Antiserum to lucifer yellow: preparation, characterization, and use for immunocytochemical localization of dye-filled retinal neurons

We present a new method for the preparation of antisera to Lucifer Yellow, and these antisera are here shown to be particularly suitable for immunocytochemical localization of multiple dye-injected cells in large pieces of vertebrate retina. The method involves the preparation of covalent conjugates of the VS isomer of Lucifer Yellow with keyhole limpet hemocyanin (KLH) or rabbit serum albumin (RSA), and their use as immunogens in rabbits. Both carrier protein conjugates yielded robust antibody responses. Antiserum to the KLH-LY conjugate contained precipitating antibodies against LY and KLH, although activity to the latter did not interfere with immunocytochemical staining. Rabbit antiserum to the RSA-LY conjugate contained precipitating antibody only against LY. When used for immunocytochemical staining of large retinal pieces containing many LY-filled cells, both antisera yielded well-stained, darkly filled cells similar to those seen with the Golgi technique; even very fine dendritic processes of retinal ganglion cells could be followed for long distances. LY immunocytochemistry provides a useful alternative to photooxidation for the analysis of multiple dye injected cells, especially in whole mounts. This approach may also be useful for immunocytochemical identification of cells filled with LY after tissue fixation.     

Trypanosoma lewisi: effects of immunosuppression on the serological reactivities of exoantigens and cellular antigens of bloodstream parasites.

Groups of rats were immunosuppressed with antithymocyte serum (ATS) and infected with Trypanosoma lewisi. Immunodiffusion studies were performed which demonstrated that trypanosome exoantigens, present in the plasma of these animals, were precipitated by antibodies in the sera of rats undergoing a typical primary T. lewisi infection; extracts of trypanosomes which had been collected from ATS-treated rats contained antigens which also were precipitated by antibodies in these sera. These precipitating antibodies could not be detected using either the plasma of untreated infected rats or extracts of trypanosomes which had been collected from untreated rats. With the exoantigens, precipitating antibodies were detected in serum samples collected from rats 14 to 250 days after infection. With the extract, precipitating antibodies were found as early as 5 days after infection and could be detected as late as 90 days after infection. Antigens of trypanosome extracts partially blocked the precipitin reactions between antisera and exoantigens, suggesting the presence of common antigens in the two preparations. Intact trypanosomes were serologically more reactive when collected from immunosuppressed rats. Trypanosomes collected from ATS-treated rats were agglutinated by antisera at titers fourfold higher than trypanosomes collected from untreated hosts. Absorption with exoantigens from immunosuppressed infected rats blocked trypanosome agglutination, indicating that these antigens are of cell surface origin. The experiments suggest that a likely result of immunosuppressing the host is a trypanosome antigen preparation that is a more reactive serodiagnostic reagent.     

Elicitation of the reproduction-inhibiting antibody ablastin by serum exoantigens of Trypanosoma lewisi

Serum exoantigens of Trypanosoma lewisi were collected 5 days after infection from immunocompetent (untreated) rats and rats immunosuppressed by treatment with either hydrocortisone acetate or dexamethasone. Normal rats were then immunized with pooled, whole exoantigen-containing serum from 1 of these 3 sources plus alum as an adjuvant, and the immune sera produced were tested individually. All contained agglutinating (trypanocidal) antibodies to both antigenic variants of T. lewisi, but only about two-thirds showed precipitating activity with exoantigens in gels. More importantly, however, when these antisera were thoroughly adsorbed with living trypanosomes (from immunocompetent hosts) to remove agglutinating antibody only and then tested for ablastic activity in vitro, all showed significant (P less than 0.01) reproduction-inhibiting activity, comparable to that shown by ablastic serum collected from rats that experienced a natural infection. Antisera from control rats similarly immunized with normal rat serum were negative in all antibody tests. The exoantigens of T. lewisi are, therefore, a complex mixture of immunogens that are related to the known immune responses to the parasite and can elicit the formation of ablastic antibody with the same biological properties as that produced during a natural infection.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

Determination of isonicotinic acid in the presence of isoniazid and acetylisoniazid Studies on isonicotinic acid formation from isoniazid in isolated rat hepatocytes

In comparison with the hepatocytes obtained from intact rats and rats pretreated with phenobarbital or 3-methylchoranthrene, the amount of isonicotinic acid (INA) formed from isoniazid (INH) increased substantially after incubation at 37°C using the pretreated hepatocytes. This suggests an oxidative pathway for INA formation from INH, apart from hydrolysis. In order to explore the exact mechanism of INA formation in the hepatocytes, an HPLC assay for INA in the presence of INH and acetylisoniazid was developed. In this assay, INA was extracted after the preparation of an ion pair with tetra-n-butylammonium hydroxide, and analysed using an ODS column and a mobile phase consisting of 0.067 M potassium dihydrogenphosphate solution-methanol (96:4 v/v). The method is simple, accurate and especially suitable for INA determination after incubation of INH in isolated rat hepatocytes.     

In vivo delayed rejection of tumors and inhibition of delayed - type hypersensitivity by HT-29 human colonic adenocarcinoma cell line

Summary Cis-diamminedichloroplatinum (II) (cis-DDP) and its structural analogue cis-diamminediaquoplatinum(II) nitrate (cis-aq) were complexed via an intermediate dextran carrier to antibodies specifically reactive with B lymphoma cells (38C-13). The potential use of these drugs in site-directed immunotargeting was evaluated. The two platinum(II) compounds were previously shown to form pharmacologically active complexes with carboxymethyl dextran (CM-dex). For the purpose of preparing drug-antibody complexes, CM-dex was first conjugated to idiotypic antibodies that recognize a specific membrane IgM on the B lymphoma cells. The conjugates were prepared by a modified water-soluble carbodiimide method in which N-hydroxysuccinimide was used to enhance the coupling reaction. The conjugation was followed by separation of the CM-dex-IgG conjugates from unconjugated CM-dex or IgG. The platinum(II) compounds were then complexed to the CM-dex-IgG resulting in complexes carrying up to 50 mole drug/mole IgG. Both cis-DDP and cis-aq complexes of CM-dex-antibody conjugates maintained most of the original cell-binding activity of the antibodies. An in vitro assay was used to demonstrate selective binding to tumor cells in which the target cells were treated with specific immune complexes and washed before culture. In this assay the specific complexes showed preferential cytotoxicity for the B lymphoma cells in comparison to the free drugs, drug CM-dex, or nonspecific immune complexes.     

Differentiation between acetylcholinesterase and the organophosphate-inhibited form using antibodies and the correlation of antibody recognition with reactivation mechanism and rate

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.     

Evaluation of the immunocytochemical method for amino acids

Free amino acids can be coupled to proteins by glutaraldehyde. Rabbits immunised with a bovine serum albumin-glutaraldehyde-amino acid conjugate form antibodies that recognise similar conjugates with brain proteins in glutaraldehyde-fixed tissue. Antisera raised against conjugated GABA (gamma-aminobutyrate), glutamate, aspartate, taurine, glutamine, or glycine were tested against a variety of small molecular compounds that had been fixed by glutaraldehyde to brain protein and immobilised on cellulose ester filters for processing together with the brain sections. This system permitted closely similar conditions for testing and immunocytochemistry. After removing antibodies against the carrier used for immunisation and against cross reacting amino acid conjugates the antisera showed a high specificity. The specific nature of the antisera was corroborated by solid phase adsorption to the homologous antigens and by inhibition experiments with free amino acids and amino acid-glutaraldehyde fixation complexes. After transection of the striatonigral pathway the ipsilateral substantia nigra was almost depleted of GABA-like immunoreactivity; this observation lends additional support to the selectivity of the GABA antiserum. A semiquantitative relation was established between the concentration of amino acid before fixation in a model system and the subsequent intensity of immunostaining. Similar model experiments suggested that the conjugation of an amino acid to brain protein with glutaraldehyde, and the immunoreactivity of the conjugates, may be significantly inhibited in the presence of high concentrations of other amino compounds.     

Interactions between precipitating and nonprecipitating antibodies in the formation of immune complexes

In the present study, we used monoclonal antidinitrophenol (DNP) antibodies to determine certain of the biophysical characteristics of precipitating and nonprecipitating antibodies. In addition, we studied the dynamics of immune complex (IC) formation when precipitating antibodies react with antigen in the presence of nonprecipitating antibodies. The antigen utilized in these studies was DNP-bovine serum albumin. All isolated nonprecipitating anti-DNP antibodies were of the IgG2b isotype, whereas all antibodies with other isotypes (IgG1, IgG3, IgM, IgA and IgE) were precipitating. Nonprecipitating antibodies did not differ significantly from precipitating antibodies in affinity, valence, or isoelectric point. Nonprecipitating antibodies inhibited the formation of precipitable IC between antigen and precipitating antibodies. In addition, preformed IC precipitates were solubilized by nonprecipitating antibodies. The solubilization of IC precipitates was influenced by the isotype of the precipitating antibody and by the antibody:antigen ratio in the IC precipitate. By isokinetic sucrose density centrifugation, we determined that solubilization of IC precipitates by nonprecipitating antibodies was associated with release of free precipitating antibody and formation of soluble IC between the antigen and the nonprecipitating antibody. In conclusion, in this study the nonprecipitating property of mouse anti-DNP antibodies is isotype-specific. Nonprecipitating antibodies compete and displace precipitating antibodies from the antigen, resulting in inhibition of IC precipitation and in IC solubilization. On the basis of the present results, we postulate that antibody-antibody interactions are important determinants of precipitating ability, and that these interactions are a characteristic of antibody isotype.     

Mimetic ligand-based affinity purification of immune complexes and immunoconjugates

We developed a simple purification method to purify alkaline phosphatase/anti-alkaline phosphatase IgG as immune complexes using mimetic affinity chromatography wherein the antibody was either a monospecific antibody, a bispecific antibody or a commercial polyclonal IgG conjugated with alkaline phosphatase (AP–IgG) covalently. The immune complexes or conjugates were efficiently bound on the mimetic Blue A6XL column and eluted under mild conditions (5–20 mM phosphate buffer). A similar strategy of purifying peroxidase/anti-peroxidase antibody complexes was also successfully demonstrated using the mimetic Red 3 column. Mimetic affinity chromatography thus appears to be a simple method to purify the desired monospecific or bispecific antibodies from the respective hybridomas and quadromas.     

Antibodies to synthetic platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) analogues with substituents at the sn-2 position.

We obtained rabbit antibodies by injecting immunogenic conjugates which were prepared by combining covalently 1-O-(15'-carboxypentadecyl)-2-O-acetyl-sn-glycero-3- phosphocholine(acetyl-CPGPC), 1-O-(15'-carboxypentadecyl)-2-O-N,N-dimethylcarbamoyl-sn-glycero-3 - phosphocholine (dimethylcarbamoyl-CPGPC), or 1-O-(15'-carboxypentadecyl)-2-O-N-butyl-carbamoyl-sn-glycero-3-pho sphocholine (butylcarbamoyl-CPGPC) with protein (BSA or KLH), respectively, and examined the specificity of the resulting antibodies by comparison with inhibition of the binding of iodolabeled CPGPC derivatives to the antibodies by corresponding or related phospholipids. Acetyl-CPGPC and dimethylcarbamoyl-CPGPC possessed haptenic activity causing production of antibodies reactive with PAF. Changes of the substituents at sn-2 in the antigens affected the specificity of the resulting antibodies. The affinity of the substituents to the antibodies decreased in the following order: acetyl much greater than dimethylcarbamoyl and butylcarbamoyl for antibodies to acetyl-CPGPC-KLH; dimethylcarbamoyl greater than acetyl much greater than butylcarbamoyl for antibodies to dimethylcarbamoyl-CPGPC-BSA; and butylcarbamoyl greater than dimethylcarbamoyl greater than acetyl for antibodies to butylcarbamoyl-CPGPC-BSA. Naturally occurring phospholipids, including lysoPAF, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, revealed no cross-reactivities with these antibodies. Anti-dimethylcarbamoyl-CPGPC-BSA IgG and anti-acetyl-CPGPC-KLH IgG inhibited a PAF-induced aggregation of washed rabbit platelets in a dose-dependent manner. In contrast, anti-butylcarbamoyl-CPGPC-BSA IgG did not affect a PAF-induced platelet aggregation, nor did preimmune IgG.     

Antigenic prenylated peptide conjugates and polyclonal antibodies to detect protein prenylation

Posttranslational modification of proteins with farnesyl and geranylgeranyl groups is a required modification for signaling proteins that includes the small GTPases in the Ras, Rho, and Rab families, heterotrimeric G proteins, and nuclear lamin proteins. To develop antibodies capable of detecting and distinguishing prenylated proteins, we synthesized two antigens, succinylglycine-(geranylgeranyl)cysteine methyl ester (SuccG-(gg)CMe, 1) and succinylglycine-(farnesyl)cysteine methyl ester (SuccG-(f)CMe, 2). These prenylated peptides were covalently coupled to bovine serum albumin (BSA) and to keyhole limpet hemocyanin (KLH) to produce polyvalent, immunogenic bioconjugates. Immunization of rabbits with these immunogens generated polyclonal antisera that contained significant titers of anti-geranylgeranyl and anti-farnesyl antibodies. The selectivity of the polyclonal antisera was examined using ELISA and dot blotting methods. The anti-farnesyl and anti-geranylgeranyl antisera crossreacted with both antigens. Attempts to purify the polyclonal antisera by either positive or negative immunoaffinity selection protocols failed to produce selective anti-geranylgeranyl and anti-farnesyl antibodies. Moreover, both crude antisera and purified antibodies also crossreacted with myristoylated and palmitoylated BSA conjugates. Immunofluorescence staining of EYFP-CVLL or EYFP-CVIM transfected CHO-K1 cells with rabbit polyclonal antisera showed colocalized membrane fluorescence. Thus, an important caveat for the use of antibodies raised against aliphatic antigens is that extensive controls must be performed to determine selectivity.     

Inhibition of the Metabolism of Streptococci and Salmonella by Specific Antisera

Streptococcal and salmonella antisera inhibited carbohydrate metabolism for groups A, B, C, and D streptococci and group E salmonella, as measured by the formation of [(14)C]dioxide from [(14)C]glucose metabolism. For salmonella, the inhibition was type specific since group E salmonella were inhibited only by salmonella E antisera and not by anti-salmonella A or C(1). For streptococci, quantitative differences were demonstrated, but major cross-reactivity was observed. At high concentrations, the antisera were bactericidal; at more dilute concentrations, for both salmonella and streptococci, carbohydrate metabolism was suppressed, but subculture on chocolate agar showed abundant growth. Cross-reacting antibodies could be absorbed by incubation with either antigen, e.g., streptococcal antisera versus heat-killed salmonella. The results suggest that the radiometric technique can be more sensitive than either capillary flocculation or visual detection of bacterial growth for detecting the inhibition of streptococci and salmonella by specific antibodies. The use of specific antisera may prove useful for bacterial species identification in an automated system for detection of bacterial growth.     

Immunocytochemical evidence of vertebrate bioactive peptide-like molecules in the immuno cell types of the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata).

An immunocytochemical investigation was carried out on round and spreading hemocytes of Planorbarius corneus by using 20 antisera to vertebrate bioactive peptides. The immunotests showed the presence of alpha 1-antichymotrypsin-bombesin-, calcitonin-, CCK-8 (INC)-, CCK-39-, gastrin-, glucagon-, Met-enkephalin-, neurotensin-, oxytocin-, somatostatin-, substance P-, VIP-, and vasopressin-immunoreactive molecules in the spreading hemocytes. The round hemocytes were only positive to anti-bombesin, anticalcitonin, anti-CCK-8 (INC), anti-CCK-39, anti-neurotensin, anti-oxytocin, anti-substance P and anti-vasopressin antibodies. No immunostaining was observed with anti-CCK-8 (Peninsula), anti-insulin, anti-prolactin, anti-thyroglobulin and anti-thyroxin (T4) antibodies. As probably in vertebrates, these bioactive peptides may modulate immuno cell function.     

Antigenic specificity of opsonophagocytic antibodies in rabbit anti-sera to group B streptococci.

An opsonophagocytic assay has been developed which requires human polymorphonuclear leukocytes, immune serum, and complement for optimal killing of Group B streptococci. Only with all three of these components was killing of greater than 1.0 log10 of the initial inoculum achieved, using rabbit antisera directed to homologous strains of each of the five known serotypes of Group B streptococci. Titers of specific antisera which opsonized the strains and resulted in greater than 1 log 10 reduction of colony-forming units, ranged from 1:100 (serotype Ib) to 1:3200 (serotype Ia). Cross-reactions between serotype-specific sera and heterologous strains were seen in certain instances. Type Ic strain and serotype Ic antiserum demonstrated cross-reactions with types Ia and Ib which were explainable by known shared antigens among these types. The only other cross-reaction which resulted in greater than 1 log 10 reduction in colony-forming units was when unabsorbed antiserum to strain Ia was used to opsonize a strain of serotype III. Opsonization of 10 serotype III strains was demonstrated with a single type III antiserum. Killing of nine of these strains required polymorphonuclear leukocytes, complement, and antiserum, but one strain, D136C, the reference strain, could be killed (greater than 1 log 10 reduction in colony-forming units) without either complement or specific antiserum. Inhibition studies were performed utilizing large m.w. polysaccharide antigens extracted from each serotype. These antigens inhibited opsonization of homologous strains by homologous antisera with 50% inhibition points ranging between 0.5 and 4 mug.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.     

Interactions of wild-type and mutant M protein of vesicular stomatitis virus with viral nucleocapsid and envelope in intact virions. Evidence from [125I]iodonaphthyl azide labeling and specific cross-linking

Four different temperature-sensitive M protein mutants (tsM) of vesicular stomatitis virus (VSV) were characterized with regard to the association of the mutated M protein either with nucleocapsids or with membranes in the intact virions. Virions were labeled with the photoreactive hydrophobic probe [125I]iodonaphthyl azide (INA) to assess interactions between viral proteins and the lipid envelope. In wild type (wt) virions, the three major structural proteins--G, M, and N--were labeled in the ratio ca. 1.0:0.4:0.2. INA labeled only the membrane-associated peptide of G protein, both in the intact virion and in reconstituted G protein--viral lipid vesicles, demonstrating the specificity of INA for lipid bilayer regions. Labeling of tsM virions with INA resulted in a 2--3-fold greater incorporation into M protein than was found for wt virions, suggesting increased M--membrane associations in the mutant virions. Temperature-stable revertants from tsM possessed wt labeling characteristics. Interaction of the M protein with nucleocapsids was assessed from the abundance of disulfide-linked M--N complexes found after disruption of the virions by sodium dodecyl sulfate solution under nonreducing conditions. The abundance of such complexes was 30--80% less from tsM virions than from wt virions, suggesting decreased M--nucleocapsid interactions in tsM virions. Temperature-stable revertants from tsM resembled wt in the abundance of M--N complex formed. We conclude that the mutations alter M protein in such a way as simultaneously to increase its association with membrane and to decrease its affinity for nucleocapsids in the intact virion.     

Solid Phase Radioimmunoassay for Identification of Herpesvirus hominis Types 1 and 2 from Clinical Materials

A solid phase radioimmunoassay (RIA) was developed for typing Herpesvirus hominis (HVH) strains isolated from clinical materials, and it also proved to be applicable to the direct detection and typing of HVH antigen in human and animal brain tissue. The procedure utilized virus-infected human fetal diploid cells or brain tissue smears in the bottom of 1-dram glass vials, antigen was detected through the use of intermediate HVH antisera produced in rabbits or hamsters and cross-absorbed with the HVH heterotype, and (125)I-labeled anti-species (rabbit or hamster) globulins produced in goats were used for detection of immune complexes. The cross-absorbed HVH antisera could be used at high dilutions in the RIA test, and they reacted with marked type-specificity in the RIA system. Specificity of the test was also improved by determining and using optimal concentrations of intermediate sera and of (125)I-labeled anti-species globulins. Results of typing HVH isolates by the RIA procedure agreed in all instances with those obtained by direct fluorescent antibody staining with cross-absorbed conjugates. The RIA procedure was effective and more sensitive than direct fluorescent antibody for demonstrating and typing HVH antigen directly in smears of infected human brain tissue.     

Immunization of cattle against modified peptides of gonadotropin releasing hormone conjugated to carriers: Effectiveness of Freund's and alternative adjuvants

Two gonadotropin-releasing hormone (GnRH) peptides with a cystein substitution of the first (C1-GnRH) or tenth (C10-GnRH) amino acid were conjugated to ovalbumin and equine serum albumin, respectively, via the sulfhydryl group of the introduced cysteine. Animals were immunized three times at 3-wk intervals with both conjugates in either saline (n = 5), Freund's complete adjuvant (FCA; n = 5), Havlogen (n = 6), Ribi adjuvant system (RAS; n = 5), dimethyl dioctadecyl ammonium bromide (DDA; n = 4), Alhydrogel (n = 5) or Regressin (n = 5). Animals immunized with conjugates in saline or RAS did not produce anti-GnRH titers. The highest anti-GnRH titers were produced by animals treated with FCA. The Alhydrogel and DDA treatments stimulated the production of GnRH antibodies in all animals treated, but titers were lower than in animals immunized with FCA. When vaccines were formulated with Havlogen or Regressin, anti-GnRH titers were low or absent. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were depressed in FCA and in Alhydrogel treated animals. The antisera raised were predominantly directed against either the carboxy- or the amino-terminal end of the GnRH peptide, or directed equally against both, depending on the individual animal. Results suggest that no epitope of GnRH dominates the immune response in cattle and show that the best alternative to FCA is Alhydrogel.     

Antigens in immune complexes from patients with breast cancer

Summary Sera and effusion fluids of patients with breast cancer (BC) contain immune complexes (IC). Antigens present in these complexes were isolated as follows: a pool of effusions from patients with BC was fractionated with ammonium sulfate. The proteins precipitating at 40% saturation were further fractionated by filtration through a Sephadex G-200 column. The material recovered in the first peak (molecules larger than monomeric IgG) was brought to pH 3.0 to dissociate the IC, and the mixture was filtered through a column of Sephacryl S-300 at pH 3.0. Proteins smaller than monomeric IgG were collected, radioiodinated, and used as antigens (¹²⁵Ag) to search for corresponding antibodies in sera of patients with BC (BCS) and of healthy individuals (NHS). ¹²⁵Ag was reacted with the sera and the immune complexes obtained were precipitated with an antiserum to human Ig and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Both NHS and BCS contained antibodies against two antigens; one of these appeared as a strong band of 17KD, the other as a doublet of approximately 25KD. It is concluded that some of the proteins in the IC from patients with BC are auto-antigens. No BC-specific antigens were identified.