Regulations of IgM immune response. IV. Effect of the hapten: carrier ratio and the nature of the carrier on the intrinsic and functional affinity of carp DNP-antibodies]

Carp anti-DNP antibodies were raised by various DNP-carrier conjugates. Their intrinsic affinity (K0) to monovalent E-DNP-L-lysine and functional affinity (DF) for binding to the multivalent DNP-T4 bacteriophages were determined. The functional affinity of antibodies elicited by T-cell-dependent DMPn-HSA (n = 3, 15, 33) is relatively high (KF):1010-1012 M-1). These KF values increase more than K0 during the immune response. The functional affinity is dependent on the molar KNP: HSA ratio. The mediate coupled DMP15-HSA elicits antibodies with the highest functional affinity. Carps immunized with T-cell-independent DNP-conjugates synthesize antibodies which have similar K0 as the antibodies elicited with DNP-HSA. However, the KF-values are in the range from 107 to 1010 M-1 only. The KF of antibodies raised with DNP-BA are 103-104 fold, those of DNP-S III and DNP-Ficoll elicited are only 4 . 101 to 4,7 . 102 fold higher than their corresponding K0-values. This means that these antibodies are not very effective in binding the multivalent DNP-T4. Specifically purified antibodies have also such low functional affinities. These strong differences in the functional affinity of carp DNP-antibodies elicited by T-cell dependent and independent DNP-conjugates are discussed with regard to stimulation of different B-cell subpopulations.     

Antigen modulation of antibody forming cells: the relationship between direct plaque size, antibody secretion rate and antibody affinity.

Using the mathematical theory of direct plaque growth, we have analyzed the expected variation of plaque size with IgM affinity and secretion rate. We use the theory to comment on recent effector cell blockage experiments and show how the theory can be used to determine the change in the secretion rate of a single antibody-forming cell subjected to blockage by a multivalent antigen. We also argue, using the mathematical theory, that under the usual experimental conditions employed in the plaque assay, cells that produce low affinity IgM antibodies will give rise to smaller plaques than cells that produce high affinity IgM antibodies.     

Interaction of monoclonal anti-peptide antibodies with lysozyme

The interaction of monoclonal anti-peptide antibodies with the free peptide and its protein counterpart has been evaluated for hen egg white lysozyme and the peptide constituting residues 38 to 45. Fluorescence methodology has been developed for the measurement of association constants based on resonance energy transfer between the excited tryptophan of antibody and bound peptide ligand conjugated to a fluorescent probe. Five antibodies, four IgM and one IgG, have been assayed by ELISA, and have demonstrated binding to the adsorbed peptide alone, to the adsorbed lysozyme alone, or to both. Multivalent interaction with the adsorbed ligand is a key factor in the efficacy of binding. Measurement of binding constants in homogeneous solution, by equilibrium dialysis and energy transfer, demonstrated that lysozyme was bound to an IgG antipeptide antibody with an association constant (4 X 10(2) M-1) 200-fold less than that for the free peptide (8 X 10(4) M-1). It was also inferred for IgM that an association constant of the order of 10(2) M-1 was sufficient to effect selective interaction in a system providing multivalent interaction. The shared conformations between protein and peptide, implied by the specific reactivity of the anti-peptide antibody with the protein, points to structural fluctuations of the surface regions and residues of globular proteins.     

Ontogeny of the liver nuclear T3-receptor during the last days of incubation and posthatch in the chick embryo.

The characteristics of the nuclear T3 receptor in the liver of the chick embryo were studied from incubation day 18 until day 1 posthatching. Treatment of the nuclei with 3 mol.l-1 MgCl2, which removed the endogenously bound hormone, was used in order to determine the total amount of receptors. The affinity constant Ka decreased between incubation day 18 (0.996 +/- 0.276.10(9) M-1) and day 19 (0.247 +/- 0.072.10(9) M-1), remained the same thereafter until hatching and increased again on day 1 posthatching (1.846 +/- 0.928.10(9) M-1). The total amount of receptors tended to increase from incubation day 18 to day 20 non-pipping (np) (from 4.40 to 11.55 fmol/micrograms DNA) and decreased thereafter to 2.38 fmol/micrograms DNA on day 1 posthatching. The amount of free binding sites reached a maximum on day 19 (6.91 fmol/micrograms DNA) and then decreased drastically until posthatching (0.19 fmol/micrograms DNA). The maximal specific binding was found on day 20 (np), just prior to penetration of the air chamber. During the time at which the level of T3 remains high in the plasma, a reduction in the amount of receptor was observed, which may be the consequence of a down-regulation by T3 itself.     

Restricted maturation of antibody-binding characteristics for hapten-specific IgM-plaque-forming cells in mice

Inhibition of plaque formation was used as a method for studying changes in binding characteristics of IgM antibody specific for the hapten, 2,4,6-trinitrophenyl (TNP). By incorporating successive concentrations of TNP ligand into specific hemolytic plaque assays, we were able to obtain inhibition curves and to determine relative ligand binding of anti-TNP antibodies secreted by plaque-forming cell (PFC) populations. The concentration of free hapten required to obtain 50% reduction in IgM PFC decreased with time after immunization, indicating that an increase in average binding affinity of their secreted antibodes occurred. The slopes of the inhibition curves also increased with time after immunization. Maturation with regard to the above two properties was virtually complete for IgM PFC 5–7 days after immunization. The manner by which the slopes of the inhibition curves changed suggested that maturation occurred as the result of a simple selective process, and it appeared to involve primarily precursor cells existing prior to administration of antigen.     

Highly effective poly(ethylene glycol) architectures for specific inhibition of immune receptor activation

Architectural features of synthetic ligands were systematically varied to optimize inhibition of mast cell degranulation initiated by multivalent crossing of IgE-receptor complexes. A series of ligands were generated by end-capping poly(ethylene glycol) (PEG) polymers and amine-based dendrimers with the hapten 2,4-dinitrophenyl (DNP). These were used to explore the influence of polymeric backbone length, valency, and hapten presentation on binding to anti-DNP IgE and inhibition of stimulated activation of RBL cells. Monovalent MPEG(5000)-DNP (IC(50) = 50 nM), bivalent DNP-PEG(3350)-DNP (IC(50) = 8 nM), bismonovalent MPEG(5000)-DNP(2) (IC(50) = 20 nM), bisbivalent DNP(2)-PEG(3350)-DNP(2) (IC(50) = 3nM) and DNP(4)-dendrimer ligands (IC(50) = 50 nM) all effectively inhibit cellular activation caused by multivalent antigen, DNP-bovine serum albumin. For different DNP ligands, we provide evidence for more effective inhibition due to (i) preferential formation of intra-IgE cross-links by bivalent ligands of sufficient length, (ii) self-association of monovalent ligands with longer tails, and (iii) higher probability of binding for bisvalent ligands. We also show that larger DNP(16)-dendrimers of higher valency trigger degranulation by cross-linking IgE-receptor complexes, whereas smaller DNP-dendrimers are inhibitory. Thus, features of synthetic ligands can be manipulated to control receptor occupation, aggregation, and inhibition of the cellular response.     

Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase.     

The kinetics of hemolytic plaque formation. II. Inhibition of plaques by hapten.

We present a mathematical theory of hapten inhibition of hemolytic plaque formation. The treatment is based upon the mathematical model for plaque growth presented by DeLisi &; Bell (1974). The lymphocyte under consideration is embedded in an infinite three-dimensional medium, and is secreting antibodies isotropically at a constant rate. As the antibodies diffuse from the source they can bind reversibly to hapten, and in the most general case reversibly to red blood cell (RBC) epitope. The model leads to a non-linear diffusion equation coupled to a set of first order differential equations. The system must, in general, be solved numerically. However, in many cases of experimental interest simplifications arise which permit closed form solutions to be obtained. In this paper we have developed solutions for three special cases.In the first example antibodies can bind only univalently to RBCs, as would be expected if the epitopes are sparsely distributed. In this case reaction between antibody site and RBC epitope is rapid ( ⪆ 1 sec) and reversible and local equilibrium is assumed. This leads to a “pure” diffusion equation in the free antibody concentration, but with a reduced diffusion coefficient.In another example univalent attachment of an antibody site to a RBC epitope is followed by a rapid irreversible intramolecular reaction. This might be expected for example if the epitope density is large. An exact solution to the resulting diffusion equation was also found in this case. In order to assess an intermediate situation, we also solved the equations for a model in which intramolecular reaction is slow and irreversible.The theory predicts that the type of information one can obtain from inhibition experiments depends critically upon the preparation of the RBC. If the cell is sufficiently haptenated so that rapid irreversible multivalent attachment is favorable, a differential plot of the inhibition curve will reflect the affinity distribution of antibody sites for free hapten. If only univalent attachment with RBCs is possible, so that antibody sites bind to RBC hapten in the same way they bind to free hapten, then a differential plot of the inhibition curve will reflect the secretion rate distribution.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.     

Restriction in IgM expression. III. Affinity analysis of monoclonal anti-lactose antibodies

A clonal analysis has been made of the murine BALB/C response to lactose-containing immunogens with respect to the affinity restriction in IgM expression. Monoclonal IgM and IgG antibodies were prepared from antilactosyl hybridomas generated from mice immunized with p-aminophenyl-beta-lactoside (PAPL) coupled to BGG or with a vaccine of Streptococcus faecalis (Strain N). Association constants for the binding of monovalent derivatives of PAPL were measured by quenching fluorescence. These derivatives carried a probe (2,4-dinitrophenyl or 1-dimethylaminonaphthalene-5-sulfonyl) that served to quench the protein fluorescence when the ligand was complexed with the protein. The central finding was that with both immunogens a restriction in the affinity of IgM was demonstrated since the highest values exhibited by IgG antibody exceeded by at least a factor of 50 the highest comparable values of the association constants for IgM antibody. It is suggested that the hypothesis of germ-line restriction, previously proposed as the basis for the affinity restriction of IgM, may also be applicable to the T cell receptor since its distinctive properties parallel those of IgM antibody.     

Test of a theory relating to the cross-linking of IgE antibody on the surface of human basophils

Recent mathematical models of bivalent hapten-induced histamine release from basophils predict that under appropriate conditions histamine release is maximum when cross-link formation is maximum, at a hapten concentration equal to 1/(2Ka), where Ka is the average affinity constant of the hapten for a single IgE binding site. To test this prediction we sensitized human basophils with a monoclonal anti-dinitrophenol IgE and generated histamine release dose-response curves with a bivalent hapten, alpha, epsilon-DNP-lysine. The monoclonal IgE has a published affinity constant of 7.1 X 10(7) M-1 for epsilon-DNP-lysine as determined by equilibrium dialysis. From the position of the maximum of the histamine dose-response curves, both in the presence and in the absence of monovalent DNP hapten, we determine that the sensitizing IgE has an intrinsic affinity constant of 6.9 +/- 0.5 X 10(7) M-1 for epsilon-DNP-lysine and 1.2 +/- 0.6 X 10(6) M-1 for alpha-DNP-lysine. The agreement between the two estimates of the epsilon-DNP-lysine affinity constant, one from histamine release experiments involving surface bound IgE and one from binding experiments involving IgE free in solution, 1) is consistent with a central prediction of the theory of cross-linking and 2) indicates that the hapten-binding properties of the IgE are unaffected by its being bound to Fc epsilon receptors on the basophil surface.     

Steroid-protein interactions. Stopped flow fluorescence studies of the interaction between steroid hormones and progesterone-binding globulin.

Stopped flow fluorometry, measuring changes in the intrinsic fluorescence of progesterone-binding globulin (PBG), was used to determine the association and dissociation rates of the interaction of PBG with seven delta4-3-ketosteroids. The rates of formation and dissociation of the PBG-progesterone complex were measured as a function of concentration and temperature. At 20 degrees, kon = 8.7 X 10(7) M-1 S-1 and koff = 0.060 S-1. The association rate constants for progesterone, deoxycorticosterone, testosterone, testosterone acetate, and medrogestone were found to be the same within experimental error. The different affinities of PBG for these steroids result from the dissociation rate constants of the steroids which ranged from 0.43 S-1 for testosterone to 0.024 S-1 for medrogestone. Two corticosteroids, corticosterone and cortisol, were both bound somewhat more slowly (approximately 5 X 10(7) M-1 S-1). Reflecting their very low affinity for PBG both steroids dissociate very rapidly: corticosterone at 1.4 S-1 and cortisol at 90 S-1. The ratio of association to dissociation rate constants gave affinity constants in agreement with independently determined constants.     

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity.

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.     

Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

The aggregation of IgE anchored to high-affinity Fc epsilon receptors on rat basophilic leukemia (RBL) cells by multivalent antigens initiates transmembrane signaling and ultimately cellular degranulation. Previous studies have shown that the rate of dissociation of bivalent and multivalent DNP ligands from RBL cells sensitized with anti-DNP IgE decreases with increasing ligand incubation times. One mechanism proposed for this effect is that when IgE molecules are aggregated, a conformational change occurs that results in an increase in the intrinsic affinity of IgE for antigen. This possibility was tested by measuring the equilibrium constant for the binding of monovalent DNP-lysine to anti-DNP IgE under two conditions, where the cell-bound IgE is dispersed and where it has been aggregated into visible patches on the cell surface using anti-IgE and a secondary antibody. No difference in the equilibrium constant in these two cases was observed. We also measured the rate of dissociation of a monovalent ligand from cell surface IgE under these two conditions. Whereas the affinity for monovalent ligand is not altered by IgE aggregation, we observe that the rate of ligand dissociation from IgE in clusters is slower than the rate of ligand dissociation from unaggregated IgE. These results are discussed in terms of recent theoretical developments concerning effects of receptor density on ligand binding to cell surfaces.     

Structural requirements for in vivo and in vitro immunogenicity in hapten-specific delayed hypersensitivity

Affinity changes of hapten-specific IgM antibodies were analysed by plaque inhibition assays and by inhibition with varying quantities of monovalent inhibitor. The resulting data were used for cell population studies, based on the affinity of plaque-forming antibody. Maturation of IgM plaque-forming cell populations was time- and antigen dose-dependent. Maturation was characterized by a consistent increase in the proportions of high affinity and medium affinity cells and a decrease in the proportion of low affinity cell populations. Thus progressive selection of high affinity cells occurred over the entire dose range of immunization. The hapten concentration at which 20% of cells was inhibited decreased with time at all administered antigen concentrations. The inhibitory hapten concentration at which 50 or 70% of cells was inhibited did not change after administration of very large immunizing doses. Thus recruitment of this type of plaque-forming cell depended on dose. In the secondary response, high affinity cells appeared initially but very transiently, and the subsequent rate of population changes was faster than that observed in the primary response. The short productive life of IgM plaque-forming populations may be responsible for the time restriction in IgM as compared to IgG maturation.     

Recovery of B lymphocyte responsiveness after complete radioactive antigen suicide, and the affinity of antibody made after incomplete suicide.

The adoptive secondary anti-DNP antibody response of primed spleen cells was specifically and lastingly inhibited by exposure in vitro to highly radioactive DNP conjugates. When the cells were treated with multivalent DNP-TGL¹²⁵I, their responsiveness was almost completely inhibited, and took about 2 months to recover. The recovery was attributable to the transferred cells, since the contribution of the recipients was estimated to be small. Similarities to the recovery after tolerance are discussed.Incomplete suicide, after exposure to radioactive multivalent or monovalent DNP conjugates, affected the high- and low-affinity secondary antibody responses impartially. In a primary system, however, there was some indication that the antibody was of lower affinity after partial suicide than in control groups. The results are discussed in terms of the very high avidity of multipoint binding, and of the lower density of receptors on nonimmune B cells already postulated by Klinman.     

Hydrophobic agaroses: basis for a model of multivalent effector-receptor interactions.

An increase in the density of butyl residues bound to Sepharose 4B leads to an enhancement of the affinity of these gels for phosphorylase b in the presence of 1.1M ammonium sulfate. A Hill coefficient of 2.9 indicates that a minimum of ca. 3 binding sites is involved in the positive cooperative adsorption of this enzyme. Binding studies of phosphorylase b on butyl-Sepharose of a specific degree of substitution demonstrate that the affinity of the gel for this ligand decreases as a function of fractional saturation. A Hill coefficient of 0.44 indicates negative cooperativity as a result of multivalent binding. From these observations a multivalent, mobile receptor model is derived which can explain such characteristics of effector-receptor interactions as: positive and negative cooperativity, high binding constants and low dissociation rate constants. The application of this model to experiments taken from the literature on the binding of the multivalent effectors concanavalin A and cholera toxin to fat cells shows that the postulated mode of interactions is probably realized in nature.     

Properties of passive binding of calcium to endoplasmic reticulum from adipocytes.

Calcium binding to isolated adipocyte microsomes enriched in endoplasmic reticulum has been characterized. Binding was concentration-dependent, saturable, and totally dissociable. Steady state was reached within 20 min at all calcium concentrations tested. Three apparent classes of binding sites were identified in kinetic and steady state studies using calcium concentrations from 1 muM to 10 mM. The affinity constants (and maximum binding capacities) as determined by computer analysis for the three classes were 2.1 X 10(5) M-1 (0.28 nmol of calcium/mg of protein), 1.3 X 10(4) M-1 (1.1 nmol/mg), and 1.3 X 10(2) M-1 (35 nmol/mg). The dissociation rate constants for the high and intermediate affinity classes of sites were 1.6 X 10(-3) S-1, respectively, and the association rate constant for the high affinity sites was 8 X 10(2) M-1 S-1. The affinity constant calculated from the rate constants was 5.0 X 10(5) M-1 for the high affinity sites in agreement with the value obtained in studies at steady state. The three classes of binding sites were specific for calcium. Magnesium was a noncompetitive inhibitor of calcium binding to all three classes of sites with a Ki of 9 to 12 mM. Calcium binding at 1 muM calcium was 50% inhibited by 18 muM La3+, 600 muM Sr2+, or 2.7 mM Ba2+. These data represent the first analysis of passive calcium binding to endoplasmic reticulum from nonmuscular cells and the first report of corresponding rate constants for either endoplasmic or sarcoplasmic reticulum. The characteristics of the binding are consistent with the properties of calcium transport by endoplasmic reticulum of adipocytes. The characteristics and specificity of the calcium binding constitute further evidence that endoplasmic reticulum plays an important role in cellular calcium homeostasis.     

Thymus-dependent network responses to a monoclonal cross-reactive antiidiotypic antibody.

BALB/c mice were inoculated i.p. with a cross-reactive anti-Idiotypic mAb (designated FD5-1) in the absence of Ag or adjuvants. Injection with unmodified FD5-1 resulted in the induction of serum antibodies reactive with both FD5-1 (Ab3) and the hapten DNP (Ab1'). Endpoint titers of the Ab3 response showed an increase in serum IgM, which was dose-responsive to both the number of injections and the amount of FD5-1 antibody injected. The serum IgM Ab3 response was found to be thymus dependent and idiotypically specific for FD5-1. Athymic mice injected with FD5-1 were unable to produce a serum IgM Ab3 response, whereas their euthymic littermates produced strong Ab3 responses. Serum Ab3 responses and Ab1' were detectable only in the IgM isotype; no specific IgG responses were observed. Indeed, IgG recognized by FD5-1 appeared to be suppressed by FD5-1. Injection of mice with FD5-1 modulated serum IgM responses to DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one (OX), phosphorylcholine (PC), and alpha 1,3-dextran (DEX) in a thymus-dependent manner. FD5-1 injection induced IgM responses against DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one, and DEX but decreased IgM binding to PC. No detectable Ab1' responses to any of the aforementioned molecules were found when the same sera were probed for IgG. The specificity of serum Ab1' from FD5-1-injected mice was evaluated by antigenic inhibition. Binding of serum Ab1' to DNP-BSA was inhibitable by DNP-lysine, whereas equivalent concentrations of lysine alone had no inhibitory effect. The antigenic specificity of IgM from normal serum binding to PC-BSA was demonstrated by inhibition with free PC, and the binding of Ab1' from FD5-1-injected mice to DEX-coated plates was shown to be inhibitable by DEX. We have described in vivo network perturbation in adult BALB/c mice injected with anti-Id antibody in the absence of Ag or adjuvants. Our findings show that injection of the cross-reactive anti-Id FD5-1 can induce thymus-dependent Ag-specific responses. In two systems where FD5-1 functions as an anti-anti-anti-Id antibody (PC and DEX), thymus-dependent responses were also observed. FD5-1 injection suppressed antibodies binding to PC, whereas DEX-specific responses were induced.     

Determination of binding constants of antibodies fractionated according to their affinity to insulin

The number of populations of antibodies differing in their affinity for insulin was determined in guinea pig antiserum. The antibody fractions differing in their affinity were obtained by affinity chromatography on an antigenic sorbent. Elution with a stepwise pH gradient from 4 to 2 differing in the number and form of the steps resulted in 10 fractions of antibodies obtained from individual animal sera. The binding constants of fractionated antibodies were determined by a bioluminescent immunocofactor method. With a change in elution pH from 4 to 2, the equilibrious binding constant of antibodies increased from 10(6) to 5 X 10(9) M-1.