Immunoneutralization of inhibin modifies hormone secretion and sperm production in bulls.

The objective of this study was to examine the role of endogenous inhibin in the regulation of FSH, LH, and testosterone secretion and sperm production in bulls. Bulls were actively immunized against bovine inhibin alpha 1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG) or HAG alone (controls) and emulsified in Freund's complete adjuvant. Primary immunization was at 14 wk of age, followed by booster immunizations in Freund's incomplete adjuvant at 28, 30, and 34 wk of age. Ten days after each booster immunization, scrotal circumferences and body weights were measured, and blood was sampled for determination of bINH antibody titer. Ten days after the third booster, blood was sampled at 1-h intervals for 8 h to quantify serum concentrations of FSH, LH, and testosterone. After this blood sampling period, bulls were castrated and testicular sperm production was determined. Serum diluted 1:4,000 from bINH-immunized bulls bound 36%, 52%, and 53% of radioiodinated bINH after the first, second, and third boosters, respectively. Serum from controls bound less than 1% radioiodinated bINH. After the third booster, serum concentrations of FSH and testosterone were increased (p less than 0.05) and LH concentrations were decreased (p less than 0.001) in bINH-immunized bulls compared with controls. After the third booster, daily sperm production per gram of testicular parenchyma was increased (p less than 0.05) in bINH-immunized bulls compared with controls. Scrotal circumferences and body weights were similar between treatment groups throughout the experiment. We concluded that inhibin has a role in regulation of secretion of gonadotrophins and testosterone and testicular sperm production, but not testicular growth, in bulls.(ABSTRACT TRUNCATED AT 250 WORDS)     

The generation of monoclonal antibodies in mice: influence of adjuvants on the immune response, fusion efficiency and distress

The objective of this study was to find a reliable alternative to Freund's adjuvant in order to reduce the distress imposed on the animals without impairing the fusion efficiency for immune-positive clones. For this purpose several commercially available adjuvants and adjuvant formulations representing different classes of molecules were compared. Humoral responses and animals' distress evaluated by clinical assessment and histopathological examinations were investigated and compared to fusion efficiencies. In a first set of experiments seven adjuvants were tested essentially to determine their potential to induce distress. Poly(A).poly(U) and GERBU were selected for further investigations due to their low overall toxicity. They were combined with five different antigens and compared to the classic Freund's adjuvant system (CFA/IFA) and to control immunizations without adjuvant. The results showed that adjuvants of very low toxicity could induce a high fusion efficiency. According to a standardized immunization protocol, GERBU induced polyclonal titres similar to Freund's whereas animals treated with poly(A).poly(U) did not attain titres higher than mice immunized with antigen in saline. Poly(A).poly(U) however, exhibited the best fusion efficiency, Freund and GERBU were slightly less efficient. Therefore poly(A).poly(U) and GERBU may serve as valuable alternatives to Freund's adjuvant for generating monoclonal antibodies. Furthermore, these two adjuvants are very easy to use.     

Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in long-term HIV-1 neutralizing humoral mucosal and systemic immunity

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.     

Production of large amounts of antibodies in individual mice.

A method is described for the elicitation of substantial amounts of anti-hapten and anti-protein antibodies in ascitic fluids of individual mice of various strains. The method utilizes repeated i.p. inoculations of u.5-mg quantities of antigen in complete Freund's adjuvant. A high volume ratio of adjuvant to antigen solution, and the volume used per inoculation, were shown to be critical factors. Ascitic fluids developed after three to five inoculations and were subsequently tapped repeatedly over a period of several weeks. All strains tested produced substantial amounts of anti-KLH antibodies, but marked differences were noted with respect to response to a hapten. Titers were enhanced in low responders by priming before the induction of ascites. The method is also useful for the production of "nonspecific" IgG and other serum components.     

The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits.

Although Freund's adjuvant has been used for decades as an immune enhancer in rabbits, adverse physiologic side effects have prompted the search for more suitable alternatives. We used osteocalcin, a bovine bone protein (M.W. 5,800), as the test antigen to evaluate four adjuvant regimens: a) primary inoculation with complete Freund's adjuvant (CFA) followed by three boosts with incomplete Freund's adjuvant (IFA), b) four serial inoculations with RIBI MPL+TDM+CWS adjuvant, c) four serial inoculations with TiterMax #R-1, and d) primary inoculation (only) with TiterMax #R-1. The antibody yield associated with the CFA/IFA regimen (mean OD = 2.152) was at least sixfold that of either TiterMax (mean OD = 0.358) or RIBI (mean OD = 0.239) multiple injection regimens. No antibody response was observed after the single injection of TiterMax antigen emulsion. Maximal antibody production occurred rapidly in response to Freund's adjuvant (day 31) as compared with TiterMax (day 74) and RIBI (day 66).     

Isotype-specific resistance against tolerance induction in SJL mice

Age-related changes in antibody response of SJL mice were examined in terms of isotype expression after treatment with immunogen or with immunogen, preceded by the molecule in normally tolerogenic form. We report here that tolerance induction and resistance to down regulation are isotype specific. Tolerance can be induced in terms of all detectable isotypes at the age of 5 weeks. In older SJL mice, tolerance to the carrier is found in IgM antibody, whereas there is resistance against down regulation in terms of IgG2a and IgG2b isotypes, and sensitization in terms of IgG3, IgG1, and IgA antibody. Furthermore, the degree of down regulation is determinant dependent. This was observed when older SJL mice, pretreated with the carrier in a normally tolerogenic form, were immunized with haptenated carrier and tested for their response to hapten and carrier determinants. In this case, IgA antibody shows tolerance to the hapten and sensitization by carrier determinants.     

An evaluation of distress following intraperitoneal immunization with Freund's adjuvant in mice

Intraperitoneal immunization with Freund's adjuvant is frequently used to stimulate antibody production in mice. To evaluate the clinical and pathological effects of this technique, mice were immunized intraperitoneally with complete Freund's adjuvant and albumin, and the injection repeated 3-4 weeks later using incomplete Freund's adjuvant. This regimen induced a mean antibody titer against albumin of 1:280 within 7 days after booster immunization and increased the abdominal width, abdominal circumference and spleen weights of immunized animals. Food intake and body weight decreased after immunization, but returned to control levels within 1-2 weeks. Open-field activity was not affected. Neutrophilia, eosinophilia and monocytosis were present 7 days after immunization and persisted for the duration of the study. Gross and histopathological lesions included multiple granulomatous abdominal adhesions and lymphoid hyperplasia. Thus, intraperitoneal immunization with Freund's adjuvant and albumin produced some adverse effects in the animal (weight loss, neutrophilia and granulomatous peritonitis). However, the animals did not appear to be severely or chronically impaired, since food intake, body weight and locomotor activity were within normal limits for most of the post-immunization period.     

A comparison of antibody titres in mouse uterine fluid after immunization by several routes, and the effect of the uterus on antibody titres in vaginal fluid

Measurements of specific antibody titres in uterine fluid of mice immunized by different routes indicated that two immunizations in the pelvic presacral space using aluminium hydroxide as adjuvant was a simple and effective way to elicit a significant IgA and IgG response. Higher IgA and IgG titres were produced in uterine fluid by subcutaneous immunization with antigen in Freund's complete adjuvant followed by intravaginal boosting without adjuvant, but this immunization involved both a toxic adjuvant and repeated applications of large doses of antigen in the vagina. Intragastric immunization produced an IgA response in the uterus but no IgG. Local intravaginal priming and boosting with large doses of antigen without adjuvant produced an IgA response in uterine fluid, but was less effective for IgG and was inefficient in terms of time and the amount of antigen used. Hysterectomy reduced the concentration of specific IgA in vaginal fluid of immunized mice to no more than 5% of normal, indicating that most of the IgA in vaginal fluid originates in the uterus. In contrast, IgG titres were not significantly different in hysterectomized and intact mice. IgA titres in vaginal fluid were at least partly restored to normal levels in sham-hysterectomized mice.     

Antigen recognition in delayed-type hypersensitivity.

It is still uncertain if cell-mediated immune reactions are more or less specific than antibody-mediated reactions. Accordingly, hapten and carrier specificity were examined in delayed hypersensitivity in guinea pigs. Hapten specificity was demonstrated with 2,4-dinitrophenyl (DNP)-guinea pig albumin (GPA), 2,6-DNP-GPA, 2,4,6-trinitrophenyl (TNP)-GPA, and dansyl (DNS)-GPA. Guinea pigs immunized with each of these conjugates were tested 7 days later with the immunogen and the other conjugates. Strong delayed skin responses were highly specific for the immunogen; there were some weak cross-reactions among the nitrophenyl conjugates, no crossre-actions between the DNS and nitrophenyl conjugates, and no responses to unconjugated GPA. Conjugates carrying different numbers (1–45) of 2,4-DNP groups per molecule were all able to elicit specific responses to 2,4-DNP.Carrier specificity in delayed hypersensitivity was confirmed by immunizing with 2,4-DNP-GPA, and challenging with the immunogen, with 2,4-DNP coupled to bovine albumin (BSA), rabbit IgG, ovalbumin, and hemocyanin. Strong responses were seen to the immunogen, a weak response to 2,4-DNP-BSA, and no response to the other conjugates. Specific immune recognition of both hapten and carrier determinants is therefore required for expression of delayed hypersensitivity. These cell-mediated reactions thus appear to be more specific than those of antibody-mediated reactions in solution.     

Increase in ovulation rate after immunization of Merino ewes with a fraction of bovine follicular fluid containing inhibin activity

Affinity chromatography of bovine follicular fluid using Matrix gel Red A resulted in a 20-fold increase in inhibin-like specific activity assessed in a mouse ovulation inhibition test system. When this material was emulsified with Freund's adjuvant and injected into adult Merino ewes their mean ovulation rate was increased from 1.2 to 2.3 (P less than 0.01). Follicles of diameter greater than or equal to 3.5 mm and 2-3.4 mm were also increased (4.33 vs 2.25 and 5.39 vs 2.44 per ewe respectively). The ovulation rate response was variable and transient. Length of oestrous cycles, number of granulosa cells per follicle and seasonal oestrous patterns were not affected. Plasma from the immunized ewes contained antibodies to the immunogen and reversed the ovulation-inhibiting effects of bovine follicular fluid in mice.     

The influence of various adjuvants on antibody synthesis following immunization with an hapten.

For the production of specific antibodies to the hapten MATP (4-Amino-1,2,2-trimethyl-phenylphosphonate) in Balb/c mice various non-toxic adjuvants were compared to Freund's complete adjuvant (FCA). For immunization the hapten MATP was coupled to the carrier human serum albumin (HSA). The immunostimulating effect of the synthetic lipopeptides Pam3Cys-OH, Pam3Cys-Ser-Ser-Asn-Ala and different concentrations of the lipohexapeptide Pam3Cys-Ser-(Lys)4 (Pam3Cys = S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N- palmitoyl-(R)-cysteine as well as of aluminium hydroxide were tested. IgG antibody titers in serum were determined in ELISA. In dose-response studies 50 micrograms Pam3Cys-Ser-(Lys)4 per mouse was the most effective dose with a long period of high antibody levels after the second booster. Pam3Cys-Ser-Ser-Asn-Ala provoked only low antibody titers. Immunostimulation with Pam3Cys--OH did not result in an increased production of specific antibodies. Compared to the control group an enhanced antibody synthesis could be provoked with aluminium hydroxide. However, the increase was much smaller than by using FCA. The lipopeptide Pam3Cys-Ser-(Lys)4 turned out to be a very potent adjuvant. One week after booster injection into mice 50 micrograms of this substance helped to elicit a higher antibody titer than FCA. Hence, as far as the degree of antibody production is concerned, Pam3Cys-Ser--(Lys)4 represents an alternative adjuvant to FCA.     

Anti-tumor vaccine adjuvant effects of IL-2 liposomes in mice immunized against MCA-102 sarcoma.

MCA-102, a murine sarcoma previously reported to be non-immunogenic in C57/BL6 murine tumor models was used in a tumor vaccine preparation which included liposome encapsulated IL-2 as an adjuvant. C57/BL6 mice were immunized in the right hind footpad with irradiated MCA-102 murine sarcoma cells on days 0, 7, and 21 with or without IL-2 liposome adjuvant at 25,000 IL-2 units/injection. Mice were challenged with live tumor in the right flank on day 35. Survival of mice given IL-2 liposomes with irradiated MCA-102 cells was significantly prolonged over mice given tumor antigen with saline, and non-immunized mice. In addition, mice which received the IL-2 liposome adjuvant also had prolonged survival over those mice immunized with the additional control adjuvants of free IL-2 or dimyristoyl phosphatidyl choline (DMPC) lipid in the form of empty liposomes. IL-2 liposome plus tumor antigen also yielded a significant local protective response against live MCA-102 tumor challenge. When live tumor was injected into the site of previous immunizations on day 21 after two immunizations, the IL-2 liposome adjuvant group showed significantly delayed local growth of tumor compared to animals immunized without adjuvant, or with the adjuvants of empty liposomes or free IL-2. Finally, immunized mice were challenged with irradiated tumor cells and saline intradermally in the ears and delayed type hypersensitivity (DTH), an indicator of helper T cell response, was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune responses to small nuclear ribonucleoproteins: antigen-dependent distinct B cell epitope spreading patterns in mice immunized with recombinant polypeptides of small nuclear ribonucleoproteins

Complex patterns of autoantibody reactivities with the small nuclear ribonucleoproteins (snRNPs) are observed in systemic lupus erythematosus. To investigate the role of individual snRNP components in the initiation and diversification of anti-snRNP Ab responses, we immunized A/J mice with recombinant Smith D (SmD), Smith B (SmB), and A ribonucleoprotein (A-RNP) with alum as adjuvant. Sera at different time points after initial immunizations were analyzed by Western blot and immunoprecipitation assays. In SmD-immunized mice, specific Abs to A-RNP and SmB were generated by 2 mo postimmunization, in addition to the detection of cross-reactive Abs between the immunogen and other snRNPs. Whereas Abs reactive with the immunogen decreased by 5 mo, Abs capable of immunoprecipitating A-RNP and SmB increased. In SmB-immunized mice, specific Abs to A-RNP were readily detectable, in addition to cross-reactive Abs. In contrast, A-RNP-immunized mice had only cross-reactive Abs to SmB without detectable Abs to SmD. However, in these mice, specific Abs to the 70-kDa protein were generated. Abs, which precipitated the native snRNP particle, were generated in all three groups of the immunized mice. Our results show that different initiating Ags from the same multiprotein antigenic complex induce distinct patterns of epitope spreading to proteins within that complex. These data have significant implications for the mechanisms of autoantibody diversification in systemic lupus erythematosus.     

Active immunization of gilts against gonadotropin-releasing hormone: effects on secretion of gonadotropins, reproductive function, and responses to agonists of gonadotropin-releasing hormone

Sexually mature gilts were actively immunized against gonadotropin-releasing hormone (GnRH) by conjugating GnRH to bovine serum albumin, emulsifying the conjugate in Freund's adjuvant, and giving the emulsion as a primary immunization at Week 0 and as booster immunizations at Weeks 10 and 14. Antibody titers were evident by 2 wk after primary immunization and increased markedly in response to booster immunizations. Active immunization against GnRH caused gonadotropins to decline to nondetectable levels, gonadal steroids to decline to basal levels, and the gilts to become acyclic. Prolactin concentrations in peripheral circulation were unaffected by immunization against GnRH. The endocrine status of the hypothalamic-pituitary-ovarian axis was examined by giving GnRH and two agonists to GnRH and by ovariectomy. An i.v. injection of 100 micrograms GnRH caused release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in control animals, but not in gilts immunized against GnRH. In contrast, administration of 5 micrograms D-(Ala6, des-Gly-NH2(10] ethylamide or 5 micrograms D-(Ser-t-But6, des-Gly-NH2(10] ethylamide resulted in immediate release of LH and FSH in both control and GnRH-immunized gilts. Circulating concentrations of LH and FSH increased after ovariectomy in the controls, but remained at nondetectable levels in gilts immunized against GnRH. Prolactin concentrations did not change in response to ovariectomy. We conclude that cyclic gilts can be actively immunized against GnRH and that this causes cessation of estrous cycles and inhibits secretion of LH, FSH, and gonadal steroids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of the virulent RH strain of Toxoplasma gondii by passages in mice immunized with Toxoplasma lysate antigens

The virulent RH strain of Toxoplasma gondii was attenuated after a few passages or just one long passage in mice immunized twice with a four-week interval between immunizations with an emulsion of Toxoplasma lysate antigens and complete Freund's adjuvant. Three avirulent strains, RH-cyst III, IV and VIII were established from the RH strain. The RH-cyst III strain was effective for vaccination against challenge with the original, virulent RH strain. The attenuation of T. gondii is an expression of the innate attributes of this parasite necessary to maintain its parasitic life cycle in nature.     

Evaluation and separation of steroid-bovine serum albumin conjugates by high-performance liquid chromatography

The conventional methods for characterization of steroid immunogens are based on the determination of the total amount of hapten bound to the protein carrier either by the UV spectroscopy or titration of unsubstituted amino groups. These methods do not allow more detailed insight into the immunogen composition. HPLC of the immunogen combined with UV detection is a relatively rapid and convenient method enabling determination of the hapten content in each fraction and, eventually, separation of individual fractions differing in the hapten content or purification of crude product.     

Effect of adjuvants on the humoral immune response to congopain in mice and cattle

ABSTRACT: BACKGROUND: We investigated several adjuvants for their effects on the humoral immune response in both mice and cattle using the central domain of congopain (C2), the major cysteine protease of Trypanosoma congolense, as a model for developing a vaccine against animal trypanosomosis. The magnitude and sustainability of the immune response against C2 and the occurrence of a booster effect of infection, an indirect measure of the presence of memory cells, were determined by ELISA, while spectrofluorometry was used to determine and measure the presence of enzyme- inhibiting antibodies. RESULTS: Mice immunized with recombinant C2 in AdjuphosTM, TiterMaxTM, purified saponin Quil ATM or GERBUTM showed the best response according to the evaluation criteria and these three were chosen for the cattle vaccination study. The animals were challenged with T. congolense four and a half months after the last booster. Cattle immunized with recombinant C2 in purified saponin Quil ATM showed the best antibody response according to the measured parameters. CONCLUSIONS: We identified purified saponin Quil ATM as a good adjuvant for immunizations with C2. The results from this study will be useful in future attempts to develop an effective anti-disease vaccine against African trypanosomosis.     

金霉素单克隆抗体的制备及检测方法的建立

采用羰基二咪唑法,将半抗原金霉素(AM)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制备金霉素免疫抗原AM-BSA和检测抗原AM-OVA,通过紫外光谱扫描检测偶联产物。采用细胞杂交瘤技术,制备抗金霉素单克隆抗体杂交瘤细胞株,建立了金霉素竞争ELISA检测方法,其灵敏度达到50ng/ml,且呈现良好的线性关系(r=0.9812),并且与其他抗生素无交叉反应。     

Potentiation of antibody responses by specific IgM: specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.     

Hyperimmune hens as a novel source of anti-Cryptosporidium antibodies suitable for passive immune transfer.

Leghorn hens were subcutaneously immunized with 25 micrograms of Cryptosporidium parvum oocyst emulsified in Freund's complete adjuvant. A booster dose was injected 5 weeks later. Anti-Cryptosporidium activities of yolks and sera measured by an enzyme-linked immunosorbent assay (ELISA), demonstrated high levels in both sera and egg yolks which persisted for at least 17 wk. Preparations from yolks with high, medium and low anti-Cryptosporidium ELISA activities were used in a neonatal mouse model to assess their biological activities. A significant parasite reduction (P less than or equal to 0.001) was found between the high and all other groups. Hyperimmune eggs could be used as a source for passive immunity in cryptosporidiosis.