Development and characterization of monoclonal antibodies against a mucin-type glycoprotein.

The preparation of greater than 30 different hybridomas, all secreting IgM class antibodies against epiglycanin, a glycoprotein at the surface of the mouse mammary carcinoma cell line TA3-Ha, is described. The specificities of 10 of the antibodies, with affinity constants in the range of 10(8)-10(10) l/mol were compared in an enzyme competitive binding assay. The affinity of epiglycanin was strongly reduced for all antibodies tested by incubation with periodate (10 mM, 4 degrees C) and was reduced for most of the antibodies by endo-alpha-N-acetyl- D-galactosaminidase. This suggested that carbohydrate, and specifically the Gal beta (1----3)GalNAc disaccharide, formed an integral part of the epitopes of most of the antibodies. The isolated disaccharide, however, exhibited 250,000 times less inhibitory activity in the competitive binding assay than epiglycanin. The binding capacity of epiglycanin was also reduced by incubation with trypsin or pronase, suggesting a high molecular weight dependency for binding. Incubation with sialidase increased its affinity for the antibodies. The binding of the antibodies to epiglycanin was strongly inhibited by peanut agglutinin, and to a lesser extent by lectins from Triticum vulgaris, Ricinus communis, Pisum sativum and Phaseolus vulgaris. None of the antibodies bound to any of eight different gangliosides immobilized on HPTLC plates. Mono- (Fab) and divalent [F(ab')2] fragments of the antibodies possessed very low affinity for epiglycanin. The results demonstrated that the specificities of the antibodies are related, but distinguishable, and they suggest that this epiglycanin-IgM model may be useful for studies on the general principles of the interaction between IgM antibodies and mucin-type glycoproteins.     

Presence of antibodies against cyclic citrullinated peptides in patients with 'rhupus': a cross-sectional study

'Rhupus' is a rare condition sharing features of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). If rhupus is a distinctive entity, an overlap between RA and SLE or a subset of SLE is currently debated. This study was performed to explore the prevalence of antibodies against cyclic citrullinated peptides (anti-CCP antibodies) in rhupus. Patients meeting American College of Rheumatology criteria for RA, SLE, or both were included. Clinical and radiographic features were recorded and sera were searched for anti-CCP antibodies, rheumatoid factor, antinuclear antibodies, anti-extractable nuclear antigens, and antibodies against double-stranded DNA (anti-dsDNA antibodies). Seven patients for each group were included. Clinical and serological features for RA or SLE were similar between rhupus and RA patients, and between rhupus and SLE patients, respectively. Values for anti-CCP antibodies obtained were significantly (p < 0.05) higher in RA (6/7) and rhupus (4/7) than in SLE patients (0/7) and healthy subjects (0/7). Our data support the possibility that rhupus is an overlap between RA and SLE, because highly specific autoantibodies for RA (anti-CCP) and for SLE (anti-dsDNA and anti-Sm) are detected in coexistence.     

False-Positive Anti-Toxoplasma Fluorescent-Antibody Tests in Patients with Antinuclear Antibodies

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.     

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.     

Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies.

Polyclonal and monoclonal antibodies to individual herpes simplex virus (HSV) glycoproteins were tested for ability to inhibit adsorption of radiolabeled HSV type 1 (HSV-1) strain HFEMsyn [HSV-1(HFEM)syn] to HEp-2 cell monolayers. Polyclonal rabbit antibodies specific for glycoprotein D (gD) or gC and three monoclonal mouse antibodies specific for gD-1 or gC-1 most effectively inhibited HSV-1 adsorption. Antibodies of other specificities had less or no inhibitory activity despite demonstrable binding of the antibodies to virions. Nonimmune rabbit immunoglobulin G and Fc fragments partially inhibited adsorption when used at relatively high concentrations. These results suggest involvement of gD, gC, and perhaps gE (the Fc-binding glycoprotein) in adsorption. The monoclonal anti-gD antibodies that were most effective at inhibiting HSV-1 adsorption had only weak neutralizing activity. The most potent anti-gD neutralizing antibodies had little effect on adsorption at concentrations significantly higher than those required for neutralization. This suggests that, although some anti-gD antibodies can neutralize virus by blocking adsorption, a more important mechanism of neutralization by anti-gD antibodies may be interference with a step subsequent to adsorption, possibly penetration.     

Prevalence and determinants of antibodies to hepatitis C virus and markers for hepatitis B virus infection in patients with HIV infection in Aquitaine. Groupe d'Epidémiologie Clinique du SIDA en Aquitaine.

OBJECTIVE: To evaluate the prevalence of antibodies to hepatitis C virus and serological markers for hepatitis B virus infection in patients with HIV. DESIGN: Cross sectional survey. SETTING: Aquitaine, southwestern France, 1991-94. SUBJECTS: 1935 HIV positive patients seen at least once since June 1991. MAIN OUTCOME MEASURES: Presence of antibodies to hepatitis C virus were detected by second or third generation enzyme linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA) and markers for hepatitis B virus detected by ELISA. RESULTS: The prevalence was 42.5% (823) for antibodies to hepatitis C virus, 56.4 (507) for antibodies to hepatitis B core antigen, 6.9% (133) for hepatitis B surface antigen, 30.2% (584) for antibodies to hepatitis B core and surface antigen with no detectable surface antigen, 26.2% (507) for antibodies to core antigen only, and 4.8% (92) for antibodies to surface antigen only. The prevalence of antibodies to hepatitis C virus was 86.1% (726/843) in subjects who had bloodborne HIV infection and 7.3% (66/899) in those with sexually acquired infection. The prevalence of markers for hepatitis B was higher among homosexuals than in the other groups of patients, except for antibodies to surface antigen alone. The relation between markers for hepatitis B and hepatitis C virus was negative among men but positive among women. CONCLUSIONS: The results favour the hypothesis that hepatitis C virus is sexually transmitted much less commonly than either HIV or hepatitis B virus.     

Kinetics of Inhibition of Influenza Virus Hemagglutination by Homologous Antihemagglutinin and Antineuraminidase Antibodies as Measured by Means of the Photometric Antibody Concentration Unit Method

The kinetics of the reactions of antihemagglutinin (AH) and antineuraminidase (AN) antibodies with homologous influenza virus were examined by use of a photometric hemagglutination inhibition test (antibody concentration unit [ACU] test). The following results were obtained. (i) The isotherms describing the reaction of AN antibodies with homologous virus were found to have much steeper slopes than did the corresponding AH antibody isotherm. This finding indicates that the photometric ACU method can discriminate objectively between AH and AN antibodies. (ii) The reaction of mixtures of AH and AN antibodies with homologous virus was examined and found that AH antibodies combine with virus independently from the presence of AN antibodies, whereas AH antibodies were found to reduce greatly the measurable degree of hemagglutination inhibition by AN antibodies. (iii) A formula was developed and experimentally confirmed by means of which the binding of virus by mixtures of AH and AN antibodies can be predicted. Thereby, the influence of the relative concentration of AH and AN antibodies on the establishing of isotherms and on their subsequent use for antibody titration by means of the photometric ACU method was investigated. It was found that the procedure routinely employed for establishing isotherms yielded data reflecting the reaction of AH antibodies only. As a consequence, the use of these isotherms will identify AH antibodies.     

Serodiagnosis of infection with verocytotoxin-producing Escherichia coli

Human sera (167) were screened for antibodies to lipopolysaccharide (LPS) prepared from strains of Verocytotoxin-producing Escherichia coli (VTEC) belonging to a range of serogroups, secreted proteins expressed by attaching and effacing VTEC, enterohaemolysin and H = 7 flagellar proteins. Twelve sera (about 7%) contained antibodies to the LPS of E. coli 05 (one), 026 (two), 0115 (two), 0145 (one), 0163 (one) and 0165 (five). Sera containing antibodies to the LPS of E. coli O26 and O145 also contained antibodies to secreted proteins of 100 and 40 kDa. An additional 34 sera, known to contain antibodies to the lipopolysaccharide of E. coli O157, were examined for antibodies to enterohaemolysin, H = 7 flagellar antigens and bacterial cell surface-associated proteins of 5, 6 and 22 kDa. Three sera contained antibodies to enterohaemolysin and one serum contained antibodies to flagellar proteins. Antibodies to membrane-associated proteins were not detected. It was concluded that enterohaemolysin, H = 7 flagellar proteins and the cell surface-associated proteins were unsuitable for use in immunoassays for providing evidence of infection with VTEC.     

Development of humanized antibodies as cancer therapeutics

Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described.     

Synergistic neutralization of human immunodeficiency virus type 1 by combinations of human monoclonal antibodies.

The ability of antibodies to the V3 region and the CD4-binding domain (CD4bd) of human immunodeficiency virus type 1 (HIV-1) to act in synergy to neutralize HIV has been demonstrated previously. However, synergy between antibodies to other HIV-1 epitopes has not been studied. We have used 21 combinations of human monoclonal antibodies (MAbs) directed against different epitopes of the gp120 and gp41 proteins of HIV-1 to evaluate their ability to act in synergy to neutralize HIV-1. Combinations of anti-V3 and anti-CD4bd antibodies, anti-V3 and anti-gp120 C-terminus antibodies, anti-CD4bd and anti-C-terminus antibodies, anti-V3 and anti-gp41 antibodies, and anti-CD4bd and anti-gp41 antibodies were tested. Our results show that some, but not all anti-V3 antibodies can act in synergy with anti-CD4bd antibodies. In addition, for the first time, antibodies to the C-terminus region have been found to act in synergy with the anti-CD4bd antibodies. Various anti-CD4bd MAbs also act in synergy when used together. The use of such cocktails of human MAbs for passive immunization against HIV-1 may prove to be important for therapy in postexposure settings and for prevention of maternal-fetal transmission of the virus. The results also provide information on the types of antibodies that should be elicited by an effective vaccine.     

Serologic survey for canine infectious diseases among sympatric swift foxes (Vulpes velox) and coyotes (Canis latrans) in southeastern Colorado

Swift foxes (Vulpes velox) and coyotes (Canis latrans) are sympatric canids distributed throughout many regions of the Great Plains of North America. The prevalence of canid diseases among these two species where they occur sympatrically is presently unknown. From January 1997 to January 2001, we collected blood samples from 89 swift foxes and 122 coyotes on the US Army Pi?on Canyon Maneuver Site, Las Animas County, SE Colorado (USA). Seroprevalence of antibodies against canine parvovirus (CPV) was 71% for adult (> 9 mo old) and 38% for juvenile (< or = 9 mo old) swift foxes. Adult (<1 yr old) and juvenile (<1 yr old) coyotes had a seroprevalence for CPV of 96% and 78%, respectively. Presence of antibodies against canine distemper virus (CDV) was 5% for adult foxes and 0% for juvenile foxes. Seroprevalence of CDV was 46% for adult coyotes and 18% for juvenile coyotes. No swift foxes had canine adenovirus (CAV) antibodies, whereas 81% and 63% of adult and juvenile coyotes, respectively, had antibodies for CAV. Seroprevalence of antibodies against Yersinia pestis was 68% among adult foxes and 34% among juvenile swift foxes. Seroprevalence of Y. pestis antibodies was 90% and 70% for adult and juvenile coyotes, respectively. No swift foxes had antibodies against Francisella tularensis, whereas seroprevalence was 4% among both adult and juvenile coyotes. Antibodies against CPV and plague were common in both species, whereas antibodies against CDV and CAV were more prevalent in coyotes compared to swift foxes.     

Nonneutralizing HIV-1 gp41 envelope cluster II human monoclonal antibodies show polyreactivity for binding to phospholipids and protein autoantigens

HIV-1 gp41 envelope antibodies, which are frequently induced in HIV-1-infected individuals, are predominantly nonneutralizing. The rare and difficult-to-induce neutralizing antibodies (2F5 and 4E10) that target gp41 membrane-proximal epitopes (MPER) are polyspecific and require lipid binding for HIV-1 neutralization. These results raise the questions of how prevalent polyreactivity is among gp41 antibodies and how the binding properties of gp41-nonneutralizing antibodies differ from those of antibodies that are broadly neutralizing. In this study, we have characterized a panel of human gp41 antibodies with binding specificities within the immunodominant cluster I (gp41 amino acids [aa] 579 to 613) or cluster II (gp41 aa 644 to 667) for reactivity to autoantigens, to the gp140 protein, and with MPER peptide-lipid conjugates. We report that while none of the gp41 cluster I antibodies studied were polyspecific, all three gp41 cluster II antibodies bound either to lipids or autoantigens, thus showing the propensity of cluster II antibodies to manifest polyreactivity. All cluster II gp41 monoclonal antibodies (MAbs), including those that were lipid reactive, failed to bind to gp41 MPER peptide-lipid complexes. Cluster II antibodies bound strongly with nanomolar binding affinity (dissociation constant [K(d)]) to oligomeric gp140 proteins, and thus, they recognize conformational epitopes on gp41 that are distinct from those of neutralizing gp41 antibodies. These results demonstrate that lipid-reactive gp41 cluster II antibodies are nonneutralizing due to their inability to bind to the relevant neutralizing epitopes on gp41.     

IgM antibodies to Epstein-Barr virus-associated membrane antigen in sera of infectious mononucleosis patients

By the indirect immunofluorescence technique, IgM antibodies to the cell surface of an Epstein-Barr virus (EBV) producer cell line, P3HR-1, were detected in sera from infectious mononucleosis (IM) patients but not in sera from patients with Burkitt lymphoma or nasopharyngeal carcinoma nor in sera from healthy adult donors having antibodies to EBV-specific viral capsid antigen (VCA). Titers of the IgM antibodies were higher in the earlier stages of IM, a pattern similar to that for IgM antibodies to VCA. The IgM antibodies to the cell surface were identified as being those against the EBV-specific membrane antigen (MA) by the following criteria: (1) The antibodies were reactive to MA-positive cell preparations but to MA-negative cell preparations. (2) Titers of the IgM antibodies were not significantly affected after absorption of sera with sheep red blood cells which could completely eliminate heterophil antibodies in the same sera. Detection of the IgM antibodies to MA may have a particular diagnostic value for providing evidence of a recent EBV infection.     

Australian Antigen and Autoantibodies in Chronic Hepatitis

The sera of 110 patients with chronic hepatitis and adequate controls were examined for antibodies to smooth muscle (S.M.), mitochondria (M.), and for antinuclear factors by the immunofluorescence method, and for Australia (Au(1)) antigen by a modified micro-Ouchterlony immunodiffusion technique. Twelve out of 13 patients with primary biliary cirrhosis had M. antibodies, two had antinuclear factor, and none had Au(1) in their sera. In chronic aggressive hepatitis 23·5% of the sera contained antinuclear factor, 13% S.M. antibodies, 10·5% M. antibodies, and 22% Au(1) antigen. Of the 12 patients with chronic persistent hepatitis, one had antinuclear factor, one S.M. antibodies, and three Au(1) antigen.The most striking finding was a mutual exclusion between Au(1) antigen and M. and S.M. antibodies. None of the 33 patients with one or the other form of chronic hepatitis and M. or S.M. antibodies had Au(1) antigen; 22 out of 77 (28%) patients without such antibodies were positive.     

Prevalence of Leptospira and Brucella antibodies in wild boars (Sus scrofa) in Tuscany, Italy

Five hundred sixty-two blood samples were collected from wild boars (Sus scrofa) shot in six districts of Tuscany, central Italy, between 1997 and 2000. Sera were examined for antibodies specific for Leptospira interrogans by microagglutination test and Brucella spp. by the Rose Bengal test and indirect enzyme-linked immunosorbent assay. Thirty-four (6.0%) samples tested positive for anti-Leptospira antibodies, 29 (5.1%) sera were positive for anti-L. interrogans serovar bratislava antibodies (titres ranging from 1:100-1:400), and 5 (0.9%) sera were positive for anti-L. interrogans serovar icterohaemorrhagiae antibodies (titres 1:100). All the examined sera were negative for anti-Brucella antibodies.     

Antibodies to synthetic platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) analogues with substituents at the sn-2 position.

We obtained rabbit antibodies by injecting immunogenic conjugates which were prepared by combining covalently 1-O-(15'-carboxypentadecyl)-2-O-acetyl-sn-glycero-3- phosphocholine(acetyl-CPGPC), 1-O-(15'-carboxypentadecyl)-2-O-N,N-dimethylcarbamoyl-sn-glycero-3 - phosphocholine (dimethylcarbamoyl-CPGPC), or 1-O-(15'-carboxypentadecyl)-2-O-N-butyl-carbamoyl-sn-glycero-3-pho sphocholine (butylcarbamoyl-CPGPC) with protein (BSA or KLH), respectively, and examined the specificity of the resulting antibodies by comparison with inhibition of the binding of iodolabeled CPGPC derivatives to the antibodies by corresponding or related phospholipids. Acetyl-CPGPC and dimethylcarbamoyl-CPGPC possessed haptenic activity causing production of antibodies reactive with PAF. Changes of the substituents at sn-2 in the antigens affected the specificity of the resulting antibodies. The affinity of the substituents to the antibodies decreased in the following order: acetyl much greater than dimethylcarbamoyl and butylcarbamoyl for antibodies to acetyl-CPGPC-KLH; dimethylcarbamoyl greater than acetyl much greater than butylcarbamoyl for antibodies to dimethylcarbamoyl-CPGPC-BSA; and butylcarbamoyl greater than dimethylcarbamoyl greater than acetyl for antibodies to butylcarbamoyl-CPGPC-BSA. Naturally occurring phospholipids, including lysoPAF, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, revealed no cross-reactivities with these antibodies. Anti-dimethylcarbamoyl-CPGPC-BSA IgG and anti-acetyl-CPGPC-KLH IgG inhibited a PAF-induced aggregation of washed rabbit platelets in a dose-dependent manner. In contrast, anti-butylcarbamoyl-CPGPC-BSA IgG did not affect a PAF-induced platelet aggregation, nor did preimmune IgG.     

Lack of pokeweed mitogen-induced IgE formation in vitro by human peripheral blood mononuclear cells: detection of cross-reacting idiotypic determinants on polyclonal Ig by antibodies to a single IgE myeloma protein

The ability of human peripheral blood mononuclear cells to synthesize IgE in vitro in response to pokeweed mitogen (PWM) is controversial. To determine whether the conflicting results obtained by different laboratories could be due to inherent qualitative differences in the anti-IgE antibodies used to measure low concentrations of IgE in culture supernatants, we compared the specificities of anti-IgE reagents prepared by various methods. Immunoadsorbent-purified antibodies were isolated from a goat antiserum to the lambda, IgE myeloma protein PS and a rabbit antiserum to the kappa, IgE protein Bed in three ways: 1) antibodies to IgE PS (anti-PS) were isolated from the goat antiserum by affinity chromatography with PS coupled to Sepharose 4B; these antibodies consisted of anti-epsilon chain-specific and anti-idiotypic antibodies to protein PS; 2) antibodies specific for the epsilon-chain (anti-epsilon) were purified by affinity chromatography with IgE myeloma proteins that were not used for immunization; and 3) antibodies to idiotypic determinants of proteins PS (anti-id PS) and Bed (anti-id Bed) were isolated on affinity columns with the respective myeloma proteins after absorption of the epsilon-chain-specific antibodies. These three types of antibodies were then used in a solid phase radioimmunosorbent test to quantitate the amount of "IgE" synthesized by peripheral blood mononuclear cells from nonatopic and atopic donors cultured for 7 days in the presence and absence of PWM. ANTI-PS antibodies detected a PWM-induced "IgE formation" in cell culture supernatants of both non-atopic and atopic donors. (ABSTRACT TRUNCATED AT 250 WORDS)     

Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies

Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity.     

California serogroup and Powassan virus infection of cats

One hundred and seventy five sera from cats in Ontario, Canada, were tested for hemagglutination inhibition (HI) antibodies to three arboviruses; namely, Powassan (POW) of the Flavivirus serogroup, and Snowshoe hare (SSH) and Jamestown Canyon (JC) viruses of the California (CAL) serogroup. All sera were negative for antibodies to POW virus. Twelve cats possessed CAL serogroup antibodies including 3 with antibodies to SSH alone, 6 with antibodies to JC alone, and 3 with antibodies to both SSH and JC antigens. POW virus was inoculated into seven cats, one intracerebrally and six intravenously. Neurologic signs were not detected in any of the cats. Histologic lesions of a nonsuppurative encephalitis and encephalomyelitis were observed in the intracerebrally inoculated cat and in one of the intravenously inoculated cats, respectively. POW virus was not isolated from the brain or spinal cord of either of these two cats. HI antibodies were detected in the sera of all inoculated animals. HI antibodies were not detected in the CSF of any animal.     

Production of monoclonal antibodies to lactate dehydrogenase (LDH) isoenzymes for immunohistochemical study on fixed tissue section

Summary Purified lactate dehydrogenase (LDH) isoenzyme 1 (H or B subunits) and isoenzyme 5 (M or A subunits) were used to prepare monoclonal antibodies (MAb) suitable for immunohistochemical detection on formalin fixed paraffin-embedded tissue sections. In the initial fusions, screening of the antibodies was based on enzyme linked immunosorbent assay (ELISA) against the immunogens. None of the antibodies obtained was satisfactory. There were various problems related to specificity, crossreactivity, affinity and also the properties of the monoclonal antibody itself. Using a combined system involving more than one method for screening, two suitable monoclonal antibodies, MAb65 (to H-type LDH) and MAb25 (to M-type LDH) were selected. Both antibodies reacted specifically with corresponding LDH isoenzymes as shown in a series of tests. Their reactivity in sections of formalin fixed paraffin-embedded tissue indicated that both antibodies are suitable reagents for immunohistochemical studies.