Concepts in antibody phage display.

This paper introduces the reader to antibody phage display and its use in combinatorial biochemistry. The focus is on overviewing phage display formats, library design and selection technology, which are the prerequisites for the successful isolation of specific antibody fragments against a diverse set of target antigens.     

Monoclonal antibody engineering in plants.

Techniques for plant transformation have been developed to such an extent that a number of foreign genes are currently being introduced into transgenic plants. Tobacco plants that produce monoclonal antibodies are of interest, because in addition to synthesis of two gene products (i.e. the heavy and light chains), the two polypeptides need to be assembled correctly, in order to result in a functional antibody. The studies on a catalytic antibody suggest that this is the case, and that the antibody functions identically to the native murine-derived antibody. The only difference observed was in the glycosylation of the heavy chain. Further transgenic plants are being generated to produce monoclonal antibodies that may be used therapeutically (and are therefore required in large quantities), or to provide disease resistance in plants. In addition, the ability of plants to assemble antibody complexes is being investigated further, to study the possibility of generating secretory IgA, which consists of heavy and light chains as well as two additional polypeptide units.     

Studies on the control of antibody synthesis. XIV. Role of T cells in regulating antibody affinity.

The effect of limiting the number of helper T cells on the affinity of the primary antibody response to a T-dependent antigen (DNP-BGG) was evaluated in a cell transfer system. Lethally irradiated, thymectomized mice were reconstituted with either bone marrow or anti-brain θ antiserum plus complement-treated spleen as the source of B cells. In addition, they received various numbers of thymus cells as a source of helper T cells. The animals were immunized with DNP-BGG 1 day after cell transfer and their splenic anti-DNP PFC response was assayed for magnitude and affinity 3 weeks later. A marked restriction in helper T-cell activity resulted in a primary response which was of low magnitude, which lacked indirect PFC, and which had a very low affinity and restricted heterogeneity. When sufficient thymus cells were given to permit a switch to indirect plaque formation, a highly heterogeneous, high-affinity primary response was elicited. Further increase in the number of thymic cells resulted in a progressive increase in the magnitude of the primary response but had no effect on affinity. Thus, a reduction of 50% in the magnitude of the response as a consequence of limiting the number of T-helper cells had no effect on the affinity of the PFC. The results are consistent with the interpretation that the effect of restriction in T-cell help on antibody affinity is not due to a direct effect on precursors of high-affinity PFC but is secondary to inefficient selection for high-affinity cells when the degree of cell proliferation is markedly reduced.     

Immunodiffusion test in avian encephalomyelitis. II. Detection of precipitating antibody in infected chickens in comparison with neutralizing antibody.

The sensitivities of double-immunodiffusion (DID) and neutralization tests to detect avian encephalomyelitis (AE) antibody in chickens were studied. Two antigens were employed in the tests. Concentrated antigen gave a higher titer of antiserum than crude antigen, which reacted only to serum having a neutralization log-index (NI) of 3.4approximately4.0 or more. Antibody responses were examined in four growing chick groups inoculated with AE virus by the intracerebral, subcutaneous and oral routes by the DID test with concentrated antigen and by the neutralization test for 1 or over 2 years after inoculation. When concentrated antigen was used, most sera having an NI of over 1.0 were positive for precipitating antibody. Therefore, the sensitivity of the DID test was nearly equal to that of the neutralization test. The DID test was considered to be applicable to the diagnosis of AE and an antibody survey in the field.     

The antibody response in Lyme disease.

We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis.     

Reaginic antibody formation in the mouse. X. Possible role of suppressor T cells in transient IgE antibody responses.

The kinetics of IgE antibody response to alum-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) was dependent on the dose of immunogen. A persistent IgE antibody response was obtained when high responder BDF1 mice were immunized with a minimum (0.05 microgram) dose. An increase of the immunogen to 10 microgram depressed IgE antibody responses but enhanced IgG antibody responses of both hapten and carrier specificities. Determination of T helper cell activity and B memory cells after immunization with different doses of antigen indicated that minimum immunogen was favorable for developing helper activity, whereas 1 to 10 microgram immunogen were more favorable than a 0.05-microgram dose for developing both IgE and IgG B memory cells. Nevertheless, neither helper T cells nor B memory cells in the spleen explains a transient IgE antibody response to a high (10 microgram) dose of DNP-OA. Evidence was obtained that immunization with 10 microgram OA induced generation of antigen-specific suppressor T cells, which were not detectable after immunization with 0.05 microgram OA. Transfer of suppressor T cells to DNP-OA-primed mice depressed both anti-hapten and anti-carrier IgE antibody responses. The results suggested strongly that suppressor T cells are involved in a transient IgE antibody response to a high-dose immunogen.     

Opsin shift in an aldolase antibody.

An antibody-retinal assembly that mimics the opsin shift (OS) of the naturally occurring visual pigments is reported. Both experiments and calculations show that the aldolase antibody 33F12 covalently binds all-trans retinal via a protonated Schiff base with a lysine residue. This chromophore, which exhibits a remarkable opsin red shift (140 nm), represents a useful model system for studying the factors that contribute to the OS.