Determination of antibody affinity using ELISA

Some new approaches for the determination of antibody affinity were proposed. It was pointed out that the proposed methods are more simple, convenient, precise, and informative than that of Friguet et al. (1985). The approach that allows determination of two-valence antibodies affinity was also proposed. The example of two monovalent antibodies presented in the examined mixture was considered. It allows to estimate the affinity of both kinds of antibodies as well as to determine their concentration relations in the mixture.     

Determination of antibody affinity by ELISA. Theory

New approaches for the measurement of antibody affinity by ELISA are suggested and considered theoretically. It was shown that not only more precise and more convenient in comparison to that suggested earlier, but also more informative graphical representation of the experimental data in the appropriate coordinate could be used for evaluation of antibody affinity. The following cases were considered: (i) determination of antibody affinity for one kind of univalent antibodies, (ii) determination of antibody affinity for one kind of bivalent antibodies, (iii) determination of antibody affinity for two kinds of univalent antibodies, which are in a mixture, and (iv) determination of antibody affinity for two kinds of bivalent antibodies, which are in a mixture. Advantages and disadvantages of the different approaches are discussed.     

Alterations in monoclonal antibody affinity and antigenic receptor site expression on mycoplasma-infected human colorectal cancer cells

The affinity of MoAb CO 17-1A and expression of its antigenic target were studied on uninfected and mycoplasma-infected colorectal cancer cell lines SW 1116 and SW 948. Binding of 125I-labeled CO 17-1A to SW 1116 cells was quantified at 37 degrees C by determination of the affinity constant (Ka) and the number of antigenic receptor sites (r) per cell using Scatchard plots. When mycoplasma-free SW 1116 cells were used as targets, Ka was 0.92 +/- 0.06 x 10(8) M-1 and r = 1.32 +/- 0.14 x 10(6) at 37 degrees C. One batch of unspeciated, mycoplasma-infected SW 116 cells had reduced affinity and a decreased number of antigenic receptor sites per cell for 125I-labeled 17-1A, while another batch of infected SW 1116 cells had a 4- to 5-fold increase in r and diminished Ka for the antibody compared with uninfected cells. When unspeciated, mycoplasma-infected SW 948 cells were exposed to 125I-labeled 17-1A and the data subjected to Scatchard analysis, the affinity of the antibody deviated markedly from linearity and rendered analysis for Ka and r meaningless. These data indicate that mycoplasma infection can produce variable effects on the cellular expression of antigenic receptor sites and the affinity of antibody for its target, and emphasize the importance of using mycoplasma-free cell lines in studies of these parameters.     

Purification of urokinase by monoclonal antibody affinity chromatography

Matrix-bound monoclonal antibodies against urokinase have been used to purify this enzyme by affinity chromatography. In a single-step procedure, urokinase can be isolated from crude preparations with high yield and high purity, and without loss of enzymatic activity.     

Isolation of chromosaponin I-specific antibody by affinity chromatography

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, modulates several developmental processes of plant roots and activates the sugar taste receptor cells in blowflies. CSI is a unique saponin for its reducing power and biological activities in both plants and insects. In the present paper, we described the method of preparation for CSI-specific antibody using CSI-affinity and soyasaponin I-affinity columns. The antibody's-specific binding activity to CSI was confirmed by a bioassay using Arabidopsis roots and a ligand-molecule interaction analysis using BIAcore 3000. Because of the lability of CSI, the CSI-affinity column was made only by a moderate reaction condition in which CSI was coupled to EAH Sepharose 4B in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). The special control of the reaction temperature was essential to complete the coupling reaction; the reaction with EDC at 0 degrees C followed by a gradual increase in temperature.     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

Modulation of antibody affinity by a non-contact residue.

Antibody LB4, produced by a spontaneous variant of the murine anti-digoxin monoclonal antibody 26-10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640-4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26-10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26-10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of monoclonal antibody affinity

In experiments with Pasteurella pestis monovalent capsular antigen and hybridoma monoclonal antibodies obtained after cloning and recloning the heterogeneity of the active centers of antibodies with respect to their affinity was revealed. In ascitic fluids obtained from the animals inoculated with different hybridoma clones 5 groups of antibodies, differing in their affinity, were determined in each fluid sample.     

Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody

We previously described the production of four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG2a, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes [125I]iodocyanopindolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and R9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9 [k-1(+R9) = 0.025 min-1 vs. k-1(-R9) = 2.04 min-1], consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor radioimmunolocation: differential antibody retention by antigenic normal tissue and tumor

On the assumption that a high specificity would be required for in vivo tumor location and drug targeting, much labor has been expended to produce monoclonal antibodies directed to antigens found primarily on tumors. However, for at least one tumor-associated antigen, stage-specific embryonic antigen 1 (SSEA-1), the concentration of the target antigen need not be higher in the tumor than that found in normal tissues for successful use of the corresponding monoclonal antibody in in vivo tumor location; rather, accessibility and antibody metabolism are more important.     

Mapping protein interactions by combining antibody affinity maturation and mass spectrometry

Mapping protein interactions by immunoprecipitation is limited by the availability of antibodies recognizing available native epitopes within protein complexes with sufficient affinity. Here we demonstrate a scalable approach for generation of such antibodies using phage display and affinity maturation. We combined antibody variable heavy (V_H) genes from target-specific clones (recognizing Src homology 2 (SH2) domains of LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 10⁸ to 10⁹ new variable light (V_L) genes. Improved binders were isolated by stringent selections from these new “chain-shuffled” libraries. We also developed a predictive 96-well immunocapture screen and found that only 12% of antibodies had sufficient affinity/epitope availability to capture endogenous target from lysates. Using antibodies of different affinities to the same epitope, we show that affinity improvement was a key determinant for success and identified a clear affinity threshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinity capture using matured antibodies to SHC1 with mass spectrometry, we identified seven known binding partners and two known SHC1 phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitation can be generated by chain shuffling, providing a scalable approach to mapping protein–protein interaction networks.     

Absorption of anti-placenta serum by means of immunosorbents]

An anti-placenta serum was absorbed by means of immunosorbents to remove antibodies against human serum proteins. The absorption met with some difficulties, because the anti-placenta serum contained antibodies against several human serum proteins. 12 different methods were compared for their suitability to adsorb these antibodies against human serum proteins. Most suitable is human serum cross-linked by glutardialdehyde. Good results were obtained too with human serum linked to Enzacryl, Polyaminostyrene or CNBr-activated Sephadex.     

Location of antigenic epitopes on antibody molecules

Using X-ray crystallographic co-ordinates of immunoglobulins, surface regions accessible to a large spherical probe, comparable in size to an antibody domain, were computed. Locations of these exposed regions were compared with those of experimentally determined antigenic sites, i.e. idiotypic, allotypic and isotypic serological markers. In all cases, an excellent agreement was found. The most prominent computed epitopes correspond to convex parts of antibody surface made by reverse turn segments of the polypeptide chain. The computed epitopes occur in homologous positions in all the immunoglobulin domains, and most of the beta-sheet surfaces on the domains are poorly antigenic. The CH2 domain (Fc fragment) has many more antigenic sites than the Fab fragments (antigen-binding fragments). Variable domain epitopes (idiotypes) involve both hypervariable and framework residues, and only about 25% of the hypervariable residues are strongly antigenic. The results indicate that, in a vertebrate body, each antibody molecule may be recognized, and its concentration regulated, by at least 40 complementary anti-immunoglobulin antibodies; therefore, a possibility of an "immune network" with much higher connectivity than is generally assumed should be seriously contemplated.