Introduction

Human immunoglobulins are broadly classified into five major serologically different heavy chain isotypes (IgM, IgG, IgA, IgD and IgE) and two distinct light chain isotypes (Kappa and Lambda) (1). Each heavy chain isotype has different biological characteristics (2) and a particular profile of effector function (3). Antibody isotypes are differently expressed in response to different antigenic stimuli (4-6). Thus, different patterns of antigen specific Igs (7,8) have been reported in a variety of diseases.
IgG is the most abundant immunoglobulin present in human serum and extravascular fluids and there might be a relationship between the IgG level and severity of diseases (9-11). Therefore, IgG has a great diagnostic value (12). Furthermore, analysis of the IgG profiles to different antigens of a microorganism might reveal different profiles of T helper associated responses, (13) and hence, is helpful for designing a potential vaccine (14). Quantification of total and antigen specific IgG levels depends on the availability of specific MAbs and sensitivity of assays could be increased with assay optimization, standardization (15) and application of high affinity MAbs. This is the advantage of MAbs in comparison to polyclonal anti IgG antibodies.
In the present study, four MAbs with specificity for different epitopes shared by all human IgG subclasses are produced and characterized.

Materials and Methods

Preparation of purified human IgG subclasses: 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.

Animal sera: 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.

Production and selection of hybridomas: 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 polyethyleneglycol (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 (1×10-4 M), aminopterin (4×10-7 M) and thymidine (1.6× 10-5 M) (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).

Analysis of specificity of MAbs by indirect ELISA: Specificity of MAbs was determined by ELISA technique as described elsewere (20). Brifely, microtiter polystyrene plates (Maxi-sorp, 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 (pre-pared in our lab) was then added and the reaction revealed with O-phenylenediamine dihydrochloride (OPD) (Sigma, USA) sub-strate. Finally, the reaction was stopped with 20% H2SO4 and the Optical Density (OD) measured by a multiscan ELISA reader (Or-ganon Teknika, Boxtel, Belgium) at 492 nm.

Isotype determination of MAbs by capture ELISA: 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.

Affinity constant (Kaff) determination by ELISA: We determined the Kaff 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):
Kaff =(n-1)/2(n([Ab′]t − ([Ab]t)
Where n= [Ag]/[Ag′]

Immunoblotting technique for analysis of specificity of MAbs: 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 (Schlei-cher & 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 visualized with diaminobenzidine tetrahydrochloride (DAB) (Sigma) substrate.

Results

Screening and selection of IgG specific hybridomas: 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.

Characterization of MAbs: 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 (Figure 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).

Determination of affinity of MAbs:The ascitic fluids of two MAbs (3F2D8 and 5F9G11) were prepared and subsequently purified by affinity chromatography using SPG column. The affinity constant (Kaff) 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 Kaff values using the formula given in the Materials and Methods. Representative curves obtained for the MAbs are illustrated in figure 4 (A, B) and the calculated average Kaff values are presented in table 4.

Discussion

In this study, four MAbs specific for isotypic epitope(s) of human IgG were produced and characterized. These MAbs were specific for Fc-dependent isotypic epitopes common to all IgG subclasses. As shown by immunoblotting studies, three of these pan-IgG-Fc specific MAbs (3F2D8, 8F9A8 and 5F19G11) reacted with linear epitope(s) and one MAb (1F4C4) reacted with a conformational epi-tope located to all human IgG subclasses.
Several MAbs specific for a variety of epitopes located to human IgG heavy chain have so far been produced by other investigators (24-27). A few anti IgG MAbs were shown to be IgG-Fab specific (25), whereas most IgG-specific MAbs were found to recognize Fc-associated epitopes, a specificity similar to all of our MAbs (Figure 3). This is in agreement with another study (26) in which a large number of MAbs were produced with specificity for conformational epitopes located on the CH2-CH3 border of the hinge region, indicating the high immunogenicity of this region. Those MAbs are very similar to one of our MAb (1F4C4) which recognizes a conformational epitope located to the Fc region of IgG molecules. Nevertheless, three of our MAbs recognized linear epitopes.
The four MAbs produced in this study displayed different profiles of cross-reactivity with a variety of animal sera. The most abundant cross-reactivity was observed with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen sera. The animals' sera used in this study were prepared from healthy normal adult animals which naturally have normal antibody levels. Moreover, some of these sera such as sheep, goat and rabbit sera were checked with the corresponding HRP-conjugated anti-Ig antibodies as positive controls which showed strong positive signal (data not presented). Thus, lack of reactivity of the MAbs to these serum samples could not be attributed to the absence of Ig in the sera.
Cross-reactivity of anti human IgG MAbs with IgG of animal species has already been reported by other investigators (22,24,25). Our MAbs displayed a restricted pattern of cross-reactivity. In addition to cross-reactivity with monkey serum, one of the MAbs (3F2D8) was cross-reacted with dog serum and another one (1F4C4) cross-reacted with horse serum. This pattern of cross-reactivity reflects to some extent the degree of amino acid sequence homology observed between human and other animals IgG molecules. Blasting of the human IgG protein sequence with those of the other animals reveals approximately 90% homology with monkey, 50-70% homology with other mammals and 30% with avian immunoglobulin (http://blast.ncbi.nlm.nih.gov/ blast.cgi). Similar pan-IgG specific MAbs cross-reacting with horse and pig IgG, have been reported by other investigators (26). Cross-reactivity with pig serum was not tested in our study. In another study, 13 pan-IgG Fc specific MAbs were evaluated, all of which cross-reacted with other animal sera and showed a wider pattern of cross-reactivity (24).
The affinity constant (Kaff) of two of our MAbs (3F2D8 and 5F19G11) was found to be 0.74×108 M -1 and 0.96×107 M -1, respectively. A similar affinity constant has already been reported for some human IgG specific MAbs using other methodologies such as agglutination inhibition (26) or fluorescent sequential saturation assay (28). We determined the affinity of our MAbs by ELISA.

Conclusion

Pan-IgG specific MAbs are useful tools for quantitative measurement of human total or antigen specific IgG in serum in different diseases. IgG specific MAbs with relatively high affinity like 3F2D8 MAb, could enhance the sensitivity and specificity of ELISA assay for quantification of IgG. Currently, enzyme conjugated anti-IgG MAbs are used for measurement of specific IgG in a variety of ELISA kits designed for diagnosis of various infectious diseases, such as Toxoplasma (29). Employment of a combination of pan-IgG specific MAbs with different specificities may increase the sensitivity of these assays as they complement each other via reactivity to different epitopes located to the same IgG, as described before (30). These MAbs could also be used for epitope mapping of the Fc region of the IgG molecule.

Acknowledgement

We thank Professor Mahmood Jeddi-Tehrani, Dr Soheila Gharagozlou, Roya Ghods, Jalal Khoshnoodi and Azam Roohi for scientific consultations and preparation of antigens. This study was supported in part by a grant from the Research and Technology Undersecretary of the Ministry of Health, Treatment and Medical Education of Iran.