A panel of 30 different purified human Ig myeloma proteins of different isotypes and IgG myeloma proteins of known IgG subclasses and light chain types was employed in this study. These myeloma proteins, obtained from patients with multiple myeloma, were either purified by diethyl aminoethyl (DEAE) cellulose (Whatmann, UK) chromatography or by affinity chromatography using Staphylococcal protein A (SPA) or Streptococcal protein G (SPG) Sepharose 4B (Pharmacia, Sweden). The heavy chain and light chain isotypes and subclasses of myeloma proteins were identified using specific mouse MAbs including: AF6 (IgM), 8a4 (IgG), 2D7 (IgA), JA11 (IgD), C4 (λ), 6el (κ), JL512 (IgG1), GOM2 (IgG2), ZG4 (IgG3) and RJ4 (IgG4), kindly provided by Professor R. Jefferis and Dr. M. Goodal (Dept. of Immunology, University of Birmingham, UK).

Polyclonal IgG were prepared as described previously (16). Briefly, polyclonal IgG was isolated from normal serum with SPG-Sepharose column. Fc and Fab fragments were produced from several purified human myeloma proteins of the IgG isotypes, by papain digestion (17). Digested fragments were isolated by affinity chromatography with SPG- or SPA-Sepharose column.

Sera from human and nine animals were prepared from their clotted blood. The animal species used in this study were chicken, rabbit, guinea pig, cat, dog, sheep, goat, horse and monkey. The human serum was used as a control.

Balb/c mice were used for hybridoma production as described else where (18). Briefly, Balb/c mice (8-12 weeks of age) were immunized with four intraperitoneal injections of Fc fragments of IgG myeloma proteins emulsified in Freund’s complete adjuvant (Sigma, USA) (first injection) or incomplete adjuvant (Sigma, USA) (other injections) (50 µg, every 2 weeks). Three days after the last injection, spleen cells were fused with SP2/0 myeloma cells (NCBI 129, National Cell Bank of Iran, Pasteur Institute of Iran, Tehran), using poly-ethyleneglycol (PEG 1500) (Sigma, USA). Hybridomas were grown in DMEM culture medium (Sigma, USA) containing 20% Fetal Calf Serum (FCS) (Seromed, Germany), penicillin (100 IU/ml) and streptomycin (100 µg/ml) and supplemented with hypoxantine (1×10⁻⁴M), aminopterin (4×10⁻⁷M) and thymidine (1.6× 10⁻⁵M) (HAT) (Sigma, USA). Ten to 14 days after fusion, secreting hybrids were identified by analysis of culture supernatants by the ELISA technique described below. Selected antibody producing cultures were cloned by limiting dilution assay (19). Clones secreting antibody of desired reactivity were expanded in 25 and 75 cm² flasks (Nunc, Denmark), harvested and cryopreserved in 40% FCS and 10% dimethylsulfoxide (DMSO) (Sigma, USA).

Specificity of MAbs was determined by ELISA technique as described elsewere (20). Brifely, microtiter polystyrene plates (Maxisorp, Nunc and Denmark) were coated with 1-10 µg/ml of purified myeloma IgG subclasses, including the immunogen and its fragments (Fab and Fc) or polyclonal IgG in PBS (0.15 M, pH = 7.2). Then 0.05 ml of culture supernatant was added. Appropriate dilution of HRP-conjugated sheep antimouse Ig (prepared in our lab) was then added and the reaction revealed with O-phenylenediamine dihydrochloride (OPD) (Sigma, USA) substrate. Finally, the reaction was stopped with 20% H2SO4 and the Optical Density (OD) measured by a multiscan ELISA reader (Organon Teknika, Boxtel, Belgium) at 492 nm.

Goat antimouse IgG1, IgG2a, IgG2b, IgG3, IgA and IgM at 1/1000 dilution, were adsorbed on to the wells of a microtitre ELISA plate (Nunc, Denmark). Isotypes of MAbs in culture supernatants were determined according to the ELISA technique mentioned above.

We determined the K_aff by ELISA technique as described elsewhere (21). Brifely, ELISA plates (Nunc, Denmark) precoated with four different concentrations of human IgG ([Ag], [Ag′] and [Ag′']) were separately incubated with serial concentrations of each MAb. Sigmoid curves were constructed using the OD values obtained for different concentrations of each MAb. Four non-overlapping curves were selected for each MAb to calculate the affinity constant. The half maximum OD (OD-50) was assigned for all selected curves from which the corresponding anti-body concentration ([Ab], [Ab′] and [Ab′']) was extrapolated. Accordingly, [Ab] and [Ab′] are the measurable total Ab concentrations at OD-50 and OD′-50 for plates coated with [Ag] and [Ag′], respectively. The affinity constant was determined using the following equation (22): K aff = ( n - 1 ) / 2 ( n ( [ A b ′ ] t - ( [ Ab ] t ) Where n= [Ag]/[Ag′]

Specificity of MAbs was assessed by immunoblotting technique as described elsewere (23). Brifely, affinity purified myeloma IgG subclasses, polyclonal IgG and their fragments were electrophoresed under native and denaturing conditions in 10% polyacrylamide gel (SDS-PAGE) (Sigma) and then transferred to a nitrocellulose membrane (Schleicher & Schuell, Germany). After blocking with 2.5% skim milk (Merck, Germany), the membrane was incubated with culture supernatants of MAbs for 1.5 hr at 37°C, followed by HRP-conjugated sheep anti-mouse Ig. The bands were finally visuallized with diamino-benzidine tetrahydrochloride (DAB) (Sigma) substrate.

Culture supernatants from growing hybridomas were screened by ELISA using a panel of five Ig heavy chain isotypes and four IgG subclasses. Hybridomas secreting MAbs reactive with all IgG subclasses with no reactivity to IgM, IgD, IgA or IgE were selected. Results obtained for the selected hybridoma clones are illustrated in table 1.

Following cloning and subcloning, culture supernatants from the selected hybridomas were further characterized. All MAbs belonged to IgG1 isotype (Table 2). Specificity of these MAbs was determined, using a panel of purified myeloma proteins, including IgM, IgG, IgA, IgD, IgE, IgG1 (n = 9), IgG2 (n = 4), IgG3 (n = 7) and IgG4 (n = 6) subclasses.

According to ELISA and immunoblotting studies, all MAbs were pan-IgG specific (Figure 1). Three MAbs (3F2D8, 8F9A8 and 5F 19G11) recognize linear epitopes, while the remaining MAb (1F4C4) reacts with a conformational epitope located on heavy chain of all IgG subclasses. Representative immunoblotting results are shown in figure 2. All these MAbs reacted only with Fc, but not Fab fragments of their immunogens (Fig. 3).

Cross-reactivity studies employing whole sera from a range of animal species indicated the most abundant cross-reactivity (100%) with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen serum. Two MAbs cross-reacted with either dog (3F2D8) or horse (1F4C4) serum Igs (Table 3).

The ascitic fluids of two MAbs (3F2D8 and 5F9G11) were prepared and subsequently purified by affinity chromatography using SPG column. The affinity constant (K_aff) of these two MAbs was determined by ELISA. Triple serial concentrations of the antigens and MAbs were selected to construct the corresponding curves and to extrapolate the K_aff values using the formula given in the Materials and Methods. Representative curves obtained for the MAbs are illustrated in (Figure 4A, B) and the calculated average K_aff values are presented in table 4.