Titer Of Antiserum Research Articles

Development of Competitive Direct Enzyme-linked Immunosorbent Assay for the Detection of Gentamicin Residues in the Plasma of Live Animals

Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities (CR50) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/B0 ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/B0 ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life (t1/2) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 10 : 1498-1504)

Expression of nitroreductase gene NOR1 in E.Coli and the preparation of antiserum

Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested withBamHI andXhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested withBamHI andXhoI, then the NOR1, gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1, was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analyzed with immunoblot. Results: The 1.25 kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1, is obtained in JM 105, and its antiserum can be prepared successfully.

Alzheimer β-Amyloid Homodimers Facilitate Aβ Fibrillization and the Generation of Conformational Antibodies

We reported previously that stabilized beta-amyloid peptide dimers were derived from mutant amyloid precursor protein with a single cysteine in the ectodomain juxtamembrane position. In vivo studies revealed that two forms of SDS-stable A beta homodimers exist, species ending at A beta 40 and A beta 42. The phenomenon of the transformation of the initially deposited 42-residue beta-amyloid peptide into the amyloid fibrils of Alzheimer's disease plaques remains to be explained in physical terms, i.e. energetically and structurally. We therefore performed spectroscopic analyses revealing that engineered dimeric peptides ending at residue 42 displayed a much more pronounced beta-structural transition than corresponding monomers. Specifically, the single chemically induced dimerization of A beta peptides significantly increased the beta-sheet content by a factor of 2. The C-terminal residues Ile-41 and Ala-42 of dimeric forms further increased the beta-sheet content by roughly one-third. In contrast to A beta 42, the beta-sheet content of the alpha- and gamma-secretase-generated p3 fragments did not necessarily correlate with the tendency to form fibrils, although p3/17-42 had a pronounced thread forming character with fibril lengths of up to 2.5 microM. Electron microscopic images show that forms of p3/17-42 generated smaller granular particles than forms ending at residue 40. We discuss these findings in terms of A beta 1-42 dimers representing paranuclei, which self-aggregate into ribbon-like ordered fibrils by elongation. Based on A beta 42 dimer-specific titers of a polyclonal antiserum we propose that the A beta homodimer represents a nidus for plaque formation and a well defined novel therapeutic target.

Optimization and validation of an enzyme immunoassay for the insect growth regulator fenoxycarb

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the insect growth regulator fenoxycarb. Polyclonal rabbit antisera, raised against protein conjugates of four haptenic derivatives of fenoxycarb, were utilized in immobilized antigen-based, competitive immunoassays. With ELISA systems that were both hapten- and carrier-heterologous, most antiserum titers fell in the range of 1:1000–1:30,000. Assay conditions, including concentrations of antisera and coating antigens, were optimized. The effect of pH, organic solvents, and various blocking agents was also investigated. A hapten-homologous and two hapten-heterologous indirect ELISAs allowed fenoxycarb determination in the range of 0.1–85 ng ml −1 with apparent IC 50 values of 1.2–2.8 ng ml −1. Cross-reactivities with a number of compounds (e.g. pesticides of related structure, hapten synthesis intermediates, fenoxycarb metabolite, photodegradation products) were determined, and the assay proved highly selective for fenoxycarb. In particular, no significant interference was found with selected pyrethroid and juvenile hormone analog insecticides, phenoxyacetic acid herbicides, and photodegradation products of fenoxycarb. Using spiked water samples, assay performance was validated by SPME/GC-MS.

New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten, and evaluation of polyclonal antiserum.

The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol in water samples at trace level.

BmTI antigens induce a bovine protective immune response against Boophilus microplus tick

Boophilus microplus trypsin inhibitors (BmTIs) present in larvae were preliminarily characterized as active proteins, approximately 10–18 kDa, by SDS-PAGE. BmTIs showed trypsin inhibitory activity on reverse zymography containing gelatin (0.03%) and also inhibited others serine proteinases (human neutrophil elastase and human plasma kallikrein). Bos indicus, Nelore breed calves, previously sensitized with BmTIs and challenged with tick larvae (20,000 larvae/animal), showed 72.8% efficacy to interfere in tick development with 69.7% and 71.3% reduction of both tick number and egg weight, respectively. Cattle BmTIs antiserum titer was approximately 1:8000. The maximum level of BmTIs antibody production was detected 40 days after the first immunization by ELISA. Our preliminary results suggest that B. microplus serine proteinase inhibitors may play a role in the tick larvae fixation and feeding processes. Therefore, the development of antibodies against BmTIs might impair the normal parasitism.

Developmental stage-related expression and identification of murine erythroid differentiation-denucleation factor (MEDDF)

Using MEDDF cDNA fragment in plasmid pBS-SK-MEDDF as template the coding sequence was cloned into pGEM-T-Easy plasmid by PCR method to delete non-coding sequence. After DNA sequencing it was confirmed that the clone sequence was correct, the coding region then was inserted into the vector pET-30a betweenBamH I andHind III to construct eukaryotic expression vector. It was found that the specific protein was up to 40% of total bactorial proteins in certain high-expressionE. coli. High titer of anti-sera was detected by inoculating New Zealand rabbits with purified MEDDF protein as an antigen. By using immunocytochemical staining it was demonstrated that the expression of MEDDF was exhibited in a developmental stage-specific manner, suggesting that MEDDF may play a certain role in the initiation of murine erythroid terminal differentiation and nuclear condensation. As for the expression of MEDDF appearing in granulocytes and megakaryocyter in murine bone marrow, it may indicate that there is an original relationship between the proteins and differentiation of murine myelogenous lineage.

Identifikasi Spesies Daging Secara Imunologi

ABSTRACT To detect the meat or meat product adulterated in beef products, the research was carried out with using immunological tecnique (agar gel precipitation test). The purpose of this study was to investigate various antigen preparation, to determine the optimum method for producing antiserum of the desired titer and specificity, and to know whether antiserum from fresh meat still capable to determine extract from cooked meat. Six kinds of extract antigen from beef and 3 kinds of meat proteins were used in the research. Those were 6 kinds of extract antigen from fresh meat, wet dendeng, dry dendeng, frest bakso, cooked bakso and abon, and 3 kinds of meat protein from globulin, myoglobin and actomyosin. The rabbit were injected intramusculary and intravenausly on alternate weeks for eight injections and were tested every week to detect titers of antiserum. The results showed that intramuscular and intravenous injection of extract antigen from frest meat, frest bakso and cooked bakso emulsified in Freund Complete Adjuvant causad antibodies production higher titers thant wet dendeng and dry dendeng, whereas extract from abon showed a negative gel diffusion response after 8 weeks injection. Serum corresponding to the extract from the other cooked meat, except the extract from abon. The extract from abon always indicated a negative response on gel diffusion test. Injection of globulin and myioglobin showed a strong positive gel diffusion test after 8 weeks with extract from frest meat, whereas actomyosin resulted in negative response to extract from fresh meat by gel diffusion test.Key Word : Antigen; Antibody; Intravenous; Intramuscular and

Immunocytochemical detection of Ig-positive cells in blood, lymphoid organs and the gut associated lymphoid tissue of the turbot (Scophthalmus maximus)

The present study was designed to search for the sites of the B-cell lineage in the different lymphoid organs of turbot (Scophthalmus maximus) by immunoperoxidase staining with a rabbit polyclonal antiserum against deglycosylated turbot IgM (TUDG-6). A turbot immunoglobulin (Ig) fraction, isolated by protein A, was checked for purity by gel filtration and SDS-PAGE under reducing conditions. The turbot IgM was deglycosylated and used to raise an antiserum. The antiserum titre was evaluated in ELISA. It was then used to analyse turbot peripheral blood leucocytes for membrane and cytoplasmic Ig and for immunohistochemistry with turbot lymphoid tissues. Very low numbers of Ig+cells were found in thymus sections. In sections of spleen, Ig+cells were observed in white pulp, around ellipsoids but were mostly concentrated and associated with melanomacrophage centers (MMCs). The lymphoid Ig+cells in the kidney tended to be dispersed among haematopoietic and granulopoietic cell populations and were in intimate association with the MMCs and blood vessels. This association between MMCs and Ig+cells in the spleen and the kidney, is discussed with respect to the role played by these organs in the immune system of fish. Last, the lymphoid population in the gut associated lymphoid tissue (GALT) of turbot was characterised with respect to staining for Ig. Immunoreactive cells were rarely detected in the epithelial layer although many lymphocytes were present, but they were frequently observed in the lamina propria, presumably as part of the GALT and involved in mucosal immune responses.

Detection of Antibodies to Avian Pneumovirus by a Micro-Indirect Immunofluorescent Antibody Test

A micro-indirect immunofluorescent antibody (micro-IFA) test with a 96-well, flat-bottomed microplate was developed for measuring avian pneumovirus (APV) antibodies. Two Japanese APV strains (MM-1, 8597/CV94) isolated at different places and times and Vero cells were used for antigen preparation in this test. The test results were compared with those of a serum neutralization (SN) test. By the micro-IFA test, specific immunofluorescent antigens were observed in the cytoplasm of cells infected with either strain, and the antibody titers of antisera to these strains were quite similar. In most cases, the results were obtained within 3 hr. Antibody titers between the micro-IFA and SN tests were highly correlated, with correlation coefficients of 0.873 (MM-1 strain) and 0.889 (8597/CV94 strain). We also investigated APV antibody status in two farms for a period of about 2 yr by the micro-IFA test and revealed that APV infections were repeated within these farms. On the basis of these results, we conclude that our micro-IFA test is useful for routine serologic surveys of APV infections, particularly when a large number of samples are to be treated, because this test was time and labor saving relative to SN tests or conventional IFA tests utilizing embryo tracheal organs or coverslip cell cultures.

Influence of the amino acid differences between the hemagglutinin HA1 domains of influenza virus H1N1 strains on their reaction with antibody.

For influenza H1N1 strains, including some of their escape variants, the association of amino acid differences located at their hemagglutinin HA1 domains with their antigenic relationship was examined. The antigenic relationship was recorded in terms of the ratios of hemagglutination inhibition (HI) titers, the concentration of antibody molecules recognized by the virus, and the equilibrium constant of epitope-paratope interaction determined with heterologous virus compared to that found with homologous virus. The HI titers of antisera were found to depend primarily on the concentration of antibody molecules recognized by the virus and much less on the equilibrium constants. The avidity of antibody in sera raised against historically later strains with earlier strains was higher than vice versa. In contrast to the results obtained with antisera, the same concentration of monoclonal antibody directed to the Sb site of A/Brazil virus was recognized by both heterologous and homologous viruses, and the differences in HI titers observed were due to avidity changes only. Some of the amino acid differences located at each of the antigenic sites were found to be associated with a reduction in the HI titers and in the concentration of antibody molecules recognized by heterologous virus, whereas other differences in addition decreased the avidity of epitope-paratope interaction. Further amino acid differences decreased the avidity only. The strains tested differed also in their amino acids located outside the antigenic sites. However, an influence of these differences on the reaction of virus with antibody could not be evidenced. For the strains tested, the antigenic hemagglutinin drift occurred by reduction of the concentration of antibody molecules recognized by the virus and by avidity changes, which, in turn, were caused by exchanges of some key residues located at the antigenic sites.

Low cost production and purification of polyclonal antibodies to staphylococcal enterotoxin A

An immunization scheme for production of antiserum to staphylococcal enterotoxin A (SEA) is proposed. The reference method of Robbins and Bergdoll was modified to reduce the number of doses and the amount of toxin used per animal. The best immunization scheme used injections in days 0, 8, 24, 59, 62 and 67. The amount of toxin at each injection was 5, 6, 20, 50, 100 and 200<FONT FACE="Symbol">m</font>g, respectively. The total amount of toxin was 381<FONT FACE="Symbol">m</font>g, which corresponded to a reduction of 107<FONT FACE="Symbol">m</font>g in the amount of toxin for each animal when compared to the reference method. The average antiserum titer using the Optimum Sensitivity Plate - OSP was 1:60 and using ELISA the titer was 1:100.000. The lack of cross-reactivity with other staphylococcal enterotoxins indicated high specificity of the antibody to SEA. The proposed immunization scheme was adequate to produce specific SEA antisera, with high titers and the aditional advantage of reducing the amount of purified SEA required for immunization.

Production and specificity of polyclonal antibodies to hexanal-lysine adducts.

Hexanal content is a widely used index of lipid oxidation in foods. The objectives of this study were to develop antibodies to hexanal-lysine adducts, devise an ELISA, and characterize antibody specificity. Hexanal was made immunogenic by covalent attachment to lysine side chains of bovine serum albumin via reductive alkylation. Polyclonal antibodies had antiserum titers as high as 6.15 x 10(5). A competitive indirect ELISA was developed with a detection limit of 0.7 ng of hexanal/mL. Antibodies were carrier-independent, reacting with hexanal conjugates of several proteins but not with the corresponding native proteins. Cross-reactivities with chicken serum albumin conjugates of n-heptanal, n-pentanal, and n-octanal were 86. 3, 11.8, and 2.2%, respectively. Antibodies reacted strongly with hexanal-modified lysine and hexanal-modified epsilon-aminocaproic acid but did not recognize free amino acids or free hexanal. It may be feasible to use this ELISA to monitor lipid oxidation in food provided hexanal is alkylated to a carrier protein prior to analysis.

Radioimmunoassay of regulatory peptides in the presence of acetonitrile: marked improvement of cholecystokinin assays

Radioimmunoassay has made it possible to measure the levels of many hormones. However, samples for some hormones, such as cholecystokinin (CCK), need to be purified by reverse phase chromatography before assay. Usually, samples are eluted from cartridges or HPLC columns in about 50% acetonitrile, dried on a vacuum centrifuge, and then reconstituted in buffer. Drying and reconstituting samples is time consuming and introduces additional sources of error and peptide loss. The present study investigated the effect of acetonitrile on radioimmunoassays for CCK to see if samples containing acetonitrile could be assayed directly. The non-specific binding of a radiolabeled peptide, the zero binding (B 0), and the fall in the presence of 2.5 fmol unlabeled CCK were determined in the presence of various proportions of acetonitrile with 0.1% TFA. Additionally, standard curves were compared in the presence and absence of 200μl of 50% acetonitrile, ( n=5). For assays using two separate CCK antisera, increasing amounts of acetonitrile gave progressively higher zero binding and fall, thereby increasing sensitivity and antibody titer. The use of 200μl 50% acetonitrile, chosen to represent typical sample conditions, increased antiserum titers by three to four-fold, as well as increasing sensitivity considerably. For one antiserum (CCK2), the IC 20 was 0.36±0.02 fmol CCK/tube in the presence of acetonitrile and 1.45±0.08 fmol/tube in its absence ( P<0.001). For the other antiserum (Dino 7), the IC 20 was 0.40±0.02 fmol CCK/tube in the presence of acetonitrile and 0.63±0.01 fmol/tube in its absence ( P<0.001). A similar increase in sensitivity was seen with a gastrin assay. However, no significant change in the gastrin antibody titer was evident. Assays for several other hormones were unaffected by 200 μl of 50% acetonitrile. At volumes encountered in samples following chromatography, acetonitrile did not adversely affect radioimmunoassays for a number of hormones, and the sensitivity and antibody titer of the CCK assays were improved. Measurement of CCK samples without drying and reconstitution increases assay efficiency and sensitivity.

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

SummaryA Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)‐ELISA, triple antibody sandwich (TAS)‐ELISA, antigen coated plate (ACP)‐ELISA, and protein‐A coated antibody sandwich (PAS)‐ELISA. TAS‐ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP‐ELISA and PAS‐ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV‐infected plants from asymptomatic plant samples than did TAS‐ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS‐ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV‐indexing of field samples, tissue sampling should be done during the rainy season when most BSV‐infected plants express severe symptoms.

Immunomethods for detecting a broad range of polychlorinated biphenyls

Aroclor 1254 (consisting of a mixture of over 100 different congeners of polychlorinated biphenyls (PCBs) was adsorbed on to the surface of polystyrene microbeads and used to immunize mice and rabbits. High titre antisera were produced. PCBs were also adsorbed on to antimony pentoxide microbeads which were then used effectively as immobilized antigen in microbead‐based ELISA assays. Antisera raised with Aroclor 1254‐coated beads contained antibodies with high affinity (as determined by BIAcore analysis) against tetracholoro‐biphenyls even though these congeners represent only a minor fraction in Aroclors.

Banana Die-Back Virus - a New Virus Infecting Banana in Nigeria.

Two viruses naturally infect Musa in Nigeria: banana streak badnavirus (BSV) and cucumber mosaic cucumovirus (CMV). During a recent field survey at Ibadan (Nigeria), some severely stunted banana plants (cv. Valery) were found that tested negative for CMV, banana bunchy-top virus, and BSV. The plants had symptoms of leaf crinkling, leaf necrosis, and cigar-leaf die-back. Subsequent suckers from the same mats were progressively more stunted. A 28- to 30-nm isometric virus was purified, and used for the production of antibodies, from the affected plants with (NH4)2SO4 to precipitate the virus. The antiserum (titer of 1:10,000) was used in enzyme-linked immunosorbent assay and immunosorbent electron microscopy to detect the virus. Mechanical inoculation with partially purified virus preparations resulted in stunting and development of pinpoint chlorotic lesions on Vigna unguiculata TVu-76 and symptomless systemic infection of Nicotiana occidentalis. The virus was not mechanically transmissible from N. occidentalis to banana. A serological relationship between this virus, banana die-back virus (BDBV), and tobacco ringspot, tomato ringspot, and cacao necrosis nepoviruses was found. The nematode species around the affected banana plants were isolated: Helicotylenchus multicinctus (Cobb) Golden was the dominant species, low numbers of H. dihystera (Cobb) Sher were present, but no virustransmitting nematodes were found in soil or banana roots. Further studies are needed to determine the mode of spread of BDBV, the implications for banana/plantain production in sub-Saharan Africa, and the safe international movement of germplasm.

Generation and neutralization of pseudovirions of human papillomavirus type 33.

Since human papillomaviruses (HPV) cannot be propagated in cell culture, the generation of infectious virions in vitro is a highly desirable goal. Here we report that pseudovirions can be generated by the assembly of virus-like particles (VLPs) in COS-7 cells containing multiple copies of a marker plasmid. Using recombinant vaccinia viruses, we have obtained spherical VLPs of HPV type 33 (HPV-33) which fractionate into heavy and light VLPs in cesium chloride density gradients. VLPs in the heavy fraction (1.31 g/cm3) carry the plasmid in DNase-resistant form and are capable of transferring the genetic marker located on the plasmid to COS-7 cells in a DNase-resistant way (pseudoinfection). The minor capsid protein L2 is not required for encapsidation but is essential for efficient pseudoinfection. Antiserum to HPV-33 VLPs inhibits VLP-mediated DNA transfer with high efficiency. Antisera to VLPs of HPV-18 and HPV-16 are not neutralizing, although the HPV-16 antiserum exhibited some cross-reactivity with HPV-33 VLPs in an enzyme-linked immunosorbent assay. In a cell binding assay, the titer of the HPV-33 VLP antiserum was 1:200 compared to the neutralization titer of 1:10(5). This indicates that neutralization is essentially due to the inhibition of cellular processes after VLP binding to cells. The encapsidation of marker plasmids into VLPs provides a sensitive and fast assay for the evaluation of neutralizing potentials of antisera against papillomavirus infections.

Cellular distribution of the rat D 1B receptor in central nervous system using anti-receptor antisera

Polyclonal antisera have been generated against two unique polypeptide fragments in the rat D 1B dopamine (DA) receptor, as deduced from the cDNA sequence. Antisera titers were monitored using solid-phase ELISA. Once the titers were established, antisera specificity was determined using Chinese Hamster ovary (CHO) cells, stably transfected with the full-length cDNA for the rat D 1B DA receptor. Immunoreactivity following staining with either anti-D 1B DA receptor antisera was equivalent, selective for the D 1B DA receptor-transfected CHO cells, and expressed at their membrane and within the cell cytoplasm. Minimal immunofluorescent staining for D 1B DA receptor proteins was detected in untransfected CHO cells, or in D 1A DA receptor-transfected CHO cells. The regional and cellular distribution patterns for the D 1B DA receptor subtype were examined in various brain areas and illustrated significant protein levels within the frontal and parietal cortices and in the hippocampus and dentate gyrus. Lesser amounts of receptor protein staining were seen in the dorsal striatum, olfactory tubercle, and cerebellar vermis. D 1B DA receptor protein staining was correlated with the cellular expression of D 1B DA receptor mRNA transcripts in these same brain regions using concurrent fluorescent analyses. The homologous coincidence in staining patterns for the D 1B DA receptor transcripts and encoded proteins in identified neurons of the frontal cortex and striatum showed variations in receptor expression in these identified basal ganglia pathways.

Characterisation of the Antibody Response to the Extracellular Region of Recombinant Thyrotropin Receptor

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable” of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.