Immune Rabbit Serum Research Articles

Comparative analysis of bovine extracts by immunoblotting and ELISA inhibition

Bovine epithelial and urine antigen extracts were compared using enzyme-linked immunosorbent (ELISA) inhibition assay and sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblotting. According to ELISA inhibition results, the two extracts share about 2% of their antigenic components. Urine-derived antigens seem to be antigenically closer to serum proteins than epithelial antigens, as determined by rabbit immune sera. The IgG responses of allergic farmers against epithelial antigens were directed mainly against a protein of 20 kD, while non-allergic farmers had only very weak reactions. Generally, the IgG response against urinary antigens seemed to be more heterogeneous than against epithelial antigens. Almost all cow-allergic farmers reacted with a band at 20 kD in IgE immunoblotting against urinary and epithelial antigen while all non-allergic farmers showed negative results.

Isolation and Characterization of Rat Carcinoembryonic Antigen

A rat tumor-associated antigen with properties similar to those of human carcinoembryonic antigen (CEA) has been detected with rabbit immune sera in extracts of transplantable rat colonic adenocarcinoma, RCA-1. This antigen, termed rat CEA, was also detectable by a monkey antihuman CEA serum and a rat monoclonal antibody to rat CEA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of perchloric acid extracts of RCA-1 tumor, followed by immunoblotting with the above-mentioned anti-CEA reagents, revealed that rat CEA activity resided in components with a molecular weight of approximately 350 kD. The glycoprotein nature of these components was indicated by positive staining with periodic acid-Schiff. Sephadex G-200 chromatography, as well as Sepharose 4B chromatography with and without sodium dodecyl sulfate indicated that the 350-kD components existed in the extracts as molecular aggregates. The 350-kD material, which had been purified by an affinity column containing rat monoclonal antibodies to rat CEA, reacted with the rabbit and monkey anti-CEA sera. This provided strong evidence that serological activity of rat CEA was confined to the 350-kD components.

Structural and antigenic analysis of Chlamydia pneumoniae

Several isolates of Chlamydia pneumoniae were compared with each other and to Chlamydia trachomatis and Chlamydia psittaci by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. Protein profiles of the C. pneumoniae isolates appeared to be the same and were distinct from the other Chlamydia species. A 39.5-kilodalton (kDa) protein, similar in molecular weight to the major outer membrane proteins (MOMP) of C. trachomatis and C. psittaci, was found in the Sarkosyl-insoluble fraction, demonstrating its association with the outer membrane complex. In the outer membrane complex, the MOMP was shown to exist in disulfide-linked protein complexes. Electron microscopy of the Sarkosyl-extracted elementary bodies showed that the structural rigidity and pear-shaped morphology remained intact. Rabbit immune sera prepared against C. pneumoniae demonstrated immunoreactive proteins of 98-, 77-, 75-, 66-, 60-, 39.5-, 28-, and 17.5-kDa proteins. Cross-reactivity experiments revealed that most of the antigenic reactivities shared between C. psittaci and C. trachomatis extend to C. pneumoniae and that the 98-kDa protein recognition appeared to be C. pneumoniae specific. In contrast to the other Chlamydia spp., the recognition of the C. pneumoniae MOMP by homologous immune sera was weak and was cross-reactive with the MOMPs of the other Chlamydia species. These results suggest that the C. pneumoniae MOMP is less immunogenic and antigenically complex than are the MOMPs of C. trachomatis and C. psittaci.

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Improvement of the fermentation-inhibition (FI) test for Mycoplasma pneumoniae was attempted. The sensitivity of detecting the FI antibody to M. pneumoniae in the homologous immune rabbit serum was notably elevated, when such FI medium containing gamma globulin-free horse serum and guinea-pig complement in substitution for unheated horse serum was used. The M. pneumoniae suspension was filtered to remove aggregates of the organisms and used as the antigen for this new FI test. In detection of the serum antibody of patients with M. pneumoniae infections, the new FI test showed much higher sensitivity than conventional FI test and strongly correlated (r = 0.84) with high density particle agglutination test known to detect both IgG and IgM antibodies.

Acinetobacter baumannii serotyping for delineation of outbreaks of nosocomial cross-infection.

A total of 152 clinical isolates of Acinetobacter baumannii from 152 patients were identified by carbon source utilization tests and examined serologically. Polyclonal rabbit immune sera against A. baumannii strains were used in checkerboard tube agglutination tests, and 20 serovars were identified. One (serovar 19) cross-reacted with genospecies 3 (serovar 3), a closely related member of the genus Acinetobacter. Several outbreaks of nosocomial cross-infection caused by serovars 4 and 10 were delineated.

Cloning and Expression of Treponema pallidum Antigens in Escherichia coli

A library of Treponema pallidum genomic DNA fragments produced by partial Sau3A digestion was established in Escherichia coli K12 using the plasmid vector pAT153. The library was screened using immune syphilitic rabbit serum and six recombinant phenotypes expressing eight treponemal polypeptides were detected. With two exceptions, all the recombinant gene products were the same size as polypeptides detected on Western immunoblots of T. pallidum. The genes encoding three novel gene products, with molecular masses in SDS-PAGE of 42, 17 and 15.5 kDa, which had not been cloned previously from T. pallidum were also identified. Monoclonal antibodies which reacted with four of the eight recombinant polypeptides were generated.

Ichthyophthirius multifiliis Has Membrane‐Associated Immobilization Antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify cell surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-114 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1- and 2-dimensional SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.

Epitope mapping employing antibodies raised against short synthetic peptides: a study of the nicotinic acetylcholine receptor

Antibodies were raised against eight synthetic peptides matching preselected portions of the amino acid sequence of nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata. To increase the probability of obtaining antibodies specific for the exact sequence of the immunizing peptide, peptides of only five to seven amino acids in length were employed. Even under these limiting conditions some of the polyclonal rabbit immune sera showed cross-reactivity with other peptides and/or other sequence regions of the receptor. Further studies with polyclonal and monoclonal sera suggested that conformation and charge pattern rather than linear sequence are the essential determinants of antibody epitopes. Application of antibodies for topological studies therefore requires that the antibody specificity for a particular region of the antigen has been firmly established. Epitope mapping with the eight anti-peptide immune sera provides information on the accessibility to antibody of matching sequences within the receptor molecule. We find the sequence portions alpha 81-85, alpha 127-132, and alpha 190-195 to be freely accessible both at membrane-bound and at purified receptor. Binding of anti-alpha 387-392 serum does not prove accessibility of this region as the serum cross-reacts strongly with peptide fragments corresponding to the regions alpha 165-200 and beta 190-200 of nAChR from Torpedo californica. To permit binding of anti-alpha 137-142 immune serum, treatment of the receptor with endoglycosidase is required, showing that Asn-141 indeed is glycosylated in native nAChR. The homologous sequence of the other subunits differing only in one sequence position from alpha 137-142 is not accessible in native nAChR to antibody, indicating clear differences in folding of the receptor polypeptides. Sequence portions alpha 395-401 and alpha 161-166 must first be exposed by appropriate treatment to permit binding of respective serum. These results and previous epitope mapping studies by other laboratories are discussed with respect to the limited sequence specificity of antibodies.

Adult Ixodes dammini on rabbits: development of acute inflammation in the skin and immune responses to salivary gland, midgut, and spirochetal components.

Rabbits exposed to female Ixodes dammini (both uninfected and infected with Borrelia burgdorferi) or injected with B. burgdorferi showed an acute inflammatory response in the skin. Granulocytes and monocyte-histiocytes were the predominant infiltrating cells. Spirochetes were detected in the tick feeding cavities in the deep dermis. The inflammatory process was accompanied by polyclonal antibody responses to tick salivary gland components. Western blots showed that immune rabbit serum reacted with proteins of molecular masses of 8, 24, and 36-41 kilodaltons in both unengorged and engorged tick salivary glands. Additional reacting bands in the immunoblot of the engorged salivary gland indicated that new antigenic components of the salivary gland are synthesized during engorgement. Rabbits did not produce antibodies to tick midgut components. Murine monoclonal antibody 11G1 detected outer surface protein A of B. burgdorferi in immunoblots of midguts from unengorged ticks, faintly in engorged salivary gland, and seldomly in unengorged salivary gland, findings suggesting that the spirochete is transmitted to the host via tick saliva during the later stages of feeding.

Protection of mice by a pseudodiffuse strain of Staphylococcus aureus possessing polyvalent capsular type antigen.

Staphylococcus aureus strain MC31 showed pseudodiffuse growth in serum-soft agar and reacted with immune rabbit sera to strains Smith diffuse (capsular type A), NS58D (type B) and NS41D (type C) but not with strain NS68D (type D) in serum-soft agar. Immunisation of mice with 300 micrograms of cell-surface polysaccharide extracted from strain MC31 protected against lethal infection by strain MC31 and the strains of capsular types A, B and C. Immune rabbit serum prepared against strain MC31 passively protected mice against challenge infection with the homologous strain, but approximately 30 times more anti-MC31 serum was required to protect against infection with the strains of capsular types A, B and C. Absorption of the passive protective activity of immune sera raised against the three capsular type strains required at least 10 times the quantity of MC31 cell-surface polysaccharide than the quantity of cell-surface polysaccharide from the homologous capsular strain. Electronmicrographs of strain MC31 treated with ferritin-labelled antisera to the three capsular strains showed only small amounts of ferritin granules around the cell wall.

Isolation of a gene encoding a Chlamydia sp. strain TWAR protein that is recognized during infection of humans

Chlamydia sp. strain TWAR is a unique Chlamydia sp. that causes acute respiratory disease. A gene bank consisting of TWAR isolate AR-39 DNA in pUC19 was screened with anti-AR-39 rabbit immune sera. Two positive clones were isolated that contained 7.3-kilobase (pLC1) and 14.9-kilobase (pLC2) plasmids. Restriction mapping and hybridization studies showed that both pLC1 and pLC2 contained a common 4.2-kilobase PstI fragment. Plasmids were used as templates of in vitro transcription-translation. All three plasmids had a novel protein product of ca. 75 kilodaltons not found in the vector alone. Western blots showed that this protein reacted with anti-TWAR rabbit immune sera and with human immune serum from an individual who had proven TWAR infection. Whole-cell lysates of TWAR demonstrated a protein having the same molecular weight and immunoreactivity as the recombinant gene product. This protein was also recognized by rabbit immune serum against Chlamydia psittaci or Chlamydia trachomatis. Southern hybridizations with the cloned fragment as a probe of digests of other Chlamydia spp. showed weakly hybridizing fragments. These results suggest that we have isolated a gene encoding a protein recognized during human TWAR infection that contains some sequences shared among Chlamydia spp.

Eimeria maxima: Identification of gametocyte protein antigens

The antigenicity of Eimeria maxima gametocyte proteins during the course of an infection and when injected into mice and rabbits was demonstrated using the Western blotting technique. Serum taken from chickens at various times postinfection reacted to a few gametocyte proteins, with the strongest reactivity seen with serum taken 14-days postinfection. Two major antigens having molecular weights of 56,000 and 82,000 were consistently detected by these sera. Using immune rabbit or mouse sera to whole gametocyte detergent extracts, the 56,000 and 82,000 molecular weight proteins were again the immunodominant antigens, despite their representing only a small proportion of the extract which was used to immunize the animals. These results, together with those obtained by Rose ((1971)) using recovered chicken serum to passively immunize chickens, indicate that these two gametocyte antigens may play a role in protective immunity to E. maxima.

A neutralization-inhibition enzyme immunoassay for anti-idiotypic antibodies that block monoclonal antibodies neutralizing Semliki Forest virus

In this paper we compare a solid-phase enzyme immunoassay (EIA) with a neutralization-inhibition enzyme immunoassay (NI-EIA) for the determination of anti-idiotypic antibodies against Semliki Forest virus (SFV)-neutralizing monoclonal antibodies (MAs) UM 5.1 (IgG2a) and UM 1.4 (IgG2a). Against these MAs strong immune sera were induced in female BALB/c mice by two subcutaneous injections, 3 weeks apart, with keyhole limpet hemocyanin coupled MA mixed with the saponin Quil A. Rabbit immune sera were prepared by intracutaneous injections of purified MA mixed with either FCA (first immunization) or IFA (second and third immunization). In the NI-EIA serum is preincubated with neutralizing MA, in wells of 96-well plates, before SFV is added. Binding of anti-idiotypic antibodies to MA results in a diminished capacity of that MA to neutralize SFV. After 1 h incubation with SFV L cells are added and residual infectious virus is allowed to multiply for 6 h at 37°C. Then the monolayers are fixed with glutaraldehyde and subsequently SFV is quantified with a horseradish peroxidase-labelled SFV-specific MA. Low absorbance values indicate that the neutralizing capacity of MA is intact and that blocking antibodies were not present in serum. In contrast high absorbance values indicate that blocking (anti-idiotypic) antibodies had abrogated the neutralizing capacity of MA. With the strongly neutralizing MA UM 5.1 as idiotypic antigen the NI-EIA proved to be at least as sensitive as the solid-phase EIA. Furthermore both normal mouse serum-absorbed rabbit immune sera and mouse immune sera were not cross-reactive in both solid-phase EIA and NI-EIA.

Role of Shiga toxin in the pathogenesis of bacillary dysentery, studied by using a Tox- mutant of Shigella dysenteriae 1.

A Tox- mutant of Shigella dysenteriae 1, SC501, was genetically engineered by cloning the Shiga toxin operon, inserting a cassette into the A subunit gene, and exchanging this in vitro-mutagenized sequence with the wild-type gene. SC501 produced a low amount of residual cytotoxicity which was not neutralized by a rabbit immune serum directed against Shiga toxin. Invasion of cultured cells demonstrated that Shiga toxin had no effect on the rate of intracellular growth of bacteria or on the rapid killing of invaded host cells. On the other hand, several significant differences were observed in macaque monkeys infected intragastrically with either the wild-type strain or its mutant. The production of Shiga toxin by the invading strain was correlated with the presence of blood within stools, a sharp drop in blood polymorphonuclear cells, and histopathological alterations, such as the destruction of capillary vessels within the connective tissue of the colonic mucosa, severe inflammatory vasculitis of the peritoneal mesothelium, and major efflux of inflammatory cells to the intestinal lumen. It is proposed that Shiga toxin influences the severity of bacillary dysentery by inducing colonic vascular damage, which accounts for bloody stools, intestinal ischemia, and inflation of a polymorphonuclear intestinal compartment during the infectious process.

Variable sensitivity of oral isolates of Eikenella corrodens to serum bactericidal activity: role of antibody.

Eikenella corrodens is a facultatively anaerobic Gram-negative bacterium which is among the predominant cultivable microflora of periodontal lesions characterized by loss of attachment level. In the present study, we examined the potential role of complement-mediated killing in host defense against this periodontopathic organism. Seven clinical isolates obtained from human subgingival plaque and one reference strain of E. corrodens were characterized with respect to (a) susceptibility to the bactericidal properties of pooled human serum and (b) the role of the classical and/or alternative pathway(s) of complement in effecting killing of sensitive strains. Six strains, including the reference strain, were found to be variably serum-sensitive, exhibiting 1-12.5% survival after two hr of incubation in the presence of 20% pooled human serum. The remaining two isolates were serum-resistant. Both serum-resistant and serum-sensitive strains consumed complement via the classical pathway in normal but not in hypogammaglobulinemic serum, thus ruling out an antibody-independent mechanism of classical pathway activation. Four of six serum-sensitive strains exhibited little or no loss of viability following incubation with serum depleted of the classical pathway component Clq. One strain which was resistant to killing by normal human serum was, nevertheless, highly susceptible to complement-mediated killing in the presence of rabbit immune serum. Two additional serum-sensitive strains were killed, albeit to a lesser extent, in Clq-depleted serum, indicative of a role of the alternative pathway in killing of some serum-sensitive strains. These results indicate a potential role for complement-mediated killing in host defense against Gram-negative periodontal bacteria such as E. corrodens. However, the ultimate contribution of this immune defense mechanism may be defined, at least in part, by the presence of a humoral response to key bacterial membrane constituents.

Isolation and characterization of the localized adherence factor of enteropathogenic Escherichia coli.

The binding factor of enteropathogenic Escherichia coli O111:H- responsible for localized adherence (LA) on HeLa cells was investigated. Inhibition of LA by carbohydrates and lectins showed that the reactive epitope on HeLa cells contains N-acetylgalactosamine units. Treatment of bacteria with EDTA for extraction of lipopolysaccharides eliminated these polymers as binding factors. Such treatment also caused a marked increase in adhesion suggesting steric hindrance by lipopolysaccharides of the LA factor binding capacity. Immunoblotting with rabbit antibodies showed a strong reaction with two components with approximate molecular sizes of 29 and 32 kilodaltons (kDa) present in the outer membrane preparations of bacteria. Both the absorbed rabbit immune serum and the outer membrane preparation of the bacteria inhibited bacterial adhesion by 100%. Outer membrane components were isolated from an N-acetylgalactosamine-agarose column by elution with KSCN, labeled with 125I, and immunoprecipitated with absorbed rabbit hyperimmune antiserum. The only component precipitated was the protein doublet at 29 to 32 kDa corresponding to the components detected by immunoblotting. The predominant component was always the 32-kDa polypeptide. We conclude that this component of the outer membrane is the best candidate for the LA factor in enteropathogenic E. coli.

Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits.

Four groups of protective rabbit immune sera were used to identify Pasteurella multocida outer membrane immunogens by a radioimmunoprecipitation procedure and Western blot (immunoblot) analysis. These are rabbit hyperimmune sera against KSCN extract of P. multocida (group 1) and rabbit immune sera against the KSCN extract of P. multocida (group 2), the outer membrane of P. multocida (group 3), and live P. multocida cells (group 4). Rabbits mounted an antibody response to 18 proteins found in the outer membrane of P. multocida, and the major antibody activities were directed to the 27,000-molecular-weight outer membrane protein (27K protein), as well as the 37.5K, 49.5K, 58.7K, and 64.4K outer membrane proteins. These outer membrane immunogens appear to be exposed on the cell surface and accessible to antibodies, since adsorption of these immune sera with intact P. multocida cells resulted in a significant reduction of antibody activities directed against these proteins, especially the 37.5K protein. Antibodies eluted from immune serum-P. multocida cell complexes were reactive to the 37.5K immunogen, confirming that this protein is exposed on cell surface and accessible to antibodies. Western blot analyses with group 1, 3, and 4 immune sera confirmed that the 27K, 37.5K, 49.5K, 58.7K, and 64.4K proteins are the major outer membrane immunogens of P. multocida in rabbits. Lung lavages of immunized rabbits also contained similar antibody activities directed against several outer membrane proteins, with major activities against the 37.5K and 64.4K proteins.

A simple novel approach for the purification of pertussis toxin

Pertussis toxin (PT) was purified from concentrated culture supernatants of Bordetella pertussis by a single step based on affinity chromatography on heparin-Sepharose 6B with a recovery of 36% in two fractions. The fraction of highest specific activity (0.91 mg of toxin per mg protein) constituted 15.3% of toxin content in crude material and appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibited the five bands corresponding to toxin subunits. The purified fraction induced clustering of Chinese hamster ovary cells at as little as 0.06 ng per ml. PT solution gave a single precipitation line with rabbit immune serum raised against crude Bordetella pertussis supernatant.

Immunochemische analyse der immunantwort bei spätmanifestationen der Lyme Borreliose: Immunochemical analysis of the immune response in late manifestations of Lyme Borreliosis

Compared to American strains, European Borrelia burgdorferi strains revealed considerable heterogeneity of major proteins. Four strains isolated from ticks, human skin and human CSF were selected from our 23 Borrelia burgdorferi isolates. These strains and the American type strain B31 were characterized by SDS-PAGE (Coomassie Blue staining) and Western blots (using rabbit immune sera against two of the strains and two monoclonal antibodies (H5323 and H3TS) against a major outer surface protein (OspA)). The strains showed considerable differences in SDS-PAGE pattern. Corroborating the results of a previous study, we could demonstrate that the OspA (31/32K) can change from a minor to a major protein and in reverse the pC (21/22K) from a major to a minor protein during subculturing. Moreover, European strains can antigenically differ in OspA, pC and also in a further low molecular weight protein of 17/18K. To examine whether the antigenic heterogeneity of European isolates is reflected in the immune response of European patients we examined sera from patients with late manifestations of Lyme Borreliosis by Western blot using the five strains as antigens. Sera from seven patients with acrodermatitis chronica atrophicans (ACA) showed a surprisingly strong reactivity with the skin isolate. All sera had antibodies against the 17/18K protein of the skin isolate, but none was reactive with the analogous 17/18K of the other strains. On the other hand a comparable predominance of one strain was not found testing sera from patients with Lyme arthritis. One patient even had antibodies against OspA and OspB proteins of strain B31. Contrary to findings in American Lyme Disease antibodies against the OspA were rarely observed in the sera of our patients (only one patient had such antibodies) although we tested the patients sera with five different strains. Only two patients had stronger reactions with the skin isolate. These findings suggest that ACA is caused by antigenically closely related Borreliae. This could explain the finding that ACA is rarely observed in the US (US strains are antigenically closely related to strain B31). Our findings in patients with Lyme Arthritis — on the other hand — suggest that “different serotypes” can cause Lyme Arthritis. This does not exclude the possibility that Borrelia proteins are an important factor in the pathogenesis of Lyme arthritis. Finally the differences in reactivity of sera with different strains in the Western blot led us to examine whether such differences are also found in serodiagnostic tests using different strains as antigens. Except in two ACA sera (which reacted stronger with the skin isolate) no significant differences were found using the five strains as antigens in the IgG immunofluorescent assay (we tested four sera from ACA patients, four sera from patients with neurological manifestations, four sera from Lyme arthritis patients and five sera from control persons without Lyme Borreliosis). These findings suggest that the IgG IFT can be performed by using only one strain as antigen. But in some cases antigen-dependent differences have been observed and we do not know whether different results occur using different antigens in other serodiagnostic tests (i.e. ELISA, IgM IFT).

Discrimination of the determinant specificity of two competitive Semliki Forest virus neutralizing monoclonal antibodies by anti-idiotypic antibodies

The two highly competitive, monoclonal antibodies (MAs), UM 5.1 and UM 8.1, recognize on the E2 glycoprotein of Semliki Forest virus different determinants as indicated by the absence of cross-reactive anti-idiotypic antibodies in rabbit immune sera induced against these highly neutralizing MAs.