Fractionation of polyclonal antibody by isoelectric focusing and chromatofocusing: separation of high-affinity rabbit clonotype anti-thyroxine antibody

Immunoglobulin G fractions prepared from conventional rabbit anti-thyroxine (T4) antisera were fractionated by agarose gel isoelectric focusing (IEF) in the range of pH 3 to 10, and by chromatofocusing using a Fast Protein Liquid Chromatography (FPLC) system. The clonotype antibodies were recovered from the fractions and subjected to Scatchard plot analysis. The highest affinity constants of the initial antibody (shown in parentheses) and those of the antibodies recovered were IEF, 1.8 X 10(9) to 8.3 X 10(9) M-1 (2.2 X 10(9) M-1); FPLC, 2.4 X 10(9) to 6.0 X 10(9) M-1 (2.5 X 10(9) M-1). A sensitive radioimmunoassay of T4 was achieved with the isolated high-affinity anti-T4 antibody. The minimum detectable concentration of T4 was 6.3 X 10(-15) to 1.5 X 10(-14) mol/tube, which was three to five times lower than detectable with the initial antibodies.     

Isoelectric focusing of the inbred mouse antibody to bacterial alpha-amylase

In the IgG antibody response to bacterial alpha-amylase (B alpha A) assayed by the enzymatic procedure, C3H/He (C3) mice were high and C57BL/6 (B6) mice were low responders. High responsiveness was inherited as a dominant characteristic in (B6XC3)F1 hybrid mice. In these strains, the primary antibody response was analyzed for heterogeneity by isoelectric focusing (IEF). The IEF spectra were visualized with the use of the capacity of antibody to inhibit the amylase activity of antigen. Increases in the antigen dose and in the time interval between immunization and bleeding resulted in increases in antibody titers accompanied by strong staining of focused antibodies and by the expansion of the pH range where antibodies were focused. High responsiveness in C3 and F1 hybrid mice was also associated with the increase in intensity of stain and the rapid expansion of pH range of focused antibodies. Another strain difference was noted in the isoelectric point (pI) values of antibodies taken early in the primary response. B6 antisera contained those fractions of antibodies focusing over a more alkaline area than C3 antibodies. A similar strain difference in the pI values of antibodies occurred in the response to an irrelevant antigen, Taka-amylase A (TAA), suggesting that the hypervariable regions of antibody molecules play no major part in the strain difference observed. Antisera from F1 hybrid mice displayed bands covering the combined pH ranges of B6 and C3 spectrotypes.     

Rat monoclonal antibody specific for prostaglandin E structure

Six different monoclonal antibodies directed against prostaglandin E2 were obtained from hybrid myelomas, following fusion of mouse NS-1 myeloma cells with spleen cells from a rat immunized with bovine serum albumin conjugates of prostaglandin E2. Four of them were of the IgG2a subclass and the other two were an IgG2b and an IgG2c. Affinities of antibodies for prostaglandin E2 were in the range 5.8 X 10(6)-6.7 X 10(8) M-1. Cross-reactivity experiments showed that one monoclonal antibody was directed almost exclusively against the prostaglandin E structure. The specific monoclonal antibody purified from ascites fluid was used for enzyme immunoassay, and as little as 30 pg of prostaglandin E1 and 100 pg of prostaglandin E2 were detected, which values are comparable to those obtained by radioimmunoassay. These results reveal that the hybridization technique is a reliable way to obtain prostaglandin E-specific antibody and that monoclonal antibodies can be valuable reagents for immunoassays.     

Idiotope mapping on the variable region of an antibody clonotype produced by normal (nonmalignant) human B cells

Human anti-N-acetyl-D-glucosamine (GlcNAc) antibodies were prepared by affinity chromatography from serum of a healthy donor (MSS). They were heterogeneous but contained a unique antibody clonotype (1A) representing 7% of all anti-GlcNAc antibodies. Out of a series of monoclonal anti-idiotopic antibodies (anti-Id mAb), we identified five antibodies that bound to clonotype 1A as shown by isoelectric focusing and Western blotting. Two of them were specific for clonotype 1A (10F59 and 13F15), thus indicating its clonal origin. However, three anti-Id mAb (16F433, 16F539, and 16F812) bound to various additional portions of anti-GlcNAc antibodies of donor MSS. With the exception of one mAb, all anti-Id mAb have very similar relative affinities to clonotype 1A, so results from competition experiments between the different antibodies and between each antibody and antigen should reveal spatial relationships between the corresponding Id and between each Id and the antigen-combining site. The results show a consistent topography of Id on the V-region of clonotype 1A. Id 59, 812, and 433 were found to be arranged in one cluster (cluster I), whereas Id 15 and 539 belonged to a second cluster (cluster II). Cluster I resides completely in the antigen-combining site, whereas only Id 15 of cluster II weakly overlaps with the binding site. Our study demonstrates an analysis of spatial relationships of Id expressed on a human antibody clonotype. To our knowledge, this is the first demonstration of Id mapping on antibodies produced by a normal (nonmalignant) B cell clone that should be accessible to regulatory signals. Such analysis may contribute to a more detailed characterization of anti-Id mAb, and may provide additional information for a better understanding of their immunoregulatory effects.     

Monoclonal antibody to rat apoC: multiple binding to apoC-I on lipoproteins increases its affinity constant

Using solid phase systems, the kinetics of binding of monoclonal antibody (LRB 45, IgG2b,kappa) to apoC-I and apoC-I on lipoproteins were investigated. At 25 degrees C, the association constant of LRB 45 antibody to apoC-I (3.56 X 10(6) M-1 X sec-1) was almost three times slower than the association constant LRB 45 antibody to lipoproteins (10.4 X 10(6) M-1 X sec-1). However, the dissociation constant of apoC-I from LRB 45 antibody (0.865 X 10(-4) sec-1) was also slower than the dissociation constant of lipoprotein from antibody (1.5 X 10(-4) sec-1). Thus, the calculated affinity constant (association constant/dissociation constant) of LRB 45 antibody for apoC-I was approximately half of that for lipoproteins (4.12 X 10(10) M-1 vs. 6.92 X 10(10) M-1). The intrinsic affinity constants for antibody binding to apoC-I and apoC-I on lipoproteins were determined by Scatchard analysis. The intrinsic affinity constant of antibody bound to apoC-I was estimated to be 5.49 X 10(10) M-1 whereas that of antibody binding to lipoproteins was 30 to 200 times less. Furthermore, ascites fluid from LRB 45 cell lines could immunoprecipitate serum lipoproteins. Thus, it is concluded that there is multiple binding of antibody to apoC-I on lipoproteins. This binding appears to increase the calculated affinity constant (avidity) for antibody-antigen interaction.     

Monoclonal antibody for calcitriol (1 alpha,25-dihydroxyvitamin D3)

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3 beta-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3 beta-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to 1 alpha,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3 beta-glu-25-OH-D3 were demonstrated to be 3.6 X 10(9) M-1 and 2.9 X 10(9) M-1, respectively, at 4 degrees C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.     

Applications of a monoclonal antibody to human ferritin in various immunoassays

Monoclonal antibodies to human liver ferritin were generated by an improved hybridoma technique using a semisolid medium containing methylcellulose for initial cloning after the cell fusion. Out of more than 1000 hybrid clones, only 1 was shown to secrete high-affinity monoclonal antibodies to human liver ferritin. The immunoglobulin subclass of this antibody was determined to be IgG2. The association constant between liver ferritin and this antibody was determined to be greater than 1 X 10(10) M-1. Due to the oligomeric nature of ferritin, this antibody can be simultaneously utilized as the first and second antibody in solid-phase sandwich immunoradiometric and enzyme immunoassays. This immunoassay procedure can be performed within 30-45 min and has a sensitivity of about 1 ng/ml. Under identical assay conditions, ferritin isolated from human spleen and human heart gave 50 and 30% cross-reactivity, respectively.     

Characterization of monoclonal anti-fluorescein antibodies from autoimmune New Zealand mice

Previous studies of murine IgM hybridoma protein 18-2-3, derived from an (NZB/NZW)F1 secondary response to fluorescein (FL) presented on T-dependent carrier, demonstrated a high binding affinity for FL (KA = 2.9 X 10(10) M-1) and cryoprecipitation, which could be abrogated upon FL binding. Based on these unusual properties and their possible association with defective immune regulation in lupus-prone mice, further studies were carried out to evaluate the basis of 18-2-3 cryoprecipitation, expression of characteristics related to the 18-2-3 clonotype, and structure/function aspects of additional homogeneous IgM and IgG antibodies of similar origin and specificity. Solubility experiments in which the effect of ionic strength on macroscopic aggregation was measured indicated that 18-2-3 intrinsically possessed both cryoglobulin and euglobulin properties in the absence of auxiliary gamma-globulin components. Rates of hapten fluorescence quenching by 18-2-3 were limited by factors other than diffusion and were dependent on solution temperature and ionic strength. Thirty-seven additional IgM and IgG monoclonal antibodies were shown to possess normal low-temperature solubility and hapten fluorescence-quenching properties, suggesting that 18-2-3 was derived from a relatively rare B cell progenitor. Collective results from FL binding and spectrotype analyses indicated that the majority of proteins were diverse with respect to variable region structure and binding mechanisms but unusually restricted in binding affinities (KA less than 5 X 10(6) M-1). Relative subclass frequencies for 30 monoclonal IgG proteins (IgG1 greater than IgG2b greater than IgG2a greater than IgG3) were consistent with polyclonal IgG subclass expression in normal mice in response to T-dependent immunogen.     

Sequential expansion of antibody heterogeneity during the response to bacterial alpha-amylase

By analyzing antibody heterogeneity during the primary immune response to bacterial a-amylase (B alpha A) in high-responder F1 hybrid mice between C57BL/6 (B6) and C3H/He (C3) mice with the use of isoelectric focusing (IEF), it was shown that the maturation of the primary IgG antibody response consisted of at least two stages. The response of every mouse tested was initiated with the production of specific antibody focused as a limited set of bands in a narrow pH range, and the subsequent rise in antibody titer was associated with the sequential expansion of the spectra involving the appearance of new bands in the pH gradient adjacent to the initial bands. A further rise was accompanied only by intensified staining of the pre-existing bands. These two stages were distinguishable regardless of the antigen dose, although increasing the dose led to widely distributed spectra of focused antibodies and an early shift from the first stage to the second. The sequential expansion of spectra following the appearance of initial antibodies with limited isoelectric point (pI) values was not unique to the anti-BaA antibody response, because similar results were obtained with the antibody response to an immunologically unrelated antigen, Taka-amylase A (TAA). Thus, the appearance of initial antibodies in a limited pH range, overlapping among all F1 hybrids tested, is not a direct reflection of similarity in the determinant specificities of these antibodies among different mice.     

A newly described activity of guinea pig IgG1 antibodies: transfer of cutaneous basophil reactions.

Hapten-specific delayed time course skin reactions containing predominant accumulations of basophils and eosinophils were elicited in newborn guinea pigs after i.v. transfer of small amounts of oxazolone immune serum. The immune serum was fractionated by column chromatography procedures, and the fractions were examined for their ability in transferring this form of cutaneous basophil hypersensitivity (CBH). Only the 7S IgG-containing peak from Sephadex G-200 columns, and only the IgG1-containing fractions from DEAE columns, transferred CBH. An affinity column of bound oxazolone removed the activity from immune serum, and it could be recovered from the column by eluting with soluble oxazolone. About 35 microgram of purified IgG1 anti-oxazolone antibody could systemically transfer CBH reactivity. An immunoadsorbant column of anti-IgG1 removed this activity, but a column of anti-IgG2 did not. None of the procedures were able to separate activity in transferring CBH from passive cutaneous anaphylactic (PCA) activity classically associated with guinea pig IgG1 antibody. IgG1 from 8-day immune and 31-day hyperimmune donors were both effective. The average association constant of 8-day antibody was 8 X 10(-4) M-1. Transfer of cutaneous basophil reactions can be mediated by low affinity serum 7S IgG1 antibody.     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A.

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Oligoclonal IgG bands with and without measles antibody activity in sera of patients with subacute sclerosing panencephalitis (SSPE)

Oligoclonal IgG bands from SSPE sera were isolated by combination of Protein A-Sepharose 4B column and preparative isoelectric focusing gel procedures. Each eluted fraction, when examined in analytic IEF, showed two or three individual bands with isoelectric points close to one another, compared to approximately fifteen IgG bands seen in whole serum. When the bands were tested for measles antibody activity in immunofixation with measles virus followed by peroxidase staining, the bands eluted in pH region 8.5 to 9.3 were found to be measles specific, whereas those in pH 7.0 to 8.4 lacked significant measles activity. When eluted fractions containing groups of bands were absorbed with measles virus, the bands in pH region 8.5 to 9.3 were removed, whereas those in pH 7.0 to 8.4 region remained unchanged; this indicated that a number of oligoclonal IgG bands without measles virus activities are present in SSPE. The bands lacking measles-specific activity may be synthesized against other infectious agents or they may represent nonspecific activation of B cell clones.     

Analysis of the interaction of antibodies with immunoglobulin idiotypes on neoplastic B lymphocytes: implications for immunotherapy

We have examined the reactions of a panel of nine monoclonal anti-idiotype antibodies with the surface immunoglobulin in situ on guinea pig L2C leukemic lymphocytes. Equilibrium binding constants were shown to range between 10(7) and 10(8) M-1 for univalent Fab' gamma fragments and between 10(8) and 10(9) M-1 for intact IgG. Saturation of the cell surface binding sites was achieved with 2.9 X 10(5) Fab' gamma molecules/cell and 1.2 X 10(5) IgG molecules/cell for each antibody, a result that is consistent with a bivalent mode of interaction for the IgG. Despite these overall similarities in binding characteristics antibodies showed striking differences in their ability to clear Ig from the cell surface by antigenic modulation in vitro. This suggested differences in the readiness with which the antibodies cross-linked neighboring surface Ig molecules. Such an interpretation was supported by differences in the times required to achieve bivalent binding at 0 degree C, and in the rates at which labeled antibody dissociated from the cell surface in the presence or absence of an excess of unlabeled antibody. The data are consistent with there being two functionally distinct types of anti-idiotype antibody: those that form predominantly intra-Ig bridges, with each antibody Fab being linked to an Fab on one target molecule ("monogamous" binding) and not favoring modulation; and those that form predominantly inter-Ig bridges ("bigamous" binding) and favor modulation. The nature of interaction is presumably dictated by the orientation of the particular idiotope concerned. This distinction could be of great importance in the therapeutic use of anti-idiotype to ablate B cell neoplasms.     

Apolipoproteins as the basis for heterogeneity in high-density lipoprotein2 and high-density lipoprotein3. Studies by isoelectric focusing on agarose films

A method is described for the isoelectric focusing (IEF) of lipoproteins on thin films of agarose. Within a pH gradient of 4.60-5.30 both high-density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL2 and HDL3 are similar although HDL2 appears richer in the more alkaline bands. Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide two-dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analysed by two-dimensional IEF-SDS electrophoresis and the individual apolipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D, and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins. IEF on agarose of either HDL2 or HDL3 allowed us to collect eight different fractions, which have the same pI in either lipoprotein class. The apolipoprotein composition of each isolated band was analysed by electroimmuno-assays for apolipoproteins AI, AII, CI, CII, CIII, D, and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, apoCIII and apoD. ApoCII and apoCII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII, and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins. Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pI for both HDL2 or HDL3.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Protein spot recognition on the non-denaturing and denaturing two-dimensional electrophoresis patterns using in situ immunosubtraction via Protein A agarose and antibodies.

Specific proteins in small amounts of human plasma were subtracted from patterns of non-denaturing two-dimensional electrophoresis (non-denaturing 2-DE) by layering a 7 microl aliquot of Protein A agarose-antibody complex on the top of an isoelectric focusing (IEF) gel before the loading of a plasma sample. The Protein A agarose suspension was recovered after non-denaturing IEF and was mixed with 8 M urea-5% 2-mercaptoethanol-1% NP-40 to extract the antibody and the specific plasma protein from Protein A agarose. The extract was then subjected to denaturing two-dimensional electrophoresis (denaturing 2-DE) and the location of the specific polypeptide was determined. The technique can be applied to the extraction and analysis of proteins in small amounts of samples.     

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.     

Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.     

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.