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L R Haaheim

Publications and source records attributed to L R Haaheim.

16 recordsLinked to original sources
PubMed1991

Monoclonal antibodies--their diagnostic potential.

Considering the long and excellent performance of 'classical' immune sera in a range of diagnostic applications, the advent of 'tailor-made' highly specific monoclonal antibodies has given even higher hopes of diagnostic accuracy for the future. Understanding what monoclonal antibodies are and how they compare to polyclonal sources of antibodies is absolutely essential in order to appreciate their potential. This paper will briefly focus on the production, characterization and use of monoclonal antibodies and look critically at the future prospects. Specificity and cross-reactivity are key terms which are explained with examples in this paper. It can be anticipated that diagnostic kits in the future will be composite assays using highly specific monoclonals (or oligoclonal cocktails) in conjunction with high-avidity polyclonal sera, thus taking advantage of the best features of both systems.

Antibodies, Monoclonal↗
PubMed1991

Fine molecular specificity of linear and assembled antibody binding sites in HIV-1 p24.

A set of seven murine monoclonal antibodies were generated against a chemically synthesized 11-kDa 104-mer peptide covering the C-terminal residues 270-373 of the p24 gag protein (HIV-1BRU strain). All monoclonal antibodies recognized HIV-1IIIB infected MOLT3 cells by fluorescence and gave positive Western blot signals with viral gag peptides (p55 and/or p24). Oligopeptide binding regions were located with competitive enzyme-linked immunosorbent assays. Detailed epitope scanning analyses (the Geysen technique) were performed by serological testing of the monoclonal antibodies against 99 overlapping hexapeptides which corresponded to the entire 104-mer region. The antibodies bound to p24 peptide sequences located within the 275-293 and 351-368 regions. One antibody (LH104-B) which reacted with residues 357-362 bound to p55 alone. In contrast, another antibody (LH104-I), which recognized the residues 358-363, i.e. with five out of six residues in common with antibody LH104-B for its epitope region, reacted exclusively with p24. At least two of the antibodies (LH104-C and -A) which bound to p24 alone, apparently recognized conformational epitopes. They gave positive reactions with the regions 288-293/351-356 and 284-289/351-356, respectively. This work shows that chemical synthesis of large peptides is a viable alternative approach to immunochemical studies of viral proteins.

Amino Acid Sequence↗
PubMed1989

Monoclonal antibodies reacting with the exopolysaccharide xanthan from Xanthomonas campestris.

We have prepared murine hybridomas secreting monoclonal antibodies against the exopopolysaccharide xanthan from Xanthomonas campestris pv. campestris 646 after fusing NSO myeloma cells and spleen cells from BALB/c mice immunized with xanthan. Four hybridomas, secreting antibodies designated A6 (IgM kappa), B3 (IgM kappa), D1 (IgM kappa), and D3 (IgG2A kappa), were selected for further studies. All antibodies reacted with a range of different xanthans. Competition studies using variants of the exopopolysaccharide as competitors suggested that specificity was mainly against the side-chain. One of the antibodies (B3) appeared to require the fully acylated side-chain with the pyruvylated terminal mannose as the immunodominant part. The three others were assumed to be directed against the nonsubstituted trisaccharide with the inner mannose-glucuronic acid being immunodominant. None of the antibodies reacted with cellulose (the xanthan backbone). Using immunoblotting techniques on nitrocellulose paper both a mixture of monoclonal antibodies, and also polyclonal ascitic fluid, could detect xanthan quantities of approximately 0.1 microgram.

Animals↗
PubMed1989

Antibodies in sera from Norwegian multiple sclerosis patients and the general population reacting with HTLV-1. A pilot study.

By using enzyme-linked immunosorbent assay, Western blot and passive agglutination we have performed a pilot study involving the testing of 349 sera from the general population of 3 coastal regions in southern Norway, 230 sera from multiple sclerosis patients from western Norway, 109 age- and sex-matched controls and 34 sera from patients with other neurological diseases, for antibodies to HTLV-I. About 2% and 4% of sera from the general population had antibodies reacting with HTLV-I in ELISA and Adult T-cell Leukemia Antigen by agglutination, respectively, whereas about 8% and 5% of the multiple sclerosis sera had such antibodies. In the general population antibodies were more frequent among young individuals and there appeared to be some prevalent regional differences. Western blot confirmatory tests showed that in some instances antibodies reacted with the gag proteins, in particular p19. The results obtained in this study do not indicate an association between HTLV-I and multiple sclerosis, although at this stage we cannot rule out that another retrovirus is involved.

Agglutination Tests↗
PubMed1989

Antibodies to staphylococcal peptidoglycan and its peptide epitopes, teichoic acid, and lipoteichoic acid in sera from blood donors and patients with staphylococcal infections.

Antibodies to the staphylococcal antigens peptidoglycan, beta-ribitol teichoic acid, and lipoteichoic acid, as well as to the peptidoglycan epitopes L-Lys-D-Ala-D-Ala, L-Lys-D-Ala, and pentaglycine, were found over a wide range of concentrations in sera from both blood donors and patients with verified or suspected staphylococcal infections. The patient group was heterogeneous with regard to both age and type of staphylococcal infections, being representative for sera sent to our laboratory. In single-antigen assays antibodies to pentaglycine had the highest predictive positive value (67%), although only 32% of the patients had elevated levels of such antibodies. Combinations of test antigens could yield positive predictive values as high as 100%, but then the fraction of positive sera was low. Indeed, the fraction of patient sera which was positive in multiple-antigen tests never exceeded 61%. The clinical usefulness of these seroassays for identifying Staphylococcus aureus as a causative agent was limited, owing to the considerable overlap in the range of antibody concentrations between patient and blood donor sera.

Adolescent↗
PubMed1989

Staphylococcus aureus exopolysaccharide in vivo demonstrated by immunomagnetic separation and electron microscopy.

Staphylococcus aureus strains were separated from mastitis milk samples without cultivation by using monodisperse magnetic polymer particles coated with polyclonal antiserum against an encapsulated S. aureus strain. Exopolysaccharide was verified by transmission electron microscopy and the serum soft-agar culture technique. Capsular polysaccharide was found on virtually all clinical isolates. Surface protein A and S. aureus-specific cell wall components were masked when the strains were cultured on an exopolysaccharide-promoting medium. Masking of surface determinants was dependent on their concentration on the bacterial surface as well as on exopolysaccharide abundance. The polysaccharide layer on in vivo bacteria was reduced markedly after just one transfer from milk to blood agar plates but was reexpressed after culturing was done on a capsule-generating medium.

Animals↗
PubMed1987

Monoclonal antibodies evoked by the free oligopeptide (Gly)5 reacting specifically with peptidoglycan from staphylococci.

Murine monoclonal antibodies reactive with the Staphylococcus aureus peptidoglycan (PG) epitope (Gly)5 were obtained using the synthetic oligopeptide (Gly)5 in its free form as immunogen. The selected monoclonal antibodies were of the IgM kappa isotype and reacted specifically with PG from S. aureus and Staphylococcus epidermidis, but gave no reaction with PG from Streptococcus pyogenes, Bacillus subtilis and Micrococcus lysodeikticus. Affinity chromatography showed that the antibodies were reactive with the N-terminus of the (Gly)5 peptide. These monoclonal antibodies can be used for the detection of staphylococcal PG in solution.

Antibodies, Monoclonal↗
PubMed1986

Intrathecal synthesis of antibodies to staphylococcal antigens in multiple sclerosis patients.

Human serum and cerebrospinal fluid (CSF) antibodies against three staphylococcal antigens were measured using the ELISA technique. Antibody levels in sera from patients with multiple sclerosis (MS) were lower than those found in sera from patients with other neurological diseases (OND) and from healthy individuals (N). The CSF antibody mean titres for patients with relapsing remittent MS were about twofold higher than those found for the other groups. Both serum and CSF antibodies were of the IgG class while IgM could only be detected in serum samples. Elevated IgG-indexes, indicating intrathecal immunoglobulin synthesis, were found only among individuals in the MS patient groups. Some of the patients also had elevated indexes of specific antibodies to the staphylococcal antigens used, i.e. lipoteichoic acid (LTA), peptidoglycan (PG) and beta-ribitol teichoic acid (beta-RTA). This indicates that some MS patients have synthesized staphylococcal antibodies intrathecally.

Adult↗
PubMed1985

Antibodies to lipoteichoic acid from Staphylococcus aureus. Specificity of murine monoclonal and human antibodies.

Two monoclonal antibodies against staphylococcal lipoteichoic acid (LTA) were made by fusing P3X63Ag8 myeloma cells and splenocytes from mice immunized with purified LTA. Both were isotyped as being IgM kappa. Their specificities were determined by enzyme-linked immunosorbent assays indicating that both antibodies reacted with the glycerol-phosphate backbone, while one of them also had some affinity for the alanyl substituent. Antibodies in serum from 7 multiple sclerosis (MS) patients and serum and cerebrospinal fluid (CSF) from 7 non-MS patients apparently reacted with the sugar moiety of LTA. In contrast, CSF antibodies from 6 of the 7 MS patients and 1 of the 7 non-MS patients had affinity for the alanine residue. This non-MS patient also had serum antibodies against the alanine residue. None of the other sera tested appeared to contain such antibodies.

Animals↗
PubMed1979

Antibodies to the strain-specific and cross-reactive determinants of the haemagglutinin of influenza H3N2 viruses. 1. Preparation of antibodies and in vitro studies.

The serological analysis of antibodies to the haemagglutinin (HA) of influenza A viruses of the Hong Kong (H3N2) subtype is described, using haemagglutination-inhibition, immuno-double-diffusion and single-radial-diffusion techniques. By cross-absorption of antisera to purified HA antigens, different populations of antibody molecules were obtained, which are designated strain-specific and cross-reactive and characterized in terms of their antigenic specificities for HA antigens of the homologous and antigenically variant H3N2 viruses. A narrowly strain-specific population of antibodies (SS"HK) was obtained as the residual antibody in antiserum to A/Hong Kong/1/68 HA after absorption with the closely related A/England/42/72 virus, whilst a contrasting broadly cross-reactive population (CR'HK) was obtained by absorption of the anti-A/Hong Kong/1/68 HA serum with the more distantly related strain A/Victoria/3/75 and eluting the cross-reactive antibodies from the absorbing virus. Similarly, specific and cross-reactive antibodies were derived from antiserum to A/Victoria/3/75 HA antigen by absorption with A/Hong Kong/1/68 virus. Single-radial-diffusion tests were performed, involving sequential application of different antibody preparations in the same wells in immunoplates containing intact virus particles. The cross-reactive and strain-specific antibodies differed in their property of mutual interference of attachment ot antigen. The results suggested that the cross-reactive antigenic determinants on the HA subunit may be located closer to the distal end of the molecule than the strain-specific determinants. Further tests employing single-radial-diffusion showed that there are more cross-reactive than strain-specific sites available for antibody in the intact virus particle. The strain-specific antibodies also gave higher haemagglutination-inhibition titres per microgram IgG than the cross-reactive antibodies.

Antibodies, Viral↗
PubMed1979

Surveillance of the antibody status to current influenza strains in the Norwegian population. 1: Method of serum sampling and evaluation of the A/USSR/77 (H1N1) impact in various age groups in 1978.

Every autumn approximately 900 randomly selected sera from various regional bacteriological laboratories are received at the NIPH and assayed for the presence of haemagglutination-inhibition antibodies to current influenza strains. After three years of preliminary investigations the system is now working successfully. It is possible, in retrospect, to assess the impact of various influenza strains in different age groups and regions of the country. In particular, after the reappearance of the H1N1 subtype (the "Russian influenza") in Europe in January 1978, the sera collected in Norway during a 3-week period in October 1978 clearly showed that those under the age of 25 years, and especially teenagers between 13 and 16 years of age, were struck by this virus. This was unexpected since there was hardly any influenza morbidity associated with this "new" strain recorded in the winter of 1978.

Adolescent↗
PubMed1978

Single-radial-complement-fixation: a new immunodiffusion technique. Assay of the antibody response to the type-specific antigens of influenza virus in post-infection human sera.

A new immunodiffusion technique in agarose gel for the quantification of complement-fixing antibodies is described. The test involves the incorporation of antigen and complement in a primary agarose gel. Heat-inactivated serum samples are allowed to diffuse radially from wells overnight at 4 degrees C. A secondary gel, containing antibody-coated sheep erythrocytes, is layered on top of the first gel and the system is incubated for 45 min at 37 degrees C. Where complement is fixed, i.e., around wells with positive serum samples, zones of unlysed cells appear. There is a straight line relationship between zone areas so produced and log(2) serum titres obtained with the conventional complement fixation test. The method appears to be applicable to a variety of antigens. It has been found suitable for bacterial and viral antigens. The test can also be reversed, thus allowing the quantification of diffusible antigens in a gel containing immune serum and complement. This paper describes in detail the use of this method as a diagnostic tool for the assay of complement-fixing antibodies to the type-specific antigens of influenza virus in paired human sera.

Antigen-Antibody Reactions↗
PubMed1976

Reactivity of rabbit IgG-fragments in single-radial-immunodiffusion.

The previously described method of assaying the antibody response to influenza virus by single-radial-immunodiffusion is based on the formation of opalescent zones when immune sera are allowed to diffuse into gels containing large amount of purified influenza virus. The zones are not thought to be the result of immune aggregates of virus particles and antibody since the virus is immobile in the gel and the average distance between two neighbouring particles is approximately 50 times the virus diameter. The present study investigates how different rabbit IgG-fragments react in this type of test. It was found that IgG, F(ab')2 and reduced (monovalent) IgG gave rise to distinct zones of opalescence. Fab', on the other hand, produced weak and hazy zones which could not be quantitated with any accuracy. We conclude that the most likely mechanism responsible for the zones is a light-scattering effect caused by antibodies attached to the viral surface and that the quality of the opalescence to some extent seems to be dependent on the Fc-fragment.

Animals↗
PubMed1976

Antigenic variants of influenza A virus obtained in vitro.

The aim of this study was to investigate antigenic "drift" in the haemagglutinin and neuraminidase antigens of influenza A virus in vitro under immunological pressure. Variants of the "Asian" influenza strains A/England/12/64 (H2N2) and A/Tokyo/3/67 (H2N2) were isolated in the allantois-on-shell system in the presence of homologous postinfection ferret sera. For each of these two viruses three generations of variants were isolated and characterized. It was found that the successive antigenic variants of A/Eng/12/64 did not resemble A/Tokyo/3/67. Thus it is probable that the pathway of antigenic drift in vitro was not the same as that which occurred in nature during the evolution of A/Tokyo/3/67 from A/Eng/12/64. In addition, A/Tokyo/3/67, which was the last strain to be prevalent before the A/Hong Kong subtype appeared, underwent significant antigenic drift from "junior" to "senior" variants. This finding did not support the concept that, when antigenic drift occurs, resulting in the appearence of viruses with new haemagglutinin antigen subtypes, the previously prevalent strain has no capacity for further antigenic drift. The study did not result in the production of strains that were identifiable as "bridging" viruses between the H2 and H3 haemagglutinin subtypes. The present paper includes the first report of antigenic variation in the neuraminidase antigens of influenza A viruses occurring in vitro under immunological pressure.

Antigens, Viral↗
PubMedDate not supplied

Rapid quality evaluation of hybridomas using ELISPOT and cell-ELISA techniques.

We have used the ELISPOT assay in combination with ELISA procedures for rapid evaluation of properties of three different murine hybridoma cell lines, 104-I, -B and -G, secreting IgG1 kappa monoclonal antibodies against a 104-mer synthetic peptide from the C-terminal part of HIV-1 p24. By conventional ELISA we obtained data suggesting that the three monoclonal antibodies had different affinities. By cell-ELISA we found that the IgG1 kappa secretion rate varied between cells (4,000 to 14,000 antibody molecules/cell/min), and ELISPOT showed that only 4-5% of 104-I cells gave antigen-specific spots, indicating a cell population with diverse properties. We recommend that the ELISPOT and cell-ELISA techniques should be used routinely to supplement conventional ELISA procedures for rapid evaluation of hybridoma properties.

Animals↗