Localization and verification by synthesis of five antigenic sites of bovine serum albumin.

Recently we have shown that the major antigenic sites of bovine serum albumin exhibit functional equivalence progessively increasing with the time at which antibodies are obtained after the first immunization. Analysis of our recent immunochemical findings and the known covalent structure of bovine serum albumin have enabled us to predict the locations of five antigenic sites of bovine serum albumin. The predicted locations were synthesized, and immunochemical studies with late-course antisera showed them to constitute antigenic sites of native bovine serum albumin.     

Antibodies against p-aminophenyl-beta-D-galactopyranoside-containing proteins

The antibodies were prepared from antisera of rabbits immunized with bovine serum albumin containing covalently bound p-aminophenyl-beta-D-galactopyranoside (APG) and purified by affinity chromatography on APG-containing ovalbumin immobilized by BrCN-activated Sepharose 4B. The antibodies possessed a selective specificity for APG and interacted with different APG-containing proteins, including APG-containing lysosomal alpha-glucosidase. The purified antibodies are immunoglobulins of G type as was determined from the molecular weights of native and dissociated antibodies and from the immunochemical assays with antibodies against rabbit IgG.     

Immune recognition of serum albumin.15. Localization by synthesis of antigenic site 4 of bovine serum albumin to the region around the disulfide bond 166-175

Recently, we have localized and confirmed by synthesis the regions within which reside five major antigenic sites of bovine serum albumin and proposed that the remaining sixth major antigenic site (antigenic site 4) was localized within subdomain 3 (fragment 115-184) of albumin. In the present work, antigenic site 4 was localized to fall around the disulfide bond 166-175 by synthesis and immunochemical reactivity of the region 162-179. The synthetic 18-residue peptide was shown to bind substantial amounts of antibodies from early (38-day) and late (398-day) anti-albumin antisera from two rabbits. As much as half the total anti-albumin antibodies could be bound by the peptide from the late antisera. It was concluded that antigenic site 4 resides within, but may not include all of, the region 162-179 of albumin.     

Tryptophan content of serum albumin

Tryptophan content of serum albumin was determined spectrophotometrically. The method used for the determination of tryptophan gave consistent results. Results show that the tryptophan contents of bovine and human serum albumin are significantly different from chicken serum albumin. Bovine and human serum albumins, however, are not significantly different from each other. A large difference in tryptophan content was found between two samples of chicken serum albumin. This suggests that the tryptophan content of serum albumin may not be constant for any given species. For these reasons, tryptophan content should not be used to estimate the molecular weight of serum albumin.     

Elimination of Homologous And Heterologous (Bovine) Serum Albumin from the Blood Circulation in the Dog)

The elimination of intravenously administered I131-labelled bovine serum albumin has been compared to the elimination rate of relabelled homologous serum albumin in normal and bled dogs, which had lost considerable blood volumes. The investigation shows that during the first four to five days after the administration the elimination is similar of heterologous and homologous serum albumin. This proves that bovine serum albumin can be regarded to be an equivalent plasma expander to homologous serum albumin in the dog. Elimination of homologous as well as heterologous serum albumin follows a simple exponential curve during four to five days after administration. The intravascular half-lives for homologous serum albumin were 6.4 ±1.5 days and 6.4 ± 0.6 days respectively in control and bled dogs. Corresponding values for heterologous (bovine) serum albumin were 5.0 ± 0.3 and 4.8 ± 0.4 days respectively. The quote for cencentrations of homologous and heterologous serum albumin in different tissues was found to be relatively constant approximately 1.4. An exception was the stomach wall in bled dogs which had a quote of 1.1 only.     

A fragment comprising the last third of bovine serum albumin which accounts for almost all the antigenic reactivity of the native protein.

The fragmentation of native bovine serum albumin by trypsin has been studied in aqueous solution under various conditions with regard to the yield and size of the fragments obtained. From a partial tryptic hydrolysate at pH 8.2 (40 degrees, 1 hour), a homogeneous fragment was isolated in high yield by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose. The molecular weight of the fragment by gel filtration on calibrated Sephadex G-100 columns and by sodium dodecyl sulfate electrophoresis was 22,500. After reduction of the disulfide bonds followed by alkylation of the resultant thiol groups with iodoacetamide, the fragment retained homogeneity by disc electrophoresis and its molecular weight remained unchanged, indicating that it was composed of a single polypeptide chain. From its amino acid composition, sequence of the first 20 residues, and actions of carboxypeptidases A or B, it was unequivocally assigned to positions 377-571 in albumin. The inhibitory activity of the fragment was 90 to 93% towards the immune reaction of the protein with the IgG fraction of the antisera. The IgGfraction accounted for 96% of the total antibody activity in the antisera. An immunoabsorbent of fragment 377-571 removed 89 to 95% of the antibody to albumin. A fluorescent derivative of the fragment, which retained full immunochemical activity, was found to bind 2 mol of antibody/mol of peptide. The disulfides in peptide 377-571 were essential for its immunochemical reaction because the latter was entirely abolished upon reduction and S-alkylation of the disulfides. Since this fragment comprised only a third of the albumin molecule, but accounted for 90 to 95% of its antigenic reactivity, the results indicated that native albumin carries identical repeating antigenic reactive sites.     

Antibodies elicited by defined oligodeoxyribonucleotide sequences.

Antibodies to the oligodeoxyribonucleotides d(pT)3, d(pT)4, d(pT)6 and d(pA-A-T-T) were elicited in rabbits by immunization with electrostatic complexes of the respective haptens with methylated bovine serum albumin (MBSA). The antisera were assayed by complement fixation using denatured DNA's of various sources as antigens. The specificities of the antibodies were determined by estimating the inhibition of the complement fixation reaction by defined oligodeoxyribonucleotides. The antibodies were shown to be specific for the sequence of the oligode-oxyribonucleotides or parts of it.     

Insulin-like growth factors I and II in fetal and adult bovine serum. Purification, primary structures, and immunological cross-reactivities

Insulin-like growth factors I and II (IGF-I and IGF-II) were prepared from fetal calf serum and adult bovine serum by gel filtration, immunoaffinity chromatography, chromatofocusing, and reverse-phase high pressure liquid chromatography, and their complete amino acid sequences determined. IGF-I and -II are found in both adult and fetal serum. The sequence of bovine IGF-I is found to be identical to that of human IGF-I, whereas 3 out of 67 amino acid residues are found to be different between bovine and human IGF-II. The differences are located in the C-peptide region of the molecule. Bovine IGF-II shows less than 10% immunological cross-reactivity with antisera against human and rat IGF-II, but is equipotent to human IGF-II in displacing human 125I-labeled IGF-II from human placental receptor. Bovine IGF-I was equipotent to human IGF-I in both radioimmunoassays and radioreceptor assays within the limits of the assay.     

A non-chromatographic radioimmunoassay for serum dehydroepiandrosterone using a mixture of antisera

A simplified method for evaluating serum dehydro-epiandrosterone (DHEA) without chromatography has been developed, using mixtures of two different anti-DHEA antisera, anti-3β-hydroxy-Δ⁵ antiserum and anti-11-deoxy-17-ketosteroid antiserum, in which cross-reactivity of each antiserum is reduced to a negligible amount. Serum (20 μ1) was extracted with 1 ml of n-hexane. One milliliter of 80% methanol was added to the n-hexane extract which was stirred and centrifuged. The n-hexane layer was discarded, and the methanol layer was evaporated to dryness. The residue was incubated with an antiserum mixture containing DHEA-7α-³H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEA-7α- ³H. The accuracy, precision, sensitivity and specificity were satisfactory. Good agreement was found between the serum DHEA levels obtained by the present radioimmunoassay and those obtained by radioimmunoassay with paper chromatography, making this method suitable for routine use.     

Antisera reactive directly to estrone sulfate.

Derivatives of estrone were prepared and linked to bovine serum albumin or its methyl-esterified form to produce immunogens which were effective in raising antisera to estrone sulfate. The most effective was estrone-3-methylphosphonothioate, electrostatically complexed with methylated bovine serum albumin. The ionically combined hapten functioned as an antigenic determinant as do covalently bound haptens when administered to sheep in emulsions with Freund's complete adjuvant. Estrone-3-phosphate covalently or electrostatically linked to bovine serum albumin also produced antisera reactive to estrone sulfate. Estrone sulfate itself, after electrostatically complexing to methylated bovine serum albumin and administration with Freund's complete adjuvant to sheep, was ineffective in producing antisera. The sera which had workable titres to estrone sulfate showed considerable cross-reaction with free estrone but was otherwise highly specific with little or no reaction with other steroid sulfates, glucosiduronates or other free steroids. Radioimmunoassay curves using [6,7-3H]-estrone sulfate were highly sensitive and were effective in the range of 5-250 pg estrone sulfate.     

Unfolded transferrin polypeptide chain is immunologically crossreactive with similar derivatives of serum albumin and alpha-fetoprotein

Radioimmunoassays utilizing reduced and carboxymethylated (RC) proteins as antigens reveal a cross-reactivity between alpha-fetoprotein (AFP) and albumin. Similar assays were used to study the relationships of AFP and albumin to other serum proteins. Of the several serum proteins tested, transferrin showed the most similarity with AFP and albumin. There was no cross-reactivity of the native proteins, but antisera prepared against RC-albumin and RC-AFP bound 125I-labeled RC-transferrin at high titers, and antiserum to RC-transferrin bound labeled RC-AFP but not RC-albumin. Inhibition assays utilizing binding of 125I-RC-AFP or 125I-RC-transferrin to anti-RC-albumin showed that the RC derivatives of AFP, albumin, and transferrin were equally efficient inhibitors, whereas other serum proteins inhibited much less. The serum vitamin D carrier protein (Gc protein) showed intermediate reactivity. The reactivities of the antisera to RC-albumins with RC-transferrin and RC-Gc protein were corroborated by immunostaining of proteins separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. These antisera stained the bands formed by RC derivatives of albumin, AFP, transferrin, and Gc protein, but not other proteins tested. AFP and albumin are known to have amino acid sequence homology. Our results suggest that transferrin and possibly also Gc protein may be structurally related to AFP and albumin.     

Factors affecting production of contrasensitizing antisera in mice.

Using four different protein antigens, two different strains of mice, and various immunization protocols, we have studied production in mice of immunological enhancement antibodies that specifically suppress induction of delayed hypersensitivity. Primary assay of these antibodies was in vivo, because no in vitro test used detected them dependably. Any antigen priming that favored initiation of humoral antibody responses prepared mice to make these contrasensitizing antibodies vigorously following appropriate boosting. The method of boosting usually was more important than that of priming, high titers regularly developing only when primed mice were boosted with much antigen in a short time and were bled a few days later. The presence or absence of delayed hypersensitivity was immaterial. CAF₁ mice made these antibodies better than CF-1 mice, and antigen effectiveness correlated with propensity to induce humoral antibody formation in mice, decreasing from ovalbumin through human serum albumin and bovine serum albumin to methylated human serum albumin. In certain antigenmouse combinations (e.g., ovalbumin in CAF₁ mice) immunosuppressive antibody production was vigorous and prolonged; in others (e.g., bovine serum albumin in CF-1 mice) it was moderate and brief. From our results one can predict what conditions should induce formation of strongly enhancing/contrasensitizing antisera, and speculate that these conditions also should elicit strong, active immunologic tolerance for averting induction of delayed hypersensitivity.     

Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol.

Methoxypolyethylene glycols of 1900 and 5000 daltons have been attached covalently to bovine serum albumin using cyanuric chloride as the coupling agent. When sufficient polymer is attached, the modified bovine serum albumin appears to lose its immunogenicity in the rabbit and, on intramuscular or intravenous injection, elicits antibodies neither to itself nor to native bovine serum albumin. It does not react with antibodies raised against native bovine serum albumin. Bovine serum albumin to which methoxypolyethylene glycol has been attached exhibits a blood circulating life in the rabbit rather similar to native bovine serum albumin, except that it is not removed from circulation by the eventual development of antibodies. Modified bovine serum albumins which had been iodinated with 125I, or prepared with [14C]cyanuric chloride, were injected intravenously in rabbits. Both labels appeared almost quantitatively in the urine after 30 days. The modified bovine serum albumins showed substantial changes in properties, such as solubility, electrophoretic mobility in acrylamide gel, ion exchange chromatography, and sedimentation, as compared with the unmodified protein.     

Production and characterization of antibodies against nivalenol tetraacetate.

Antibody against nivalenol tetraacetate (tetra-Ac-NIV) was prepared by immunization of rabbits with triacetyl-15-pimelate-NIV conjugated to bovine serum albumin. By using tritiated tetra-Ac-NIV as the test ligand, antibody titers were demonstrated as early as 4 weeks after immunization. Useful antibody for radioimmunoassay (RIA) of tetra-Ac-NIV was obtained 7 weeks after immunization, with one booster injection. Results of competitive RIA revealed that the antibody was most specific to tetra-Ac-NIV. The relative cross-reactivity of this antibody with tetra-Ac-NIV, deoxynivalenol triacetate, and neosolaniol triacetate was found to be 100, 2.2, and less than 1, respectively. Practically no cross-reaction was found with deoxynivalenol, fusarenon X, and NIV. The detection limit for tetra-Ac-NIV by RIA was about 5.0 ng/ml (0.5 ng per assay). The use of this antibody for quantitation of NIV in cereals after acetylation of sample extracts is proposed.     

Development of radioimmunoassay for motilin

A highly specific and sensitive radioimmunoassay for motilin has been developed. High titers of motilin antisera were produced in rabbits by repeated immunization with synthetic porcine motilin coupled to bovine serum albumin. Motilin was radioiodinated with Na[¹²⁵I] by the chloramine-T method and was purified using a CM-Sephadex C-25 column eluted with ammonium acetate gradient at pH 5.9. A specific radioactivity of 300 to 400 μCl/μg was usually obtained. Untreated plasma showed significant nonspecific interference in the assay; however, the interference could be minimized by extracting the plasma with methanol. All of the known gastrointestinal hormones were tested and showed no cross-reactivity at 10 ng/ml. Plasma motilin levels in dogs were significantly increased within 3 min, after an intraduodenal bolus injection of 50 ml of 0,1 n HCl. This increase coincided with a marked increase in myoelectrical activities of the duodenum.     

Effects of active immunization against gonadotropin releasing hormone on serum luteinizing hormone,progesterone levels and estrous cycles in the guinea pig

Mature female guinea pigs that had been observed to undergo three consecutive periods of estrus at approximately 16-day intervals were immunized with either 100 μg gonadotropin releasing hormone (GnRH) conjugated to 100 μg bovine serum albumin (BSA) or 100 μg BSA alone during diestrus (day 5–10) of the fourth cycle. Booster immunizations were administered 32 days after the first injection. Animals were bled by cardiac puncture at the time of first injection and at 16, 32, 48 and 64 days. Animals were necropsied at 64 days after first treatment.Daily observation indicated that vaginal manifestation of estrus was not apparent after a period equal to one estrous cycle in seven of ten GnRH immunized guinea pigs and after two cycles in the remaining three GnRH immunized guinea pigs. Estrous cycles persisted in BSA treated females throughout the experiment.Serum luteinizing hormone (LH) declined significantly by 32 days after the first immunization against GnRH and remained lower than both pretreatment values and levels in control animals at the same bleeding times throughout the experiment. Serum progesterone levels were significantly lower in the GnRH immunized group than in the control group at 48 and 64 days.At necropsy the weight of the ovaries of GnRH immunized guinea pigs was significantly lower than that of controls. Corpora lutea and antral follicles were present in both GnRH treated and control females. The presence of serum progesterone levels and of antral follicles in the GnRH immunized females suggests that a low level of gonadotropic support may have persisted to 64 days after initiation of treatment.Results indicate that immunization against GnRH can reduce LH and progesterone levels and induce cessation of estrous cycles in the guinea pig.     

Proteolytic Enzymes and Biological Inhibitors

The serological relationship between bovine and swine trypsins, and bovine α-chymotrypsin has been studied with rabbit antisera at different stages in the immunization period. By using paper electrophoresis to distinguish between the naturally occurring inhibitors and the antienzymes in the γ-globulin fractions, combined with the casein precipitating inhibition test (electrophoretic CPI-test) it was found that at 18 days after immunization the antienzymes inhibited only the homologous enzymes. After an additional 12 and 24 days the anti- bovine trypsin also inhibited swine trypsin and α-chymotrypsin, and anti-swine trypsin inhibited bovine trypsin, while antia-chymotrypsin inhibited only the homologous enzyme. The enzyme inhibition in the heterologous systems was about 1/10 of that in the homologous systems. Similar results were obtained by applying the Kunitz test to isolated γ-globulins. The total trypsin inhibitory activity of the whole anti- bovine trypsin serum increased 50 % from the beginning to the end of the immunization period (tested on bovine trypsin). Using the double diffusion technique, cross precipitation only occurred between anti-bovine trypsin and swine trypsin. Acetyltrypsin (bovine) was affected by the 3 antisera in a way similar to native bovine trypsin. The results are discussed in relation to other reports concerning the serological relationship of animal proteinases.     

Glycogen synthase from human and bovine polymorphonuclear leukocyte Immunochemical characterization and comparison to glycogen synthase from rat and rabbit muscle and liver cells

Glycogen synthase from human and bovine polymorphonuclear leukocytes was purified to homogeneity. Rabbit antisera were raised against the two glycogen synthases and used for immunochemical analysis. Western blotting analysis showed that the subunit of glycogen synthase in crude homogenates of human and bovine leukocytes in both cases has an M_r of 85 000. The existence of a cross-reactivity between the two enzymes and the corresponding antisera demonstrates immunological similarities between bovine and human leukocyte glycogen synthase. In addition, both antisera recognize glycogen synthase in crude cellular extracts from rabbit and rat liver and from skeletal muscle. Leukocyte glycogen synthase, therefore, cannot be classified as either muscle (M-type) or liver (L-type) glycogen synthase and our results do not support the proposed immunochemical distinction between M- and L-type glycogen synthase.     

Production of highly specific polyclonal and monoclonal antibodies using estradiol-3-O-carboxymethyl ether as hapten

The preparation of high affinity and high specificity polyclonal and monoclonal antibodies to estradiol is described. Monoclonal antibodies were derived from BALB/c mice hyperimmunized with estradiol-3-O-carboxymethyl ether conjugated to bovine serum albumin. Spleen cells were hybridized with mouse myeloma cells. Quite a few monoclonal antibodies showed very good affinity for estradiol. Extended immunization and hyperimmunization were essential for producing a greater number of positive clones secreting high affinity antibodies. Binding constants of the antisera and their cross-reactivities with related steroids were calculated. Both polyclonal and monoclonal antibodies showed very high affinity for estradiol exhibiting little or no cross-reactivities with structurally related steroids indicating that this site of linkage is a good choice for discriminating between differences at the 16-17 position in the D-ring. This monoclonal antibody (44.28.6), having negligible cross-reactivity with estriol and estrone, can be used for diagnostic purposes.     

Passive immunization of the tiger prawn, Penaeus monodon, using rabbit antisera to Vibrio harveyi

Passive immunization, toxicity neutralization and the persistence of passive protection in the tiger prawn ( Penaeus monodon ) were investigated using rabbit antisera to the formalinized extracellular products (ECP) (RαECP) and/or formalinized bacterial cells (RαBC) of luminescent Vibrio harveyi strain 820514 originally isolated from diseased tiger prawns. Rabbit antiserum to bovine serum albumin (RαBSA) or phosphate-buffered saline (PBS, pH 7·2) both served as controls. The toxicity of ECP to prawns was neutralized by pre-incubation with RαECP. Passive immunization by pre-injection of RαBC or RαECP into prawns 3 d in advance protected against a lethal dose challenge of bacteria. To determine the persistence of passive protection by rabbit antiserum in tiger prawns, the RαBC, RαECP, RαBSA or PBS were injected into prawns. At 10, 17 or 24 d post-immunization, groups of prawns were given a lethal dose challenge of bacteria. The prawns in the two control groups were all killed within the first 2 d following challenge at all three challenge dates, Pre-injection with RαBC and RαECP provided total protection for 10 and 17 d, respectively, with all treated prawns surviving for at least 2 weeks post-challenge. This is the first study using mammalian antisera to investigate toxicity neutralization, passive immunization and persistence of passive protection by rabbit antisera in prawns. The results could be useful in future studies on virulence mechanisms and disease control of vibriosis in cultured prawns.