Antibody Production In Rabbits Research Articles

Isolation and characterization of the canalicular membrane bile acid transport protein of rat liver

The aim of this study was to isolate the Na(+)-independent bile acid transporter from rat canalicular plasma membranes by affinity chromatography. The affinity matrix used consisted of lysylcholic acid covalently linked to CH-Sepharose 4B, resulting in an anionic ligand essentially identical to glycocholic acid. The protein fraction, adsorbed and eluted from the affinity column, was markedly enriched in a 100-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) compared with the total membrane and membrane extract. The 100-kDa band, further purified by preparative SDS-PAGE, was electroeluted from excised gel fragments and used as an immunogen for antibody production in rabbits. The immune serum, but not preimmune serum, specifically recognized a single, 100-kDa polypeptide on one- and two-dimensional immunoblots of canalicular membranes. In contrast, no reactivity was observed with proteins in liver basolateral or ileal brush-border membranes. The 125I-labeled protein was immunoprecipitated from membrane extracts solubilized in NP-40 and was found to migrate with a pI of 5.3 on two-dimensional electrophoresis. The apparent molecular weight of the protein was reduced by 50% after deglycosylation with N-glycanase. The 100-kDa protein was localized specifically and exclusively by immunocytochemical methods to the bile canalicular domain of the hepatocyte plasma membrane. Moreover, the immunoglobin G fraction prepared from the antiserum significantly inhibited taurocholate transport by canalicular membrane vesicles and decreased the covalent labeling of the 100-kDa protein by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus the isolation of a single 100-kDa protein by bile acid-affinity chromatography, as well as the inhibitory effects of antibodies directed against this polypeptide, provide further support for its role in the canalicular transport of bile acids.

Production of Rabbit Antibodies Against Carboxy-Terminal Epitopes Encoded by Bullous Pemphigoid cDNA

A partial cDNA clone (called BP cDNA) with coding sequences for the carboxy-terminal region of bullous pemphigoid (BP) antigen has been recently isolated and sequenced. In order to determine whether specific peptides encoded by the cDNA could be used to raise antibodies against BP antigen, fusion proteins derived from fragments of the BP cDNA and 17-mer or 19-mer synthetic peptides, corresponding to its deduced amino acid sequence, were used to generate rabbit antibodies. Three restriction enzyme fragments, 1179 bp (5' end), 264 bp (middle), and 546 bp (3' end), of the 1992 open reading frame (ORF) of BP cDNA were subcloned in frame into pEX plasmids to make beta-galactosidase fusion proteins FP1, FP2, and FP3, respectively. Fusion proteins of the predicted molecular weight, and which bound anti-beta-galactosidase antibodies, were produced, confirming the length of the predicted ORF. Rabbits immunized with FP1, but not FP3, produced antibodies, similar to authentic antibodies from BP patients, which: 1) bound the epidermal basement membrane at titers over 10,000, as determined by indirect immunofluorescence; 2) bound the basement membrane on the roof of 1 M NaCl-split skin; 3) immunoprecipitated the 230-kD BP antigen; and 4) bound the hemidesmosome, as determined by immunoelectron microscopy. Rabbits immunized with FP2 also produced lower titer BP-like antibodies. We further showed that short hydrophilic synthetic peptides, contained in FP1, could induce similar BP-like antibodies in rabbits at immunofluorescence titers up to 2560. These rabbit antibodies should prove useful for further studies on the function and structure of particular epitopes of BP antigen as well as on the pathophysiology of disease.

Vitellogenesis in the shrimp, Penaeus vannamei: in vitro studies of the isolated hepatopancreas and ovary

Abstract 1. 1. Yolk proteins were isolated from ovaries of the shrimp Penaeus vannamei and used as an antigen for antibody production in rabbits. 2. 2. Protein synthesis was measured for both the hepatopancreas and the ovary in vitro, and proteins present in both tissues were immunoreactive with the antibodies. 3. 3. Extracts of shrimp eyestalks inhibited in vitro protein synthesis by both tissues. The inhibitory factor from the eyestalks was heat stable and had a molecular weight of 3300 daltons.

Immunofluorescent localization of the Ca2+-dependent proteinase and its inhibitor in tissues of Crotalus atrox.

The native 108,000 dalton Ca2+-dependent proteinase (CDP) and its 115,000 dalton protein inhibitor (CDPI) were purified from bovine skeletal muscle using native polyacrylamide gel electrophoresis and were used to elicit antibody production in rabbits and BALB/c mice. Polyclonal antibodies were purified as IgG fractions by column chromatography; monoclonal antibodies were produced by the hybridoma technique. Indirect immunofluorescence localization of CDP and CDPI in tissues of Crotalus atrox show both proteins to be ubiquitous. Both occur in the cytoplasm and are absent from the cell membrane and the nucleus; CDPI is also present in the I-band of skeletal muscle.

Serotaxonomy of Solanum, Capsicum, Dunalia, and Other Selected Solanaceae

Serotaxonomic comparisons were made on about 50 species of Solanaceae. Immunoabsorption data were obtained from reactions of seed protein antigen systems with antibody systems absorbed by several antigen systems in numerous permutations. Relationships of the taxa were computed by newly developed methods. Twenty species of Solanum, mostly of subg. Leptostemonum, were compared using six antisera. Solanum nigrum (subg. Solanum) was serologically distinct from the others. Several groups of species belonging to different sections of Solanum were recognized. Phenetic and phyletic relationships within sect. Androceras were explored. Re-analysis of a complete cubic matrix of immunoabsorption data for eight species of Solanum rearranged relationships slightly but emphasized the divergence between different sections of this genus. A broad survey was made using an antibody system raised to Capsicum annuum. This revealed the distinctiveness of Nicandra from other Solanaceae, several possible inter-generic relationships, but also some unlikely ones. Further studies revealed closer relationships of Dunalia species to Capsicum than to Iochroma. Protein characters are valuable in plant taxonomy for assessing inter-generic and even interfamily relationships (Jensen & Fairbrothers, 1983). Within the Solanaceae several serological studies have been made by workers such as Chester, Hammond, Tucker, Gray, and Lester (see review by Lester, 1979). Because of the complex physico-chemical properties of proteins, and the even more complex phenomena of immunological reactions, many different serotaxonomic techniques have been developed (Jensen & Fairbrothers, 1983). Immunoabsorption resolves subsets of antigenic determinant sites as recognized by corresponding subsets of antibodies after the removal of common antibodies from the antibody system by absorption by other antigen systems. Recent developments in the theoretical and practical aspects of immunoabsorption techniques and the analysis of the resultant data by appropriate numerical taxonomic procedures have been discussed in detail (Lester, 1979; Lester et al., 1983). For this paper these procedures were applied to selected species of Solanaceae. MATERIALS AND METHODS Seed samples from the Birmingham University Solanaceae Collection (Tables 1-3) were used to prepare protein extracts, to induce antibody production in rabbits, and for subsequent immunological experiments using absorbed antibody systems. Procedures, especially the scoring and analysis of results, followed those described by Lester (1979) and Lester et al. (1983). Three sets of experiments were conducted. 1. Twenty accessions of Solanum, mostly of subg. Leptostemonum (Table 1) were compared using six antibody systems (data published in Lester et al., 1983). 2. Eight species from diverse sections of the genus Solanum (Table 2) were compared using eight antibody systems (data published in Lester, 1979). 3. About 25 species of Capsicum, Solanum, and other genera of Solanaceae, mostly of tribe Solaneae (Table 3) were compared using one antibody system (Lester, unpubl. data). Most of these data have now been analyzed by most of the procedures described by Lester et al. (1983), especially by using Jaccard's and similar coefficients to estimate phenetic relationships. Some of the data sets also were subjected to cladistic analysis. Many dendrograms were produced by different analyses of the various sets and subsets of data, some of which are presented herein (Figs. 1-6). I We are grateful to Sarah Marsh for growing these plants, to the Science and Engineering Research Council (U.K.) for financial support, and to Julie Brean, Barbara Klauza, and Sarah Sutton for technical assistance. 2 This paper was part of the Second International Symposium of the Biology and Systematics of the Solanaceae presented at the Missouri Botanical Garden on 3-6 August 1982. 3 Department of Plant Biology, University of Birmingham, Edgbaston, Birmingham B1 5 2TT, England. 4 Department of Nematology, University of California, Riverside, California 92521. ANN. MISSOURI BOT. GARD. 73: 128-133. 1986. This content downloaded from 207.46.13.60 on Thu, 21 Apr 2016 07:28:58 UTC All use subject to http://about.jstor.org/terms 1986] LESTER & ROBERTS-SOLANACEAE SEROTAXONOMY 129 TABLE 1. Listing and classification of the 20 accessions of Solanum species that were compared immunologically using antibody systems to CAP, ROS, SIS, QUT, TOR, and PRN.

Immunoelectron microscopic evidence for different compartments in the secretory vacuoles of the rat seminal vesicles

Immunoelectron microscopy of the rat seminal vesicle was performed using specific antibodies to secretory proteins. Proteins were precipitated from rat seminal vesicle secretion and were separated by SDS-polyacrylamide gel electrophoresis. Among the great number of bands the two most prominent bands were selected and designated SVS II and IV. Their apparent molecular weights were 48 kDa and 16.5 kDa respectively. The bands were excised from the gels and used for antibody production in rabbits. The respective antisera were used for immunohistochemical studies both at the light and electron microscopic levels in the rat seminal vesicle and the different prostatic lobes in infantile, adult and castrated animals. A positive immunoreaction was observed in seminal vesicle and lateral prostatic epithelium of the intact adult rat, while it was lacking in prepubertal and castrated animals. The subcellular distribution of both proteins was clearly different: SVS II was exclusively confined to the electron dense core of the secretory vacuoles, while SVS IV was detected only in the clear halo surrounding the central granule. It is suggested that the spatial arrangement of both proteins in the seminal vesicle secretion vacuole reflects a particular functional significance of each of these proteins. These proteins may serve as a tool in the study of regulation of androgen-dependent protein synthesis.

Studies on antigenicity of the polyethylene glycol (PEG)-modified uricase

The purified uricase (urate: oxygen oxidoreductase, EC 1.7.3.3) from Candida utilis was modified to varying degrees with monomethoxypolyethylene glycol (PEG) of different molecular weights using cyanuric chloride as the coupling reagent. The PEG-uricase conjugates were examined on their immunological properties by means of ring test and passive cutaneous anaphylaxis (PCA). As increasing amounts of PEG were attached to uricase, it showed decreasing ability to elicit antibody production in rabbits. When sufficient polymers were attached, the modified uricase was devoid of the capacity to combine in vivo and in vitro with antibodies from guinea pigs injected with the unmodified uricase, however, were still able to react with antibodies to PEG-uricase conjugate. Antibodies against PEG-uricase conjugates also reacted with PEG modified superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1). These results indicate that the coupling of PEG to uricase resulted in the loss of original antigenicity and immunogenicity, but in the appearance of new antigenicity and immunogenicity which never showed any cross-reactions against the native uricase.

Antigenic and allergenic properties of the storage mite Tyrophagus putrescentiae

The antigens and allergens in the storage mite Tyrophagus putrescentiae, commonly found in grain and hay and sometimes in house dust, were characterized by crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis. Three mite extracts, T. putrescentiae (TP) mite bodies, feces, and a combination of mites, feces, and culture medium (TP + CM) were studied. TP and TP + CM (primarily feces) extracts exhibited 20 and 18 antigens, respectively. By use of serum from two skin test-positive patients, autoradiograms demonstrated TP contained two allergens and TP + CM contained five allergens, three of which probably originated from feces. CM (whole wheat flour) did not stimulate antibody production in rabbits. TP feces shared 10 antigenic determinants with TP and 14 with TP + CM. Two antigens common to TP feces and TP were also shared allergens.

Human placental steroid sulphatase--purification and monospecific antibody production in rabbits.

Human steroid sulphatase was purified 43-fold from placental microsomes using a four step procedure: solubilization with Miranol H2M, Bio-Gel A 1.5 m chromatography, column chromatofocusing and Sephadex G-75 chromatography. The purified enzyme that appeared electrophoretically homogeneous was used to immunize rabbits. Protein blotting demonstrated that the resulting antiserum mainly reacted with a polypeptide of 63 000 dalton, which is about the size of placental steroid sulphatase. The antiserum was freed from minor impurities by absorbing it to Sepharose 4B with immobilized antigens prepared from a steroid sulphatase deficient placenta.

Alteration of capsular type of encapsulated strains of Staphylococcus aureus during freeze-drying and storage.

Remarkable alteration was shown in capsular type antigen production in encapsulated strains of Staphylococcus aureus stored by lyophilization for 10 years. This alteration was further elucidated by antibody production in rabbits immunized with the altered strain and by absorbing the antibodies with representative capsular type strains.

Affinity separation and partial characterization of serologically active Leishmania donovani antigens.

Leishmania donovani-soluble antigens capable of antibody production in rabbits were separated from the total antigenic make-up of the parasite by adsorption on to anti-Leishmania immunoglobulins coupled to CNBr-activated Sepharose 4B (ALIG). Immune rabbit sera were produced either by intravenous inoculation of living promastigotes or by intradermal and subcutaneous inoculation of soluble antigens. Approximately four times as much soluble antigen was bound to ALIG produced by i.v. than i.d. and s.c. inoculation although the percentage recovery was less. The antigenic fractions recovered were subjected to immunoelectrophoresis, isoelectric point and molecular weight determination. The results of affinity chromatography indicate high antigen-antibody interaction affinity, the recovery of at least 28 antigens (with 0.15 M NaCl, pH 11.0) and complete loss of serological activity (determined by immunoelectrophoresis) on treatment with 8 M urea.

Solid-phase immunoassays for quantitation of antibody to bovine white matter proteolipid apoprotein.

Two solid-phase immunoassays have been developed for quantitation of antibodies to bovine white matter proteolipid apoprotein. Conditions were established for optimal specific antibody binding. Water-soluble proteolipid apoprotein was bound to microtiter plates and plates were incubated with test serum. Goat anti-rabbit IgG conjugated with horseradish peroxidase was used as the second antibody for an enzyme-linked immunospecific assay and 125I-labeled protein A for a radioimmunoassay. Both procedures have been used to follow the time course of anti-proteolipid antibody production in rabbits and to compare different immunization protocols.

Immunocytochemical localization of a calcium-activated protease in skeletal muscle cells

The 80 000-D subunit of a calcium-activated protease from skeletal muscle was purified to homogeneity using preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was used to elicit antibody production in rabbits. Antiserum was purified using affinity chromatography to yield a monospecific antibody fraction (anti-80K) directed against the 80 000-D subunit. Localization studies showed that the 80 000-D subunit is present in or near the sarcolemma of cultured myoblasts and sectioned muscle tissue, in discrete areas of the cytoplasm of myoblasts, and in the Z disks of the myofibril. The location of the calcium-activated protease in the cell suggests that the enzyme may be involved in myofibril degradation and in membrane alterations in developing and mature muscle cells.

A polypeptide antigen from a strain of Staphylococcus simulans. 2. Antigenic and biological properties.

A purified polypeptide antigen from Staphylococcus simulans CCM 2705 produced one precipitation line by double diffusion in agar with rabbit antiserum against homologous whole bacteria. The purified antigen did not induce antibody production in rabbits. However, when the antigen was complexed with methylated bovine serum albumin, antibodies with specificity against the polypeptide were produced. The antigen did not sensitize normal or tanned erythrocytes for agglutination in antiserum. The polypeptide antigen induced a primary skin reaction and was toxic for mice. It also induced production of MIF as demonstrated by the migration inhibition test. The polypeptide was found to be a leukotaxigen. No difference between C4 normal and C4 deficient serum was noted. C5 was found to be necessary for the induction of chemotaxis.

The use of culture-derived metacyclic trypanosomes in studies on the serological relationships of stocks of Trypanosoma brucei gambiense

Metacyclic trypanosomes of five stocks of Trypanosoma brucei gambiense were produced in vitro in tsetse head-salivary gland explant cultures and used to infect rabbits. Sera were collected from the rabbits and monitored by agglutination tests for antibody production to nine serotype antigens of T. b. gambiense. In the case of a Nigerian stock of T. b. gambiense the sequences of antibody production were found to be similar in animals infected with the stock transmitted by tsetse flies and from culture. Many similarities were also found between the patterns of antibody production in rabbits infected with stocks of T. b. gambiense from Senegal, Nigeria, Zaire and Uganda. The occurrence of similar serotypes in geographically different stocks of T. b. gambiense provides further support for continuing efforts to develop improved serodiagnostic tests for sleeping sickness based on variable trypanosome antigens and to find techniques for immunoprophylaxis.

Selective production of rabbit antibodies against mouse suppressor cells. Analogy with mouse anti-IJ sera

Selective production of rabbit antibodies against mouse suppressor cells. Analogy with mouse anti-IJ sera

Isolation of specific and common antibodies to staphylococcal enterotoxins B, C1, and C2.

Staphylococcal enterotoxins C1 and C2 elicit the production of antibodies in rabbits that give precipitin reactions with both toxins and gel diffusion plates. These enterotoxins also elicit the production of antibodies that are specific for each of the enterotoxins. Enterotoxin B elicits the production of antibodies in some rabbits that react with enterotoxins B, C1, and C2 in gel diffusion plates and in radioimmunoassay. Antibodies specific for enterotoxin B are produced also. Enterotoxin C1 elicits the production of antibodies that cross-react weakly with enterotoxin B, indicating that the antigenic sites involved in the cross-reactions are not identical in the two toxins. The antibodies have been isolated by affinity chromatography.

Prostate-specific acid phosphatase: purification and specific antibody production in rabbits.

Demonstration of prostate-specific acid phosphatase by immunologic methods in tissue sections or in plasma necessitates a monospecific antiserum. This is produced by immunizing rabbits with pure prostate-specific acid phosphatase antigen, prepared from seminal fluid. The ejaculate is centrifuged, dialyzed against a citrate buffer, pH 4.8, and centrifuged again. The supernatant is brought onto a Sephacryl S-200 Superfine column and eluted with the same buffer. Prostate-specific acid phosphatase-positive fractions are concentrated, brought onto and stepwise eluted from a Sulphopropyl Sephadex column with citrate buffers at pH 4.8, 5.2, and 5.5. Fractions containing prostate-specific acid phosphatase are concentrated again and purified over a Blue Sepharose CL-6 B column. After elution, a new concentration step yields a protein concentration of 0.5-1.0 mg . ml-1. This sample in polyacrylamide gel electrophoresis shows a single protein band and is used to immunize female rabbits. The purity of the antiserum is then tested by immunoelectrophoresis and an Ouchterlony technic. Besides anti-prostate-specific acid phosphatase, two other antibodies against serum proteins are present, one of which is anti-albumin. Absorption of these impurities results in a monospecific antiserum against prostate-specific acid phosphatase.

Leishmania donovani: Physicochemical, immunological, and biological characterization of excreted factor from promastigotes

Leishmanial excreted factor (EF) from promastigote cultures was enriched from the crude product by differential precipitation with ammonium sulfate and perchloric acid, followed by column chromatography; and by boiling EF-antibody complex. Boiling destroyed the antibody, releasing the EF, which retained its ability to precipitate antibody. Enriched EF from Leishmania donovani promastigotes was found to be a highly negatively charged, carbohydrate-like material with a molecular weight approximating to 33,000, when monitored against a series of protein markers by gel filtration. Its ability to precipitate with antibody was unimpaired by boiling, lyophilization, pH changes from 1 to 11, treatment with high concentrations of NaCl, 10% phosphotungstic acid in 10% HCl, 0.6 M perchloric acid, 5% H 2SO 4, acetone, or dioxan. It did not absorb at wavelengths between 220 and 750 nm. Treatment with trypsin, Pronase, neuraminidase, and hyaluronidase did not affect its activity. Biochemical analysis showed that enriched EF contains carbohydrates but, at our level of detection, no protein, lipid, triglycerides, fatty acids, DNA, RNA, pentoses, amino sugars, sialic or uronic acid. Precipitation of EF by antibody was studied and the optimal molecular proportions for complete precipitation determined. EF-antibody complex, prepared at optimal proportions, and EF complexed with methylated bovine serum albumin, like EF alone, did not elicit antibody production in rabbits. EF in 0.5% phenol-saline elicited a delayed skin response of induration and erythema in guinea pigs cured of L. enriettii. Elevated temperature increased the release of EF from promastigotes, while the presence of trypsin acting at 37 C seemed to inhibit this effect slightly. Fractionation of mechanically broken promastigotes, by differential centrifugation and stepwise sucrose gradients, revealed a factor that precipitated rabbit antibody against whole promastigotes. This factor was associated with the soluble, organelle-free fraction and resembled EF when monitored by gel diffusion. This factor did not migrate when the complete extract from the broken promastigotes was run in immunoelectrophoresis. Boiling the extract for 5 min released a factor, which migrated to the anode. This factor appeared to be associated with another component in the promastigote, from which it dissociated on boiling. Boiling hamster tissues infected with leishmanial amastigotes, i.e., spleens containing L. donovani and epididymides containing L. tropica, also released factors similar to EF. These precipitated antibody in the same way, producing precipitation arcs that were continuous with those formed by EF from the homologous promastigotes. EF acted as a conditioner for culture promastigotes. Conditioned cultures showed maximal growth before similar, unconditioned cultures. However, both types of culture produced equal numbers of promastigotes per unit volume by the end of exponential growth.

Antigenic stimulation of lymphocytes: II. Immunogenicity of dinitrophenyl-polyethylene oxide antigens

Synthetic antigens consisting of dinitrophenyl groups attached to linear or branched-chain polyethylene oxide stimulate anti-DNP antibody production in rabbits and a proliferative response in vitro in immune rabbit lymphocytes. A requirement for immunogenicity is divalence. Linear DNP 2PEO is most effective at a molecular weight of 10 4, but a clear response is obtained even at 10 3. The optimal valence of DNP n PEO ( n = 4–80, MW, 4 × 10 5) is 20. Destruction of T cells with antithymocyte serum does not impair the in vitro response of the remaining B lymphocytes to DNP 2PEO, indicating that these antigens are T independent.