Gad Spira

The production of more useful monoclonal antibodies 2. The use of somatic-cell genetic and recombinant-DNA technology to tailor-make monoclonal antibodies

In last months issue(1) Matthew Scharff and his colleagues discussed recent improvements in the technique of making monoclonal antibodies by cell fusion. However, not all the monoclonal antibodies generated by hybridoma technology have all of the properties required for a particular task. This second part of a two-part review deals with ways in which these first-generation reagents can be improved by identifying somatic-cell mutants with the desired properties or by engineering new and even novel molecules using recombinant DNA technology.

Depolarization exposes the voltage sensor of the sodium channels to the extracellular region

SummaryTwo domains of Na channels were mapped with site-specific antibodies raised in rabbit against synthetic peptides corresponding to a part of the voltage sensor of internal repeat 1C 1+(amino acids 210–223) and to a region designated dipole (amino acids 1690–1699) of eel electroplax sodium channels. The antibodies bind to their respective domains in both purified and membrane-bound channels and immunoprecipitate the channels from eel electroplax and rat brain synaptosomes.Anti-C1+depresses the action potential of rat sciatic nerve in a concentration-dependent way. It binds to the external side of rat brain synaptosomal vesicle, and its binding is potentiated by depolarization. Anti-dipole binds to the inner side of the vesicle, and the binding is inhibited by depolarization.

Involvement of different S4 parts in the voltage dependency of Na channel gating.

SummaryThree synthetic peptides corresponding to parts of S4 of the first repeat of eel electroplax sodium channel were synthesized. The basic peptide was C1+which corresponds to amino acids 210–223 (eel channel numbering) and two subfractions: an external fraction, C1ex+(amino acid 210–217); and an internal part, C1in+(amino acid 218–221). Peptide C1+includes four of the charged amino acids of this domain; peptide C1ex+includes three of the charged amino acids and is closer to the external membrane surface (according to channel models) than peptide C1in+which includes the fourth charged amino acid alone.Antibodies generated in rabbits against these peptides were shown to be site specific. Using the whole-cell patch-clamp technique, we found that in rat dorsal root ganglion (DRG) cells, the antibodies against C1in+but not against C1ex+had an effect on the gating parameters. They shifted the Na-channel inactivation curve towards hyperpolarization and decreased the slope of the Na-channel activation curve. These results demonstrate that during the conformational changes associated with channel gating, the fourth charged amino acid of S4 must be accessible to antibodies given to the external solution. Furthermore, they indicate a specific involvement of S4 in the voltage dependency of the gating processes.This study was supported by a basic research grant of The Israel Academy of Sciences and Humanities (#430.87 to H.M. and G.S.).

Case Report Non Symptomatic Hypercalcemia in a Myeloma Patient

Abstract An asymptomatic hypercalcemic IgG-Kappa myeloma patient is presented. A calcium-IgG complex was isolated and purified from the serum by column chromatography. Following further purification on a Sepharose protein A column, the IgG-Kappa molecule did not contain any detectable amount of calcium, indicating that calcium is loosely bound to IgG. The monoclonal IgG demonstrated a strong calcium binding capacity in vitro. The calcium-IgG complex in the serum was shown not to be saturated and thus could absorb more ionized calcium. In view of the presence of an abnormal monoclonal IgG capable of binding excess calcium, and the fact that the asymptomatic patient did not respond to the anti-hypercalcemic drugs, the treatment was stopped.

In vitro produced monoclonal rheumatoid factor: Purification, radiolabel, and possible applications

Abstract A human monoclonal rheumatoid factor (IgM, λ) produced in vitro by Epstein-Barr virus (EBV) immortalized cell line was purified from cell culture medium. Purification, based on the binding of this antibody to protein A, was carried out by adsorption on protein A-Sepharose and elution with low pH buffer. The eluted preparation (×400 purified) was radiolabeled. The specific binding of the purified rheumatoid factor (RF) to immune complexes was kept intact. This unique human monoclonal antibody is applied as a universal reagent to detect low titers of antibodies directed toward cellular antigens.

The Generation of Better Monoclonal Antibodies through Somatic Mutation

Hybridoma technology1 and the monoclonal antibodies it provides have made it possible to apply serology to many new basic and clinical problems. Among the many benefits of hybridoma technology are the potential of obtaining pure antibodies from impure immunogens, the chemical homogeneity of monoclonal antibodies, making them reliable reagents, the large amounts of antibody that can be generated, and the ability to renew exactly the same antibody whenever it is needed.

MOLECULAR COMPARISON OF CULTURED HYBRIDOMA CELLS THAT SWITCH ISOTYPES AT HIGH AND LOW RATES

We have previously reported the isolation of variants from the 36.65 and PC1.4.1 hybridoma cell lines that spontaneously switch from γ1 to γ2a and γ2b at high and low rates. In order to further characterize the phenotype of these variants, we have now investigated the production of germline transcripts and methylation which are two of the molecular correlates of isotype switching. While some of the correlations that exist in normal cells were present in some of the clonal variants, others were not. However, the higher switching variants of both cell lines had higher recombinational activity as measured with a shuttle vector. The distinct phenotypic characteristics of each cell line provide an opportunity to dissect the roles of individual molecular events in the process of isotype switching.

A sensitive method for detecting cells coated with antibodies with human monoclonal rheumatoid factor produced in vitro

A human monoclonal rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus immortalized cell line has been used to detect and quantitate antibodies specifically bound to cells. The RF is purified from cell culture supernatant by a simple procedure and then radiolabeled. In this assay, the binding of 125I-labeled RF to cells coated with specific antibodies is determined. This RF method detects very low titers of antibodies directed against specific cellular antigens and also minorities of antibody coated tumor cells in mixtures with identical non-coated cells. The advantages of this unique human monoclonal antibody are discussed.

Generation of Mutant Monoclonal Antibodies

Since the description of the hybridoma technology in 1975 (1), monoclonal antibodies have found extensive use in almost all fields of biology and medicine. Initially it seemed sufficient to obtain a monoclonal antibody with the desired specificity. However, it soon became obvious that many of the available monoclonal antibodies were not optimally effective for the task to which they were being applied. In order to illustrate this, and present some of the solutions to these problems, we will first briefly describe the relevant structural aspects of the immunoglobulin molecule, then review the kinds of problems that have arisen, and finally present in some detail some solutions to these problems.

Detection of IgD 'hidden' lambda light chain.

Immunoelectrophoresis and immunodiffusion of some IgD myeloma proteins fail to demonstrate precipitin bands between the light chain and anti-light chain serum. Reduction and alkylation of purified IgD preparation was found to render the molecule reactive to anti-lambda serum. Iodinated IgD myeloma protein was successfully precipitated by both anti-delta and anti-lambda antibodies. Both antisera precipitated heavy and light chains of a MW of 70,000 and 24,000 daltons respectively. In addition, a band of 39,000 daltons was also evident. Reduction and alkylation of the iodinated molecule, followed by precipitation with anti-delta serum demonstrated only the 70,000 and 39,000 daltons bands. The present report indicates that by sensitive techniques, such as iodination and immunoprecipitation, non-reactive hidden light chains can be detected in IgD lambda molecules.