose column into IgG1 fraction. The suppressive activity was absorbed on a dinitrophenylated bovine gamma globulin Sepharose 4B column and was eluted by either acidic buffered solution or dinitrophenol from the affinity column. It was absorbed by antiguinea pig IgG1-Sepharose and not by antiguinea pig IgG2-Sepharose, Therefore, the suppressive activity found in the sera of 1-fluoro-2, 4-dinitrobenzene sensitized guinea pigs was attributed to IgG1 antihapten antibody.

Ontogeny of B lymphocyte function. X. Strain differences in maturation of the capacity of the B lymphocyte population to produce a high-affinity antibody response.

AbstractThe ontogeny of the B lymphocyte population to be capable of producing a high‐affinity plaque‐forming cell (PFC) response to a haptenic determinant was studied by cell‐transfer techniques in five different mouse strains. Lethally irradiated adult mice were reconstituted with liver from fetal or neonatal donors or spleen from young donors as a source of B lymphocytes. In addition, all animals received 1 × 108 adult thymus cells. Recipients were immunized with dinitrophenylated bovine gamma‐globulin (DNP‐BGG) one day after cell transfer. The heterogeneity of affinity of their anti‐DNP PFC response was assayed by hapten inhibition of plaque formation. It was established by use of congenic mice differing at the Igh locus that only donor cells responded under these conditions. By use of this transfer system, it was shown that the B cell populations of A/HeJ, AKR and C57L/J mice aquire the capacity to produce a high‐affinity PFC response between day 15 and 18 of fetal life. The B cell populations of LAF1 and DBA/1 J mice mature between 7 and 10 days after birth. The B cell population of C57BL/6 J mice matures between 21 and 28 days of age. In LAF1 and AKR mice, the B cell population acquires the capacity to generate a high‐affinity indirect PFC response at approximately the same time as the capacity to generate a high‐affinity direct PFC response. The time of maturation of the capacity to generate indirect PFC was found to differ in different strains of mice. Maturation of the capacity to produce indirect PFC appears to represent a dinstinctly different differentiation event from the acquisition of the capacity to produce a high‐affinity PFC response. The data suggest that the maturation of the B cell population to be capable of producing high‐affinity PFC is a precisely regulated differentiation event, the timing of which is strain‐related. Preliminary data suggest that this regulation is not linked to either the H‐2 haplotype or to the Igh allotype.

Radiation-Induced Alterations in Murine Lymphocyte Homing Patterns: II. Recovery and Function of Memory Cells in LBN Rats

Suspensions of lymph node cells from dinitrophenylated bovine gamma globulin (DNP-BGG)-immune LBN F/sub 1/ hybrid rats (Lewis X Brown Norway) were prepared, irradiated, and injected intravenously into unirradiated syngeneic intermediate hosts and irradiated syngeneic adoptive controls. After allowance of 24 hr for homing to occur, the intermediate hosts were killed and cell preparations from the lymph nodes and spleen were injected intravenously into separate irradiated LBN final host groups. All control and experimental groups were challenged (DNP-BGG saline iv) 24 hr after the injection of the lymphoid cells. Rats were bled on Days 7, 11, and 14 after challenge and the antigen-binding capacity (ABC) of the serum was determined. After correction for the fraction of the total cell population transferred from the intermediate host, the peak ABC of the final hosts was related to the number of memory cells present. It was thus possible to determine the relative distribution of the memory cell population to the spleen and lymph nodes of the intermediate hosts. In the intermediate control animals, irradiated memory cells provided a secondary antibody response which was delayed but not suppressed when compared to unirradiated cells. In intermediate hosts, the homing of lymph node memory cells to themore » spleen and lymph nodes was significantly reduced by an exposure to 200 R of x radiation.« less

Potentiation of cytotoxic T-cell function by virus.

Inoculation of C57BL/6J mice with allogeneic P815 mastocytoma cells in the presence of simian virus 40 (SV40), a DNA tumor virus, led to an enhanced cytolytic T-cell response to P815 in vivo. Cytotoxic function was also augmented if SV40 was given subsequent to a primary immunization, even when mice were given a suboptimal dose of immunizing cells. Although SV40 increased the cell-mediated immune response to allogeneic cells, it did not enhance the antibody response to the soluble antigen dinitrophenyl bovine gamma-globulin, a helper T-cell-dependent response. Thus it appeared that SV40 had a selective adjuvant effect on lymphocyte subpopulations, since it increased cytotoxicity but not helper T-cell function.

Desensitization of delayed-type hypersensitivity by antigen and specific antibody

The role of antibody in the desensitization of delayed-type hypersnsitivity (DTH) to dinitrophenylated bovine gammaglobulin (DNP-BGG) was studied in rats. Rats sensitized by a subcutaneous injection of DNP 32-BGG in Freund's complete adjuvant (FCA) were desensitized 14 days later with various doses of DNP 32-BGG injected intravenously. It was found that only certain doses (100–500 μg) of DNP-BGG effectively desensitized, antigen doses outside this optimum range being ineffective in suppressing DTH. In adoptive cell transfer experiments, it was shown that sensitized peritoneal cells incubated with optimum doses of the antigen in the presence of specific antiserum in vitro failed to transfer the delayed response to normal recipients, whereas the treatment of the sensitized cells with the antigen or with the antiserum separately did not impair the ability of these cells to transfer DTH. The effect of desensitization is specific and is not permanent. The DTH reappears 3–4 wk after desensitizing injection.

Immunogenicity of Lowly and Highly Derivatized Dinitrophenyl-Bovine γ-Globulin in Different Strains of Guinea Pigs

Abstract Guinea pigs immunized with lowly derivatized dinitrophenylated bovine gamma globulin produced large amounts of antibody directed against the carrier molecule and large amounts of antibody directed against the hapten. Guinea pigs immunized with a highly derivatized preparation did not produce antibody against the carrier but produced large amounts of antibody directed against the hapten. Both preparations provoked the production of about the same amount of anti-hapten antibody, but the lowly derivatized antigen provoked the production of anti-hapten antibody of higher average affinity.

Antigen modulation of the immune response. The effect of delayed challenge on the affinity of anti-dinitrophenylated bovine gamma globulin antibody produced in adoptive recipients.

Secondary immune responsiveness to dinitrophenylated bovine gamma globulin (DNP-BGG) was transferred to heavily irradiated rats by means of small lymphocytes from the thoracic duct of immunized syngeneic donors. Affinity of the antibody produced by the adoptive recipients when challenged immediately was the same as that seen in immunized controls and cell donors, suggesting that the performance of lymphocytes in the thoracic duct of immunized rats provide an accurate measure of the immunologic history of memory cells both in the intact animal and the lymphocyte-depleted donor. The relevance of this to cell cooperation in the production of antibody to hapten-protein complexes is discussed. Direct evidence on antigen modulation of the immune response was also obtained. When immune thoracic duct lymphocytes were transferred to adoptive recipients and challenged either immediately or after a delay of 6 wk, it was found that, although the amount of antibody decreased with this delay in challenge, the affinity remained constant. Thus it would seem that in control rats, the increase in the affinity of antibody with time after immunization is, in fact, due to the selection of new populations of cells by the progressively waning concentration of antigen.

Radioresistance of cooperative function of carrier-specific lymphocytes in antihapten antibody responses.

Transfer of lymphoid cells from strain-2 guinea pigs immunized to bovine gamma globulin into syngeneic recipients immunized with dinitrophenyl ovalbumin markedly enhances the secondary antidinitrophenyl response of the recipient to challenge with dinitrophenyl-bovine gamma globulin. This function of the carrier bovine gamma globulin-specific cells is resistant to irradiation with up to 5000 rads, although the capacity of the irradiated cell population to transfer immunologic memory for bovine gamma globulins or to be stimulated by antigen to synthesize DNA in vitro is abolished by this treatment.

EFFECT OF BOVINE GAMMA GLOBULIN ON SUBSEQUENT IMMUNIAZTION WITH DINITROPHENYLATED BOVINE GAMMA GLOBULIN.

Rabbits were immunized with bovine gamma globulin intravenously in saline and were reinjected with dinitrophenylated bovine gamma globulin intravenously. No secondary effect on anti-dinitrophenyl antibodies was observed by prior injection of bovine gamma globulin; on the contrary, no anti-dinitrophenyl antibody was produced in these animals whereas in controls, which had not received prior bovine gamma globulin injections, antibody production was detected, especially with highly derivated dinitrophenyl-bovine gamma globulin preparations. A secondary effect against bovine gamma globulin was observed, especially with the less derivated preparation.