Site-directed polyclonal antibodies inhibit binding of activated estrogen receptor to DNA

We have generated three polyclonal antisera to the DNA-binding domain of the human estrogen receptor (hER). Antiserum AT2A was generated against a peptide spanning amino acids 231-245 of hER, while antisera AT3A and AT3B were generated against a peptide spanning amino acids 247-261 of hER. The interaction of these three antisera with ER has been characterized by sucrose density gradient analysis. The antisera bound to the unactivated (8S), salt-activated (4S), and heat transformed (5S) ER complex. All the antisera appeared to be site-specific since binding of salt-activated ER to the antisera was blocked by the presence of excess free synthetic peptides. Antisera AT3A and AT3B inhibited the binding to DNA of the KCl-activated 4S ER and the heat-transformed 5S ER. Although antiserum AT2A bound to ER, it did not inhibit DNA binding of activated ER complexes. The ability of antisera AT3A and AT3B to inhibit ER binding to DNA was concentration dependent. Once bound to the DNA, ER complexes were not significantly affected by incubation with the antisera, suggesting that binding of DNA to ER inhibits antibody ER interaction and renders that domain inaccessible to the antibodies. These results demonstrate that site-directed antibodies to ER inhibit binding of activated ER complexes to DNA in vitro.     

Purification of human kidney angiotensin I converting enzyme using reverse-immunoadsorption chromatography

A rapid and highly efficient procedure for purification of angiotensin I converting enzyme from human kidney has been developed. Following tryptic solubilization, the enzyme was partially purified by DEAE-cellulose and hydroxylapatite chromatography. The final step consisted of “reverse immunoadsorption” on a column prepared by coupling antisera raised against contaminating proteins to CNBr-activated Sepharose CL-6B. Starting with 600 g kidney tissue, 6.1 mg of enzyme was obtained with a specific activity of 108 U/mg using Hip-His-Leu as substrate, a 3400-fold purification with an overall yield of 26%. The preparation gave a single band on 7.5% SDS-urea gels and a single arc against antisera to impure enzyme in crossed immunoelectrophoresis. A single N-terminal amino acid (leucine) was detected by dansylation. This procedure has allowed the initiation of structural studies with the human enzyme. “Reverse immunoadsorption” may be a generally useful method for protein purification.     

Immunological evidence for FGLamide- and W2W9-allatostatins in the ovary of Gryllus bimaculatus (Ensifera, Gryllidae)

Using polyclonal antisera to Gryllus bimaculatus allatostatin A1 (FGLamide) and B1 (W²W⁹‐amide), two sensitive and specific competitive enzyme‐linked immunosorbent assays (ELISAs) were developed, which allowed detection of A‐ and B‐allatostatin‐like immunoreactivity in extracts of ovaries and partially purified chromatography (HPLC) fractions. Immuno‐positive HPLC fractions inhibited biosynthesis of Juvenile Hormone in vitro in the rapid partition assay. Both antisera were also used to immunolocalize A‐ and B‐allatostatin epitopes in the ovary of larval and adult crickets. A‐ and B‐allatostatin immunoreactivity appeared in the cortical cytoplasm of the oocytes at the anterior cell poles. Controls treated with the pre‐immune sera or with antisera preadsorbed onto the respective peptides coupled to a solid phase support did not stain. There was no staining in any neural structures within the ovary.     

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of human alpha-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band pI = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.     

Xenoantisera against a murine T cell tumor product cross-react with immunoglobulin Fab determinants

Some murine monoclonal T lymphoma cells express a surface component that reacts with chicken antisera produced against the Fab fragment of normal mouse IgG. In the present study, we use a solid phase immunoadsorbent consisting of affinity-purified chicken anti-Fab coupled to Sepharose to isolate a product produced by the in vitro T cell line, WEHI-7.1. The affinity-purified T cell surface molecule (IgT) migrated on SDS-PAGE as a single band of approximately 65,000 daltons. The object of these studies was to produce xenoantisera against the purified T cell product cross-reactive with Ig determinants and to characterize the antisera. Rabbits immunized with this purified molecule produced antibodies that reacted with Fab fragments of polyclonal mouse IgG and with the myeloma proteins MOPC-104E and MOPC-41, as detected by enzyme-linked immunosorbent assay (ELISA). This binding was eliminated by adsorption of the antisera with normal polyclonal IgG; however, adsorption with fetuin did not significantly affect the reactivity of the antisera. Radioimmune precipitation assays revealed that the rabbit anti-IgT bound to normal murine spleen and thymus cells; this reactivity was abrogated by adsorption with insolubilized polyclonal IgG. Competition radioimmunoassays demonstrated that detergent extracts of the thymus and the spleen contained material that inhibited the precipitation of MOPC-41; nonlymphoid cells lacked such material. The rabbit anti-IgT serum blocked the binding of antigen by normal T cells; adsorption of the antiserum with polyclonal IgG-Sepharose abrogated this blocking capacity. A solid phase immunoadsorbent prepared from the IgG fraction of the rabbit anti-IgT isolated a single component from formic acid-solubilized mouse thymus. This molecule had an approximate mass of 65,000 to 70,000 daltons. The anti-IgT serum isolated surface IgM and IgD from lactoperoxidase-catalyzed radioiodinated B cells. The anti-IgT serum detected IgM and IgG in mouse serum with the use of immunoelectrophoresis. The anti-IgT immunoadsorbent isolated several components from normal mouse serum, that, when analyzed by SDS-PAGE under reducing conditions, revealed bands corresponding to mu-, gamma-, and light chains as well as components that migrated between mu- and gamma-chains, and another component with an approximate mass of 45,000 daltons. Our results with antibodies to a purified T cell product indicate that a surface component of normal T cells and certain monoclonal T cell tumor lines is serologically related to the Fab fragment of serum Ig and is implicated in the binding of antigen.     

Autoimmune thyroiditis with transient thyrotoxicosis: comparison between painful thyroiditis and painless thyroiditis

Clinical and laboratory findings and long-term outcomes in 8 patients (7 women) with autoimmune thyroiditis (AT), aged 34-59 years, who had a painful tender goiter and a transient thyrotoxicosis with a low thyroid radioactive iodine uptake (RAIU), were compared with those in 15 patients (13 women) with painless thyroiditis (PT), aged 23-69 years. Six painful AT and 6 PT patients had a history of prior awareness of goiter. All patients with painful AT had a moderate or marked elevation of erythrocyte sedimentation rate and a positive result for C-reactive protein, while only 3 PT patients (group B) did. There were no significant differences between the mean age, duration of symptoms, white blood cell count, serum triiodothyronine (T3) and thyroxine (T4) concentrations, serum T3/T4 ratio and duration of thyrotoxicosis after the initial examination and prevalences of positive results for antithyroglobulin and -microsomal antibodies in the two diseases. Two of 8 painful AT patients showed a histologically chronic fibrous variant and 6 others showed chronic lymphocytic thyroiditis. All PT patients examined also showed lymphocytic thyroiditis. Two and 5 painful AT patients developed transient and persistent hypothyroidism, respectively, while 8 [7 in group A (normal ESR), 1 in group B] and 3 PT patients (1 in group A, 2 in group B) did, respectively. The mean serum thyroid-stimulating hormone level in the hypothyroid phase in painful AT patients was higher than that in PT patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.     

Immunofluorescent localization of two water-soluble glycoproteins including the major allergen from the pollen of ryegrass,Lolium perenne

Summary Two major glycoproteins have been localized in sectioned grains of ryegrass pollen by direct and indirect immunofluorescence methods using Fluorescein isothiocyanate (FITC)-labelled IgC fractions of antisera. These glycoproteins are the major allergen Group 1 allergen, and a principal antigen Antigen A. Four methods of fixation were employed: freeze-drying, methanol, 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h at 4°C. The post-embedding staining technique of immunocytochemistry was used: anthers were embedded directly, or after dehydration, in JB-4 plastic resin and antibody reacted with sectioned pollen.The effects of these fixatives on the antibody combining sites of the antigens were quantified by a solid phase radioimmunoassay using [¹²⁵I]protein A to measure antibody binding. Glutaraldehyde was the only fixative to significantly depress antibody binding of both Antigen A and Group 1 allergen to their homologous antisera. This radioimmunoassay was modified to reyeal that FITC conjugation to either antibody did not impair antigen binding. In mature pollen, these antigens were located in the cytoplasm and in the complex wall. In developing grains early in the maturation period, specific fluorescence was concentrated at the periphery of the cytoplasm.     

Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

An immunoradiometric assay for the measurement of neuropeptide Y in plasma

The development of an immunoradiometric assay (IRMA) for the direct measurement of neuropeptide Y (NPY) concentrations in plasma is reported. The assay employs simultaneous addition of ¹²⁵I-labelled affinity purified sheep anti-(NPY 31–36) immunoglobulin (IgG) and a rabbit anti-NPY serum to 0.25 ml volumes of standard or unknown. After 16 hr incubation at 4°C NPY-bound labelled IgG is precipitated using sheep anti-(rabbit IgG Fc region) IgG coupled to Dynospheres solid phase. Precipitated counts are proportional to the NPY concentration in samples. Using this methodology it is possible to measure basal levels in normal human subjects (range 1–5 fmol/ml). Technical difficulties encountered in raising “site-specific” antisera to NPY during the establishment of this assay are outlined.     

Hypolipidemic therapy modulates expression of apolipoprotein B epitopes on low density lipoproteins. Studies in mild to moderate hypertriglyceridemic patients

Low density lipoproteins (LDL) of untreated moderate to severe hypertriglyceridemic patients (HTG-LDL) are smaller in size and are relatively enriched in triglycerides and proteins compared with normal LDL (N-LDL). HTG-LDL also manifest defective binding to the LDL receptors of normal cultured human fibroblasts. These structural and functional defects are reversible by effective hypolipidemic therapy. The aims of the present study were to confirm the reversibility of the structural and functional defects in mild to moderate hypertriglyceridemic patients and also to test the hypothesis that therapy improved the binding of HTG-LDL to cells by modulating epitopes of apolipoprotein B (apoB-100) on the surfaces of LDL particles. Fasting plasma samples were obtained from five mild to moderate hypertriglyceridemic patients before and 3 weeks after bezafibrate therapy when mean triglyceride levels were 436 and 157 mg/dl (P less than 0.01), respectively. LDL particles were isolated by zonal ultracentrifugation, characterized chemically, and assayed for cell association and proteolytic degradation in-up regulated normal human skin fibroblasts. LDL immunoreactivity was tested in solid phase competitive binding radioimmunoassays (RIA) using three monoclonal antiLDL antibodies (Mab). Mab 464B1B3 and Mab 465B6C3 react against epitopes in the COOH-terminal (T2/K4) fragment of apoB-100. Mab D7.1 reacts with an epitope in the midportion (T3/K3) fragment. Mab 464B1B3 inhibits the binding of LDL to the LDL receptor. Hypolipidemic treatment altered the composition of LDL. Mean LDL triglycerides fell from 9.4 to 5.8% of LDL mass (P less than 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)     

Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone human B lymphocytes and derived tumors

Two monoclonal antibodies (LN-1, LN-2) reactive with B lymphocytes in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by utilizing cell extracts from pokeweed mitogen-stimulated peripheral blood lymphocytes and diffuse histiocytic lymphoma SU-DHL-4 cells, respectively. Both monoclonal antibodies were initially identified by indirect immunofluorescence screening techniques on paraformaldehyde-acetone-fixed cell preparations. Specificity screens with 36 well-characterized human lymphoma and leukemia cell lines showed that both LN-1 and LN-2 stained cell lines of B cell lineage but were unreactive with those of T cell or, with one exception, myeloid derivation. Null cell acute lymphoblastic leukemia cell lines were found to be LN-2+ but LN-1-. The B cell specificity of these reagents was confirmed on 15 lymphoma and 17 leukemia biopsy specimens by using indirect immunofluorescence techniques. Immunoperoxidase staining of sections from B5-fixed, paraffin-embedded human lymphoid tissues showed that LN-1 bound to the cell membrane and cytoplasm of germinal center cells whereas LN-2 stained the nuclear membrane and cytoplasm of germinal center and mantle zone B lymphocytes as well as interfollicular histiocytes and thymic medullary dendritic cells. Both monoclonal antibodies failed to stain cortical thymocytes, lymph node T cells, and peripheral blood T and myeloid cells. Immunoperoxidase staining of 20 nonlymphoid human organs and tissues revealed that LN-1 reacted positively with red blood cell precursors of the bone marrow, ciliated epithelial cells of the bronchus, distal tubular cells of the kidney, and ductal cells from several organs including the breast and prostate. In contrast, LN-2 was unreactive with all human nonlymphoid organs and tissues including the bone marrow. Indirect immunofluorescence staining of a panel of 26 solid tumor cells lines showed that LN-1 was reactive with the majority of epithelium-derived cell lines, glioblastomas, and astrocytomas but was unreactive with neuroblastomas, small cell carcinoma of the lung, and sarcomas. LN-2 was unreactive with 25 of 26 of the solid tumor cell lines by these techniques. Immunobiochemical studies have shown that LN-1 recognizes a cell surface sialoantigen whereas LN-2 is directed against a 35,000 dalton nuclear membrane protein. Because of their high specificity for B cell tumors and their ability to stain B5-fixed, paraffin-embedded tissues, LN-1 and LN-2 are useful reagents for the diagnosis and classification of the human lymphomas and leukemias.     

Immunofluorescence studies of developmental changes in sea urchin eggs and embryos

By means of indirect immunofluorescence techniques, distinct sea urchin antigens were localized in eggs and embryos (Paracentrotus lividus). The specificity of the method was ascertained from controls in which the specific rabbit anti-sea-urchin sera were substituted by rabbit antiserum to an unrelated antigen (human serum albumin), by normal rabbit serum or by phosphate-buffered saline. The specificity of staining was also evaluated by comparing the different staining patterns obtained either with antisera to whole homogenates of eggs and embryos or with antisera to distinct antigens.     

Hypothalamic influence on differentiation of the immunoreactive ACTH beta-LPH, alpha- and beta-endorphin containing cells in adenohypophysial primordia of rat fetus in organ culture (author's transl)]

Adenohypophysial primordia were isolated in rat fetuses from day 12.5 to day 15.5 of gestation. The organ culture employed for maintenance of the primordia was made up according to Watanabe et al. (1973). The fixation of primordia in Bouin Hollande's solution was performed after 9, 8, 7 or 6 days of culture when the normal duration of pregnancy was achieved. The cultivated primordia were immunologically studied using different antisera: anti-alpha(17-39)ACTH, anti-beta(1-24)ACTH, anti-beta-LPH, anti-alpha and anti-beta-endorphins, with immunoperoxidase or immunofluorescence techniques, including control experiments of the specificity of the antisera. A similar study was performed on pituitaries removed from normal rat fetuses from day 16.5 of gestation and each day up to birth, and fixated immediately. In vivo the first cells reacting with all the antisera used in this study were observed on day 16.5 of gestation; their number increased during gestation (Fig. 1 A, B and C). Immunoreactive cells with the different antisera could be detected in primordia isolated on day 12.5 of gestation after 9 days of culture. Numerous groups of cells were observed in primordia of older fetuses (Fig. 2 A and B). These data indicate that the corticotropic cells in rat fetuses could start to be differentiated without stimuli from the hypothalamus since primordia were isolated before the appearance of this cell type in normal rat fetuses and before the differentiation of the hypothalamus. The presence of ACTH and other peptides such as beta-LPH or beta-endorphin would support the hypothesis of a common precursor in this cell type existing early in gestation. Similar results were obtained in human fetuses.     

Immunochemical evidence for the transmembrane orientation of glycophorin A. Localization of ferritin-antibody conjugates in intact cells.

Antibodies were raised in rabbits to a 51-amino acid cyanogen bromide-derived peptide of human erythrocyte glycophorin A which has been shown to represent the C-terminal end of the 131-residue polypeptide chain. Antibodies prepared by immunoadsorption were found to be directed against a chymotryptic-derived peptide (residues 102 to 118) of glycophorin A but were unreactive with either intact or proteolytically modified red blood cells. No cross-reactivity was observed with glycophorin B of human or sialoglycoproteins prepared from red blood cells of other mammalian species. Ferritin-antibody conjugates of such sera were applied to thin sections of intact red blood cells (frozen or protein embedded) and were found to localize exclusively to sites distributed uniformly along the inner surfaces of the membrane. No staining was seen on sections prepared from red blood cells from other species nor on sections of human red cells pretreated with unconjugated antisera. These results provide additional evidence in intact, fixed human erythrocytes that glycophorin A has a transmembrane orientation.     

A monoclonal IgG4 (lambda) with factor V inhibitory activity.

A monoclonal IgG4 (lambda) with inhibitory activity to human coagulation factor V was isolated from the serum of a patient with a fatal hemorrhagic diathesis by using a combination of DE-52 ion exchange chromatography and isoelectric focusing techniques. Using the criteria for defining a monoclonal immunoglobulin of restricted mobility on protein electrophoresis, immunoelectrophoresis, and isoelectric focusing, as well as neutralization with class, subclass, and light chain type antisera, we are the first to demonstrate a factor V inhibitor as a monoclonal IgG4 (lambda) detectable in serum or plasma.     

Genetic polymorphism of six plasma proteins in the domestic cat

Phenotypes of cat plasma apolipoprotein A4 (APOA4), antithrombin 3 (AT3), alpha 1B-glycoprotein (A1BG), transferrin (TF), vitamin D-binding protein (GC), and an unidentified pretransferrin (PTF) were determined by using simple methods of horizontal, nondenaturing gel electrophoresis followed by protein staining. The cat proteins were identified by immunoblotting using antisera for human plasma proteins. Three alleles were reported for each of TF and PTF, and two alleles were reported for each of GC, APOA4, AT3, and A1BG. The mongrels and Persians showed a high degree of polymorphism at most of the loci whereas the Birmans exhibited much less variation. Genetic evidence indicating the occurrence of a monomeric and a dimeric form of APOA4 in cat plasma was reported.     

Immunofluorescence Staining of Group B Coxsackieviruses

Studies were conducted on the sensitivity and specificity of indirect fluorescent-antibody (FA) staining for identification of group B coxsackieviruses. Antisera produced in four different species (monkeys, rabbits, horses, hamsters) and immune ascitic fluids prepared in mice were compared for suitability in FA staining. The horse antisera showed high titers of nonspecific staining, and the rabbit antisera showed relatively low homologous FA titers. Immune reagents from monkeys, hamsters, and mice were used for homologous and heterologous testing against cell cultures infected with the various group B coxsackieviruses. Antisera or immune ascitic fluids produced in these three species showed some heterotypic and nonspecific staining at low dilutions, with the monkey antisera showing the highest heterotypic titers. However, the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. Heterotypic staining appeared as diffuse, low-level staining of the cells, whereas homotypic staining revealed characteristic, brightly staining aggregates of viral antigen in the cytoplasm of the infected cells. By using hamster immune sera, appropriately diluted to eliminate heterotypic staining and yet give strong homotypic staining, it was possible to identify correctly 79 (93%) of 85 field strains of group B coxsackieviruses at the first passage level in BS-C-1 cells; the remainder of the strains were identified after two passages in BS-CS-1 cells. No incorrect identifications were made. A limited number of field strains of group B coxsackieviruses were passed into rhesus monkey kidney and human fetal diploid kidney cells, and these were all correctly identified by FA staining, even the strains which failed to produce a cytopathic effect in the human fetal diploid kidney cells. Two human heart and brain tissues from which coxsackievirus type B4 had been isolated failed to show homotypic FA staining in excess of nonspecific or heterotypic staining.