Expression of IgH chain genes in antibody response to ragweed and purified Amb a 1: evidence for differential regulation of isotype production.

A survey of the work with Ig response to allergens carried out previously reveals an allergen-specific response both by IgE and all of IgG subclasses. Response of non-sensitive people is characterized by the appearance of a variety of the IgG subclasses. We have reexamined ragweed and Amb a 1 specific Ig response in 54 nonsensitive and 147 atopic or atopic-allergic people using a new inverse sandwich immunoassay allowing discrimination based on antibody affinity. We show that non-sensitive people present no, 0 out of 54, Ig response with affinities higher than Ka 10(7) M(-1). The subpopulation of 66 atopics who never have experienced desensitization responds vigorously and solely (56 out of 66) with genes of the sequence gamma2-alpha2. Only ten showed an additional weak response from gamma1-alpha1. This suggests a possible association between the atopic state and selective activation of part of the gene sequence.

An automated method for determination of antibody affinity distribution functions with nanogram quantities.

We have developed a method to determine the binding affinity distribution functions (the probability density function for affinity constants will be referred to as affinity distribution throughout the text) for human serum antigen specific IgE for their respective allergens. This fully automated method, based on the Access technology of Sanofi Diagnostics Pasteur, is a highly sensitive two-step sandwich immunoassay requiring only nanogram quantities of antibodies. Allergen-antibody binding isotherms can be determined covering a range of 5 magnitudes of ligand (allergen) concentration. An affinity distribution function, describing the polyclonal nature of the antibody response, can be calculated from the binding isotherm. The validity of the method is assessed using mAbs against the purified allergen Der p 1. The resolving power and sensitivity of the method are demonstrated using selected sera from donors with high and low levels of specific IgE for these allergens. We are able to show that heterogeneous IgE populations can be determined using 100 microl of sera containing 150 pg of specific IgE per ml. Overall, the range of affinities that can be determined by this method is 1 x 10(6)-1 x 10(11) M(-1).

Epitope mapping and binding analysis of insulin-specific monoclonal antibodies using a biosensor approach.

Molecular interactions between the human insulin molecule and a panel of seven monoclonal antibodies (mAbs) against insulin were investigated by surface plasmon resonance. Using real-time analysis (BIAcore technology), the antigenic sites recognized on the human insulin molecule by these mAbs were mapped. Three overlapping regions could be defined on the insulin molecule coupled to the dextran matrix on the basis of its recognition by three mAbs groups: group I (mAbs 19, 10), group II (mAbs 13, 5, 25, 15) and group III (mAb P10). The antigenic determinants of the molecule in solution were mapped using a two-site assay in which insulin was presented to each mAb, bound to a rabbit anti-mouse IgG Fc gamma 1 coupled to the dextran. Four antigenic regions were defined: regions I and III were defined by the same antibodies as above whereas region II was recognized differently by mAbs 13, 5 (region IIa) and mAbs 25,15 (region IIb). The Ka value of the mAbs determined using the BIAcore were in the same range (from 3 x 10(7) M-1 to 6.6 x 10(9) M-1) whether insulin was coupled to the dextran matrix or in solution. Furthermore, mAbs 19 and P10, which recognize two distinct regions on the insulin molecule, were selected to develop a highly sensitive immunoassay for measurement of insulin in serum.

Immunoanalysis of human insulin using monoclonal antibodies reveals antigenicity of evolutionarily conserved residues.

Seven anti-human insulin monoclonal antibodies (mAb) were produced according to an efficient immunization protocol elaborated in our laboratory. Their affinity constants for the binding to the insulin molecule ranged from 5.0 x 10(8) M-1 to 1.0 x 10(10) M-1 when insulin was in solution and from 6.0 x 10(6) M-1 to 2.5 x 10(8) M-1 when insulin was adsorbed onto the microtiter plate. The antigenic sites on the insulin molecule recognized by these mAbs were mapped using two approaches. MAb pairs capable of binding simultaneously to human insulin in solution (using a two-site ELISA) or adsorbed onto a microtiter plate (using a competitive ELISA) were first sought. Three antigenic regions were defined on the surface of adsorbed human insulin and four on soluble insulin. Two distinct antigenic regions common to both the adsorbed and the soluble forms of insulin were defined by our mAbs. In a second approach, the immunological cross-reactivities of these mAbs with species variants of insulin, chemically modified insulin of known structure and a panel of 78 overlapping nonapeptides covering the entire sequence of human proinsulin were assessed. Evidence was obtained that the epitopes recognized by the mAbs included residues conserved during evolution in the insulin molecule. The epitopic specificity of one group of mAbs (group I) was precisely defined. This group recognized a highly conserved region of the insulin molecule including residues 10-17 of the A chain.

An efficient immunization protocol for production of monoclonal antibodies against soluble human insulin.

A new immunization protocol has been developed to obtain specific monoclonal antibodies (mAb) directed against the soluble form of human insulin. Various protocols differing on the basis of the nature of immunogen, the number of injections and the route of administration of the antigen were compared. Mice with the highest anti-insulin titers were selected for cell fusion. The results showed that the immunization protocol involving 2 injections of insulin followed by a boost 2 months later mainly stimulated B lymphocytes secreting IgM mAb directed against immobilized insulin. Immunization with 2 injections of human proinsulin followed by 2 injections of a human insulin-bovine serum albumin conjugate and finally with a booster injection of this conjugate on each of the last 4 days preceding fusion was necessary to obtain a high percentage of hybridomas secreting specific IgG mAb able to recognize immobilized insulin (indirect ELISA) as well as iodinated insulin (liquid-phase radioimmunoassay).