A Simple Hemolytic Plaque Technique for the Enumeration of Anti-Hapten Antibody Forming Cells

Silver, Hulbert K. B.; Miller, J. F. A. P.; Warner, N. L.

doi:10.1159/000230437

Cited by 7 publications

(1 citation statement)

References 12 publications

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“…Anti-DNP PFC assays were performed using the method of DNPCG coupled SKBC described by Silver et al [25]. Washed SRBC (10 ml, 2 %) in physiological saline were incubated at 37 "C for 1 h with 40 t o 700 p1 DNP-CG at 4 t o 6 mg protein/ml based o n absorbance measurements at 280 nm.…”

Section: Antibody Determinationsmentioning

confidence: 99%

Inhibition of secondary anti‐hapten responses with the hapten conjugated to type 3 pneumococcal polysaccharide

1972

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injection of 2,4-dinitrophenyl (Dl\iP)-lysine conjugated t o type 3 pneumococcal polysaccharide (DNP-lysine-SIII) markedly reduced the secondary IgG anti-DNP antibody response of irradiated mice injected with primed cells and the homologous dinitrophenylatcd foreign protein used for priming. Serum antibody titers against the foreign protein, hemocyanin, were not affected and the injection of DNP-lysine-SIII could be delayed until the 3rd day, but not the 4th day, after injection of the primed cells and homologous antigen. In mice in which anti-DNP antibody production was reduced by DNP-lysine-SIII, antibody isoelectric spectral analysis of residual antibody suggested that clones of antibody-producing cells were eliminated entirely o r escaped unaltered. Purified SIII is a persisting antigen which readily induces macroglobulin responses in mice and does not seem to stimulate T cells. The results suggest that antigenic determinants o n such antigens gain access to B cells and suppress IgC antibody production.

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Section: Antibody Determinationsmentioning

confidence: 99%

Inhibition of secondary anti‐hapten responses with the hapten conjugated to type 3 pneumococcal polysaccharide

1972

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Analysis of affinity of monoclonal antibody responses by inhibition of plaque‐forming cells

North

Askonas

1974

Eur J Immunol

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It has been suggested that dominant monoclonal responses in mice after transfer of small numbers of spleen cells are the result of strong selection for cell clones producing antibody of high affinity. We have attempted to measure the affinity of anti-DNP (2,4-dinitrophenyl) and anti-NIP (4-hydroxy-5-iodo-3-nitrophenacetyl) antibodies produced in monoclonal responses by inhibition of plaque-forming cells (PFC) with free hapten. PFC from individual monoclonal spleen foci and from spleens after adoptive transfer of 5 x 1 O5 to 2 x 1 O6 primed spleen cells were generally inhibited at lower free antigen concentrations than the PFC found in a heterogeneous secondary response.It was possible to compare several individual cell clones, forming monoclonal antibody to DNP, with respect to the free antigen concentration needed for 50 '% plaque inhibition. These comparisons did not correlate well with the relative affinities of serum antibody estimated by the Stupp-Farr technique.Published work analyzing PFC from plasma cell tumor MOPC 3 15 forming antibody to DNP might have predicted that the curve of PFC inhibition by free hapten for a monoclonal PFC population would be steep. Usually this was not the case, and 20-80 ' 3% PFC inhibition occurred over 100-fold concentration range of free hapten.Some of the anti-DNP clones formed large plaques and when these PFC were inhibited by free antigen, the plaque diameter decreased progressively as the hapten concentration increased. Large plaques consequently require more free antigen for inhibition than small plaques. The poor agreement between apparent affinity measured by plaque inhibition and that measured in serum antibody for monoclonal antibody responses may well be a result of the observed differences in plaque size between individual clones.We therefore have strong reservations about the use of plaque inhibition by free antigen to estimate antibody affinity at the cellular level.

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Coupling of large haptens to proteins and cell surfaces: Preparation of stable, optimally sensitized erhythrocytes for hapten-specific, hemolytic plaque assays

et al. 1973

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A Simple Hemolytic Plaque Technique for the Enumeration of Anti-Hapten Antibody Forming Cells

Cited by 7 publications

References 12 publications

Inhibition of secondary anti‐hapten responses with the hapten conjugated to type 3 pneumococcal polysaccharide

Inhibition of secondary anti‐hapten responses with the hapten conjugated to type 3 pneumococcal polysaccharide

Analysis of affinity of monoclonal antibody responses by inhibition of plaque‐forming cells

Coupling of large haptens to proteins and cell surfaces: Preparation of stable, optimally sensitized erhythrocytes for hapten-specific, hemolytic plaque assays

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