Biomedical subjects

G A Wemhoff

Publications and source records attributed to G A Wemhoff.

6 recordsLinked to original sources

PubMed1992

Kinetics of antibody binding at solid-liquid interfaces in flow.

We have developed the theoretical framework for a displacement immunoassay conducted in flow under nonequilibrium conditions. Using a repetitive displacement technique, we determined the displacement rate and apparent dissociation rate constant at different flow rates. Our data suggest that the kinetics are best described by a first-order function. The displacement efficiency, the displacement rate, and therefore the apparent dissociation rate constant were calculated and demonstrated to be flow rate dependent. The theoretical framework developed in this study was successful in predicting the behavior of antigen displacement in flow.

Antibodies, Monoclonal↗

PubMed1991

Novel trifunctional carrier molecule for the fluorescent labeling of haptens.

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

A continuous flow immunoassay for rapid and sensitive detection of small molecules.

An immunosensor operating in continuous flow and capable of detecting low molecular weight antigens is described. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of additional reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within 3 min in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. Signal detection occurs downstream of the antigen recognition event. In this study, the hapten 2,4-dinitrophenol (DNP) as DNP-lysine was used as model antigen. To generate a labeled antigen, DNP was coupled to the terminal amino group of insulin A chain (tetra S-sulfonate form) which provides two tyrosine residues for the introduction of an 125I-label (DNP-Ins-125I) or three carboxyl groups for the attachment of three fluorescein residues (DNP-Ins-Fl). The radiolabeled antigen was used to establish assay conditions. Subsequently, fluorescein was substituted for the radioisotope label in order to develop an assay independent of the restrictions associated with isotopes. Using this flow immunoassay, we were able to detect DNP-lysine down to a detection limit of 143 nM (29 pmol/200 microliters) using DNP-Ins-125I or DNP-Ins-Fl as labeled antigen. The density of immobilized antibody and the flow rate were identified to be critical parameters for the sensitivity of the assay.

Animals↗

PubMed1988

Cellular pathways for rejection of class-I-MHC--disparate skin and tumor allografts.

We have investigated the relative roles of the Lyt-2+ and L3T4+ T lymphocyte subsets in rejection of class-I-MHC-antigen-disparate skin and tumor allografts. To deplete T cells in vivo, rat anti-Lyt-2 or anti-L3T4 monoclonal antibodies (mAb) were administered to adult-thymectomized (ATX) recipient mice prior to transplantation. BALB/c (H-2d) recipient mice rejected the Ia- Sarcoma I (Sa1) (H-2a) tissue culture-derived tumor after depletion of the L3T4+ T cell subset in vivo. In contrast, depletion of the Lyt-2+ T cell subset permitted lethal tumor growth in all recipient mice. To determine the role of particular T cell subsets in rejection of Ld class-I-MHC-antigen-disparate allografts, BALB/c skin was transplanted to BALB/c-H-2dm2 recipient mice. Skin grafts were rejected by control mice with a mean survival time (MST) of 14.5 days. The MST of skin grafts for mice treated with anti-L3T4 mAb was 16.6 days. In contrast, administration of anti-Lyt-2 mAb alone (MST = greater than 47 days) or together with anti-L3T4 mAb (MST = greater than 50 days) caused prolonged or indefinite graft survival in all recipient mice. Depletion of specific T cell subsets was confirmed by flow cytometric analysis and by analysis of T cell function in vitro. These results suggest that Lyt-2+ T lymphocytes are essential for rejection of class-I-MHC-disparate allografts; indirect presentation of alloantigen to L3T4+ T cells may not be necessary for rejection.

Animals↗

PubMed1988

B-cell transplants to immunodeficient xid neonates do not imprint their helper T cells.

A great variety of Igh-restricted immunoregulatory phenomena have been described in responses to both haptens and carriers. The investigation of Igh-dependent T-cell functions is of considerable interest because it relates to issues such as the interdependence of the T- and B-cell networks, isotope- and idiotype-specific help, the role of Igh-linked products in the generation of the helper and cytolytic T-cell repertoires and Igh-linked effects on cell interactions among helper and suppressor cells. Helper T cells have been shown to be critically susceptible to B cell-mediated education and it is generally assumed that T cells become imprinted by B-cell idiotypes during development. It occurred to us that xid immunodeficient neonates, which lack T15+ B cells and T15 antibodies, might provide a suitable host to investigate the B-cell dependent imprinting of T cells providing help in T15+ anti-PC responses. We designed our experiments to test whether or not T15 deficient xid mice could be used as recipients of normal B cells capable of imprinting the host's helper cells. The results of our experiments demonstrate that unirradiated xid neonates are readily engrafted with normal B cells from both neonatal and adult donors. The transplanted B cells reconstitute anti-PC and anti TNP-FICOLL PFC responses in the xid hosts. However, the early engraftment of T15+ B cells in xid mice did not cause a detectable imprinting of their helper T cells, i.e., we could not detect any biases in the preference of carrier-/primed cells from reconstituted mice for primary or secondary T15+ or T15- B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Alterations of idiotypic profiles: the cellular basis of T15 dominance in BALB/c mice.

Phosphorylcholine (PC) is a component of cell walls and membranes from a variety of widely distributed microorganisms. It is highly immunogenic in mice and most murine strains have circulating anti-PC antibodies which are known to confer protection against certain bacterial infections. BALB/c mice offer a striking example of a high responsiveness to PC, a propensity to generate PC-binding myelomas, and a great restriction of idiotype expression in anti-PC antibodies; in fact, most BALB/c anti-PC IgM antibodies express the T15 idiotype marker. Although it has been suspected that T15 dominance is somewhat related to the continuous antigenic load presented by microorganismal flora found in conventional mice, a complete experimental account of how antigenic selection brings about such extreme idiotypic dominance is not yet available. In the studies presented below, we investigated the role played by the host environment, T cells, and antigen in affecting the generation of the anti-PC T15 idiotype profile in lethally irradiated adoptive hosts reconstituted with syngeneic neonatal liver cells. The results presented herein indicate that the transfer of mature carrier-primed T cells with neonatal liver cells does not influence the generation of the T15 idiotype profile. We also demonstrated that anti-T15 idiotype suppressed mice, used as lethally irradiated hosts of immature immunocompetent cells, allow an increased rate of reconstitution of the anti-PC response when compared to nonsuppressed hosts. Since the administration of a T15+ anti-PC antibody inhibits both reconstitution and idiotype expansion, we conclude that T15+ B cells do not self-promote themselves. In contrast, we observed that exposure of adoptive hosts to PC antigens can enhance the anti-PC response and alter the idiotypic profile in favor of T15-bearing clones. The failure of T15 idiotype and the success of PC antigens in promoting T15 dominance led us to search for an "antigen inside" which might play a role in the ontogeny of T15 expression. To this end, we produced isologous anti-T15 hybridomas and selected one BH8 binding IgM hybridoma, 15B1, as a potential carrier of an internal image of PC. In vivo tests involving the administration of this antibody to irradiated adoptive hosts immediately prior to the transfer of neonatal liver cells indicate that T15 expression in adoptive transfer can be enhanced by this antibody. Finally, we discuss a model to account for the generation of T15 dominance in normal adult BALB/c mice and its loss in adult BALB/c mice used as adoptive hosts for syngeneic immature immunocompetent cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗