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

North, John R.; Askonas, Brigitte A.

doi:10.1002/eji.1830040511

Cited by 15 publications

(4 citation statements)

References 24 publications

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“…Since the avidity measured by this method is a weighted average between the local antibody concentration and the true avidity of the antibody secreted, large differences in rates of antibody secretion will cause apparent avidity differences between two PFC secreting identical antibody molecules. Such differences have been shown experimentally with PFC from the B-cell clone E9 which secretes a homogeneous immunoglobulin specific for DNP when large variation in uninhibited plaque size was observed (~ 10-fold) (32). In our experiments, the variation in uninhibited plaque size was small (~twotbld).…”

Section: Discussionsupporting

confidence: 79%

Isolation of Antigen-Binding Cells From Unprimed Mice

Julius

Herzenberg

1974

The Journal of Experimental Medicine

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The time-dependent increase in the average affinity of serum antibody after immunization (1), is said to be a result of antigen-driven selection of those antibody-forming cell precursors having the highest affinity antigen receptors (2). This interpretation infers that the specificity and affinity of antigen-binding receptors on antibody-forming cell precursors are directly correlated to the serum antibody produced by progeny antibodysecreting cells.It has been found that changes in the affinity of serum antibody with time after immunization reflect changes in the population of antibody-secreting cells. By determining the concentration of antigen required to inhibit individual plaque-forming cells (PFC) assayed at various times after immunization, a time-dependent avidity increase at the antibody-secreting cell level is seen and is comparable to the increase in affinity found in serum antibody (3). Thus, a good correlation between the (intrinsic) affinity of serum antibody and the avidity of the antibody-secreting cell products has been established.In contradistinction, evidence correlating the avidity of antigen receptors on antibodysecreting cell precursors, with the avidity of antibody secreted by their progeny cells, is meager. Indirect evidence has been reported demonstrating that memory cells with high avidity antigen-binding receptors are required to give rise to high avidity antibody-secreting cells (4). However, the putatively high avidity precursors were not isolated and tested directly in these experiments.To directly d e t e r m i n e the relationship between the avidity of secreted a n t i b o d y a n d the avidity of antigen receptors on precursors of a n t i b o d y -s e c r e t i n g cells requires the isolation of precursor cells with different antigen avidities a n d m e a s u r e m e n t of the avidities of a n t i b o d y -s e c r e t i n g cells derived from these * This work supported by NIH grants GM 17367, CA 04681, and AI 08917. ~Abbreviations used in this paper: DNP-MGG, 2,4-dinitrophenyl mouse gamma globulin; ~DNP-MGG, fluorescein-conjugated DNP-MGG; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; F/P, fluorescein per protein; KLH, keyhole limpet hemocyanin; ~KLH, rhodamineconjugated KLH; PBS, phosphate-buffered saline; PFC, plaque-forming cell(s); TNP, 2, 4, 6-trinitrophenyl.

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Section: Discussionsupporting

confidence: 79%

Isolation of Antigen-Binding Cells From Unprimed Mice

Julius

Herzenberg

1974

The Journal of Experimental Medicine

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“…If retained antigen were released during the PFC assay procedure it might produce an effect similar to that observed in experiments on the inhibition of anti-hapten plaques by increasing concentrations of free hapten, which resulted in a gradual diminution in the size of all plaques, instead of the expected stepwise inhibition [30]. A similar effect was seen when free SIII was used to inhibit anti-SIII plaques: as the concentration of SIII increased all the plaques remained visible, but they gradually became smaller and more difficult to identify (G.G.B.Klaus, unpublished data).…”

mentioning

confidence: 72%

The immunological properties of haptens coupled to thymus‐independent carrier molecules. I. The characteristics of the immune response to dinitrophenyllysinesubstituted pneumococcal polysaccharide (SIII) and levan

Klaus

Humphrey

1974

Eur J Immunol

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The immunological properties of haptens coupled to thymus-independent carrier molecules 1. The characteristics of the immune response to dinitrophenyl-lysine-substituted pneumococcal polysaccharide (SIII) and levanThe antibody response t o the hapten 2,4-dinitrophenyl (DNP)-lysine coupled t o two thymus-independent carrier molecules (pneumococcal polysaccharide SIII and levan) has been studied in mice. The characteristics of the anti-hapten response are essentially similar to the response t o the carrier itself: both conjugates elicit a strongly dose-dependent IgM response (with little or no IgG antibody), and supraoptimal doses paralyze both anti-hapten 2nd anti-carrier antibody responses. The anti-hapten response t o DNP-lys-levan is thymus-independent; it is also carrier-independent, since levan-paralyzed mice give a normal anti-hapten response t o the conjugate and, conversely, paralysis of the antihapten reactive lymphocyte population fails t o affect the response t o the carrier. The results d o not support the concept of B cell-B cell cooperation that has been proposed t o explain the immunogenicity of hapten-polysaccharide conjugates. Instead, they indicate that these conjugates immunize separate and noninteracting B cell populations, the one hapten-specific and the other carrier-reactive. Both the numbers and the size of anti-DNP hemolytic plaques were related t o t h e immunizing dose a f DNP-lys-levan. Preliminary experiments suggest that the reduction in plaque size in partially tolerant mice may be caused by persisting antigen on the surface of immunized lymphoid cells.

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“…Such a postulate is tenable in view of the evidence for structural heterogeneity of CRI + molecules mentioned earlier, but our findings are inconclusive inasmuch as the CRI-population in hyperimmune sera could account for the observed avidity differences. Moreover, because objections to the use of hapten inhibition of PFC for avidity estimates have been raised on both theoretical (43) and practical (44) grounds, it would be necessary to directly compare primary and secondary antibodies by equilibrium dialysis before reaching any firm conclusions.…”

Section: Discussionmentioning

confidence: 99%

Idiotype profile of an immune response. I. Contrasts in idiotypic dominance between primary and secondary responses and between IgM and IgG plaque- forming cells

Jd¹,

Lewis²,

Goodman³

1981

The Journal of Experimental Medicine

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Numerous antigens are now known that can induce antibodies bearing similar or identical variable region determinants (idiotypes) in all individuals of one or more strains of inbred mice (1-3). In many cases, the immune response to such antigens is of a highly restricted character, as judged by isoelectricfocusing (IEF) 1 of induced antibodies or other criteria. A curious feature is that the fraction of antibodies bearing a particular cross-reactive idiotype (Id) varies markedly between the known systems. Characteristic values indicating the extent of idiotypic dominance in the various systems can be tabulated (4). For example, ~30-35% of induced antibodies to group A streptococcal carbohydrate in A/J mice bear a common Id, designated A5A (5). In contrast, the response of BALB/c mice to the phosphorylcholine (PC) determinant, presented on T-independent (6) or T-dependent (7) carriers, is almost entirely (>--90%) dominated by antibodies bearing the T15 Id. Many of the factors that determine the extent of dominance of a paricular Id in a given response are obscure, although the clonal heterogeneity of B cells capable of responding to the epitope is likely to be important.The enumeration of characteristic values of clonal dominance implies a certain degree of stability. However, situations are known in which the representation of particular Ids changes considerably during the course of a response or during a multiple immunization regimen. MacDonald and Nisonoff (8) reported that crossreactive idiotypic specificities, present in the sera of individual rabbits early in the course of a hyperimmunization schedule with a p-azobenzoate conjugate, were replaced, at 2-4 mo, by a new set of cross-reactive specificities. A better-defined system, the NP-b Id present on anti-(4-hydroxy-3-nitrophenyl)-acetyl (NP) antibodies of C57BL/6 mice, has been analyzed by M~ikel~i and Karjalainen (9) and Jack et al.

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

Cited by 15 publications

References 24 publications

Isolation of Antigen-Binding Cells From Unprimed Mice

Isolation of Antigen-Binding Cells From Unprimed Mice

The immunological properties of haptens coupled to thymus‐independent carrier molecules. I. The characteristics of the immune response to dinitrophenyllysinesubstituted pneumococcal polysaccharide (SIII) and levan

Idiotype profile of an immune response. I. Contrasts in idiotypic dominance between primary and secondary responses and between IgM and IgG plaque- forming cells

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