Production of rabbit polyclonal antibody against apobec-1 by genetic immunization

Circulating apolipoprotein B (apoB) exists in two forms; apoB-100 and apoB-48. ApoB-48 is a truncated form of apoB resulting from RNA editing. The editing enzyme, called apobec-1, converts a cytidine (C) at nucleotide 6666 in apoB 100 mRNA to a uridine (U) and changes a CAA codon to an in-frame stop codon, UAA. We have produced a specific rabbit polyclonal antiserum against apobec-1 by genetic immunization. The cDNA of mouse apobec-1 was inserted downstream and in-frame at the BamH I site in the last exon of human growth hormone cDNA driven by a cytomegalovirus promoter. This plasmid was injected together with another plasmid expressing granulocyte macrophage colony-stimulating factor into the thigh muscles of a rabbit. The resulting antiserum demonstrated high specificity on Western blots, and inhibited the apoB mRNA editing activity of mouse liver extract in a dose-dependent manner. This report demonstrates that DNA immunization is a powerful technique that can be readily applied to other sparse or difficult-to-purify proteins in lipid metabolism.     

Antigenic differentiation of turkey rhinotracheitis virus strains using monoclonal antibodies and polyclonal antisera

Monoclonal antibodies (mAbs) were prepared against the 8597/CV94 strain of turkey rhinotracheitis virus (TRTV). These mAbs were used to investigate antigenic relationships among three strains (8597/CV94, 1162/92 and CVL14/1 strain) of TRTV, together with polyclonal chicken and rabbit antisera to 8597/CV94 strain, and guinea pig antisera to each of the three strains. Thirty mAbs to the glycoprotein (G:3 clones), fusion (F1:6 clones), phosphorylated (P:6 clones), nucleocapsid (N:12 clones), and matrix (M:3 clones) proteins of viral antigen were obtained by cell fusion. Among these, two mAbs to F1 protein showed virus neutralizing activity. The results of ELISA test indicated that some mAbs only reacted to the 8597/CV94 strain, some reacted to 8597/CV94 and 1162/92 strains, and others reacted to all three viral strains. In neutralization tests with the three virus strains, polyclonal chicken and rabbit antisera against the 8597/ CV94 strain showed the same antibody titers. Results with four neutralizing mAbs including two previously reported mAbs [Ref. 21] indicated the titers of two mAbs (Pn2-2E and Pn3-2F) to 8597/CV94 were much higher than those to the other two viral strains. No differences were observed in the titers of the other two mAbs (Pn01-8E and Pn06-4D) against any viral strains. In cross-neutralization tests with polyclonal guinea pig antisera, there was some variations among viral strains. This work demonstrated that the Japanese isolate 8597/CV94 of TRTV is somewhat different in antigenicity from two British isolates from chickens and turkeys.     

Vaccination against feline pneumonitis

A commercially available modified live chlamydial vaccine against feline pneumonitis was tested in 26 cats for its ability to protect against aerosol challenge exposure to the feline pneumonitis strain of Chlamydia psittaci. After cats were challenge exposed (30 days after vaccination), pyrexia of greater than 40.0 C occurred in 81% of nonvaccinated (control) cats and in 13% of vaccinated cats (principals). Evidence of upper respiratory tract disease and the presence of the agent in ocular fluids were observed less frequently in principals than in nonvaccinated cats. In the cats euthanatized at intervals of 3 days after challenge exposure, C psittaci was demonstrated in 60% of tissues tested from nonvaccinated controls and in 34% of similar tissues obtained from principals.     

Specificity of rabbit antisera against lipopolysaccharide of Acinetobacter

Acinetobacter has been reported to be involved in hospital-acquired infections with increasing frequency. However, clinical laboratories still lack simple methods that allow the accurate identification of Acinetobacter strains at the species level. For this study, proteinase K-digested whole-cell lysates from 44 clinical and environmental isolates were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with hyperimmune rabbit sera to examine the possibility of developing a serotyping scheme based on the O antigen of Acinetobacter lipopolysaccharide (LPS). The antisera, obtained by immunization of rabbits with 13 of the heat-killed isolates investigated, were characterized by Western blotting and enzyme immunoassay by using proteinase K-digested whole-cell lysates and phenol-water-extracted LPS as antigens. In both assays, the antisera were shown to be highly specific for the homologous antigen. In addition, assignment of Acinetobacter LPS to the smooth or the rough phenotype was shown not to be reliable when it was based only on the results obtained with silver-stained gels. O-antigen reactivity, determined by Western blot analysis, was observed with 11 of the 31 isolates, most of which belonged to the species Acinetobacter baumannii (DNA group 2) and the unnamed DNA group 3. Interestingly, some O antigens were found in a DNA group different from that of the strain used for immunization. The results indicate that O serotyping of Acinetobacter strains is feasible and thus may provide a simple method for the routine identification of these opportunistic pathogens.     

Generation of polyclonal rabbit antisera to mouse melanoma associated antigens using gene gun immunization

Lymphocytes from patients with melanoma have been used to clone melanoma associated antigens which are, for the most part, nonmutated melanocyte tissue differentiation antigens. To establish a mouse model for the use of these 'self' antigens as targets for anti-tumor immune responses, we have employed the mouse homologues of the human melanoma antigens Tyrosinase, Tyrosinase Related Protein-1 (TRP-1), gp100, and MART-1. We sought to generate antisera against these proteins for use in the construction of experimental recombinant and synthetic anti-cancer vaccines, and for use in biologic studies. Using genes cloned from the B16 mouse melanoma or from murine melanocytes, we immunized rabbits with plasmid DNAs coated onto microscopic gold beads that were then delivered using a hand-held, helium-driven 'gene gun'. This strategy enabled us to generate polyclonal rabbit sera containing antibodies that specifically recognized each antigen, as measured by immunostaining of vaccinia virus infected cells. The sera that we generated specifically for TRP-1, gp100, and MART-1 recognized extracts of the spontaneous murine melanoma, B16. The identities of the recognized proteins was confirmed by Western blot analysis. The titers and specificities of these antisera were determined using ELISA. Interestingly, serum samples generated against murine MART-1 and gp100 developed antibodies that were cross-reactive with the corresponding human homologues. Recognition of human gp100 and murine Tyrosinase appeared to be dependent upon conformational epitopes since specificity was lost upon denaturation of the antigens. These antisera may be useful in the detection, purification and characterization of the mouse homologues of recently cloned human tumor associated antigens and may enable the establishment of an animal model of the immune consequences of vaccination against 'self antigens.     

Production of antisera to synthetic benzyloxycarbonyl-C-peptide of human proinsulin

Antisera to the C-peptide of human proinsulin were obtained by immunizing guinea pigs with synthetic benzyloxycarbonyl-C-peptide conjugated to human albumin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In three series of 10, the animals were injected with C-peptide conjugated to albumin in the molar ratio of 23 : 1, 15 : 1, and 4 : 1, respectively. Antibodies to human C-peptide were present in all the surviving 25 animals. Fifteen of the antisera were suitable for measuring C-peptide concentrations lower than 0.10 pmol/ml. The antisera demonstrated an increasing immunogenicity with increasing molar ratio of C-peptide to albumin in the conjugate. In the fourth series, ten guinea pigs immunized with benzyloxycarbonyl-C-peptide ionically bound to QAE-Sephadex A-25 did not produce detectable antibodies to C-peptide. A qualitative evaluation of the radioimmunoassay by use of the antiserum with the highest titer and sensitivity, "M 1230", revealed a mean intra-assay and inter-assay coefficient of variance of 3.2 and 9.6%, respectively.     

Detection and molecular characterisation of feline foamy virus serotypes in naturally infected cats

A total of 11 cases with trisomy 14 as the sole abnormality were found in the database of a large cytogenetic reference laboratory from 1993 to the present. Four of the 11 cases had an isochromosome 14q. In 8 cases, the trisomy 14 was a mosaic cell line. Eight cases were diagnosed with myelodysplasia, 2 cases had both myelodysplastic and myeloproliferative features similar to some atypical chronic myeloid leukemia cases, and 1 case had acute myeloid leukemia of M1 or M2 type. Nine were males and two females. The median age was 77 years. Referring physicians were contacted and clinical information was available in only 8 cases. Survival ranged from 1 month to approximately 3 years. An abnormal red cell morphology, such as elliptocytes or schistocytes or both, was observed in the majority of cases. This study along with the reported cases strengthens the hypothesis that trisomy 14 is a nonrandom cytogenetic abnormality associated with myeloid malignancy.     

Microinjected antisera against ductin affect gastrulation in Drosophila melanogaster

Ductin is a putative connexon-forming protein in gap junctions of arthropods. To analyze the role of gap-junction mediated cell-cell communication during Drosophila embryogenesis, we used two different polyclonal anti-ductin sera. One antiserum was directed against ductin isolated from gap junctions of the lobster Nephrops whilst the other was raised against a nonapeptide at the N-terminus of ductin from Drosophila. Both antisera were found to inhibit, when microinjected into Drosophila ovarian follicles, the intercellular exchange of fluorescent tracer molecules between oocyte and follicle epithelium. This result indicates that Drosophila ductin plays a decisive role in gap-junctional communication and confirms the cytoplasmic location of the ductin N-terminus in gap junctions. On immunofluorescence preparations and immunoblots, the anti-ductin sera specifically recognized ovarian as well as embryonic antigens. Following microinjections of the antisera into embryos prior to gastrulation, significantly reduced rates of hatching larvae were obtained. Moreover, microinjections into the mid-ventral region of the embryos resulted in specific ventral defects that depended on the concentration of the ductin antibodies. In particular, larvae with ventral holes in their cuticles occurred with high frequency. During gastrulation, antiserum-injected embryos often developed defects in the middle region of their ventral furrow. Here, mesodermal cells failed to invaginate correctly and, thus, no cuticle was formed. We conclude that, during Drosophila embryogenesis, gap-junctional communication is required for epithelial integrity and morphogenetic events.     

Production of IgE antibodies against the 22 kDa tegumental membrane-associated antigen of schistosomes is directed by the antigen itself

It has previously been reported that the predominant target of immunoglobulin E (IgE) recognition in sera from humans infected with Schistosoma japonicum in The Philippines or with S. mansoni in Kenya, is a 22 kDa tegumental membrane-associated schistosome antigen. In the present study, we demonstrate that the 22 kDa antigen can direct the production of antigen-specific IgE antibodies independently of schistosome infection and in the absence of any other parasite components or adjuvant. Three strains of mice were immunized using the purified, recombinant 22 kDa antigen of S. japonicum without the use of any adjuvant. Sera from all three strains of immunized mice, but not control animals, generated IgE antibodies specific for the native 22 kDa schistosome antigen in Western blots. Thus, the 22 kDa antigen itself must contain signals (presumably encoded by the primary amino acid sequence or by the secondary or tertiary structures of the molecule, or by a combination of these) which are sufficient to direct the isotype switch required for production of antigen-specific IgE.     

Effect of dual-subtype vaccine against feline immunodeficiency virus infection

Dual-subtype feline immunodeficiency virus (FIV) vaccine, consisting of inactivated cells infected with subtypes A (Petaluma strain) and D (Shizuoka strain), was developed and tested for its vaccine efficacy against FIV infection in specific pathogen free (SPF) cats. Animals were monitored for proviral DNA by FIV-specific PCR and for FIV-specific antibody profiles by ELISA and virus-neutralization assays. In addition, blood from challenged cats was inoculated into naive SPF cats to confirm the viral status of the vaccinated cats. All cats immunized with Petaluma vaccine alone were protected against homologous Petaluma challenge, but only one of four cats was protected against heterologous Shizuoka challenge. More importantly, all cats immunized with the dual-subtype vaccine were protected against both Petaluma and Shizuoka challenges. These results suggest that a multi-subtype vaccine approach may provide the broad-spectrum immunity necessary for vaccine protection against strains from different subtypes.     

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.     

Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv. trifolii R39 studied with monospecific polyclonal antisera

Monospecific polyclonal antisera raised against Rhizobium leguminosarum bv. trifolii R39, a bacterium which was isolated originally from red clover nodules, were used to study the colonization of roots of leguminous and nonleguminous plants (Pisum sativum, Lupinus albus, Triticúm aestivum, and Zea mays) after inoculation. Eight weeks after inoculation of soil-grown plants, between 0.1 and 1% of the total bacterial population in the rhizospheres of all inoculated plants were identified as R. leguminosarum bv. trifolii R39. To characterize the associative colonization of the nonleguminous plants by R.leguminosarum bv. trifolii R39 in more detail, a time course study was performed with inoculated roots of Z. mays. R. leguminosarum bv. trifolii R39 was found almost exclusively in the rhizosphere soil and on the rhizoplane 4 weeks after inoculation. Colonization of inner root tissues was detected only occasionally at this time. During the process of attachment of R. leguminosarum bv. trifolii R39 to the rhizoplane, bacterial lipopolysaccharides were overexpressed, and this may be important for plant-microbe interaction. Fourteen weeks after inoculation, microcolonies of R. leguminosarum bv. trifolii R39 were detected in lysed cells of the root cortex as well as in intracellular space of central root cylinder cells. At the beginning of flowering (18 weeks after inoculation), the number of R. leguminosarum bv. trifolii R39 organisms decreased in the rhizosphere soil, rhizoplane, and inner root tissue.     

Production and characterisation of monoclonal antibodies specific for bovine interleukin-4

This study assessed physical activity patterns in a sample of urban African Americans, whose participation in physical activity has not previously been well-described. From questions administered by interviewers during health fair screenings in 19 churches in East Baltimore, information regarding participation in regular, leisure-time activity (defined as 30 minutes of activity, 5 days per week), time spent walking on the job, and distance walked to and from work was assessed from 365 adults (69% women). Regular, leisure-time activity participation was 18% for men and 16% for women. When the definition of physical activity participation was broadened to include: (1) spending over half the day walking at work; (2) walking at least 10 blocks to and from work; as well as (3) regular, leisure-time activity, 41% of men and 38% of women were active. These data suggest that, while a small percentage of African Americans participate in regular physical activity, a substantial percentage are regularly active when non-leisure-time activity is assessed. To accurately characterize overall participation, physical activity derived from a variety of sources, including transportation and work-related activity, should be assessed.     

New polyclonal antiserum against microtubule-associated protein 2 (MAP2); preparation and preliminary characterization

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T-antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [32P]phosphorous-containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS-dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [gamma-32P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T-antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T-antigen, and the C-terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.     

Glucagon of caviomorphs and other tetrapods immunohistochemically investigated with two antisera against the N- and C-terminal portions of the molecule

Water proton nuclear magnetic resonance relaxation times and magnetization transfer (MT) parameters of rat hearts were studied 24 h or 4 weeks after ligation of the left coronary artery or sham operation. Compared with sham-operated controls, measured relaxation times (T1sat and T2) of both acute and chronic myocardial infarction increased. The MT effect significantly decreased in the infarcted myocardium. The changes in relaxation times and MT effect were significantly greater in chronic infarcts compared with acute infarcts. Improvements in calculated image contrast between normal and infarcted tissue were supported by images of ex vivo hearts with chronic infarction. Image contrast was increased at short echo times in the presence of macromolecular proton pool irradiation. Exploiting changes in tissue MT following myocardial infarction to enhance contrast between normal and infarcted tissue should allow improved identification and characterization of infarcted myocardium.     

Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor

Effective ex vivo purging techniques can decrease the likelihood of infusing bone marrow contaminated with leukemic cells during autologous transplantation. In preliminary studies, OL(1)p53, a 20-mer phosphorothioate oligonucleotide directed against p53 mRNA, decreased the number of acute myelogenous leukemia (AML) cells in vitro, suggesting a possible role for OL(1)p53 in purging bone marrow harvests of leukemia cells. To demonstrate that OL(1)p53 was nontoxic to hematopoietic progenitor cells, normal bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h, and hematopoietic progenitor cell survival was determined by in vitro colony assays. OL(1)p53 had no toxic effect on the growth of either myeloid (CFU-GM) or erythroid (BFU-E) progenitor cells. OL(1)p53 was then used to ex vivo purge bone marrow harvests from nine patients with either AML or myelodysplastic syndrome (MDS). Bone marrow cells were incubated with 10 microM OL(1)p53 for 36 h before transplantation. The median times posttransplantation for the patient to recover an absolute neutrophil count greater than 0.5 x 10(9)/L and a platelet transfusion independence were 30 days and 56 days, respectively. Incubation of bone marrow cells with OL(1)p53 had no detrimental effect on the growth of hematopoietic progenitor cells, and transplantation of autologous bone marrow cells treated with the phosphorothioate oligonucleotide, OL(1)p53, resulted in successful recovery of circulating neutrophils following high-dose therapy in patients with AML or MDS. The data show that OL(1)p53 can be used safely to purge autologous bone marrow harvests from patients with leukemia.     

Carrier-state infection of feline T-lymphoblastoid cells with feline calicivirus

The susceptibility of feline T lymphocytes to feline calicivirus (FCV) in vitro was investigated using feline T-lymphoblastoid cell lines, namely MYA-1 and FL74 cells. The virus titers of supernatants in FCV-infected MYA-1 and FL74 cell cultures increased rapidly, and FCV antigens were also detected in the FCV-infected cells. There were slight differences in the molecular weights of capsid proteins expressed in FCV-infected MYA-1, FL74 and Crandell feline kidney cells. MYA-1 and FL74 cells were productively and persistently infected with FCV, and FCV antigens were observed in the FCV-infected cells for more than one month. At 3 months post infection, FCV-infected FL74 cells that stopped producing infectious FCV could be reinfected with FCV. However, no cytopathic effects were observed.