Generation and Characterization of Mouse Hybridomas Secreting Monoclonal Antibodies Specific for Human IgG3

Abstract

Mammalians express several subclasses of the IgG molecule. In human being there are four homologous IgG subclasses, each of which is structurally unique and has different functions. Quantification of IgG subclasses is fundamental to clinical assessment and diagnosis of many diseases as such assessments depends on the availability of subclassspecific antibodies (Abs), particularly monoclonal antibodies (MAbs). In the present study, we produced and characterized two murine MAbs specific for human IgG3 molecule. These MAbs were obtained by the fusion of myeloma cells with splenocytes from Balb/c mice immunized with heavy chain of a human IgG3 myeloma protein. Fused cells were selected in hypoxanthine, aminopterine and thymidine (HAT) medium and cloned by limiting dilution assay. Ab-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. Two stable hybridomas designated 1F18G7 and 1F18A11 were obtained secreting MAbs specific for Fc fragment of human IgG3. None of these MAbs showed cross-reactivity with other immunoglobulin isotypes derived from human and nine other animals, except 1F18A11 which displayed a weak cross-reactivity with only dog serum. Immunoblotting results indicate that these MAbs react with linear epitope(s) located in the heavy chain of human IgG3 molecules. The affinity constant of 1F18G7 and 1F18A11 MAbs was found to be 0.81×10(9) Mol (-1) and 0.71×10(9) Mol (-1), respectively, as measured by ELISA. These two MAbs with relatively high affinity can be useful tools for quantification of IgG3 subclass levels in human serum.

Keywords: Heavy chain; Human IgG3; Immunoglobulin; Isotype; Light chain; Monoclonal antibody.

Figure 1

Acetate cellulose electrophoresis of ascitic fluids of 1 F18G7 and 1F18A8 MAbs. NS=Normal serum; 4F18B12=Anti nG1m (a) isoallotype MAb employed as a control

Figure 2

Reactivity of IgG3 specific MAbs with IgG subclasses Lanes 1 to 28 represent: MM1, MM11, MM24, MM46, MM50, MM53, MM63, MM65, MM13, MM12, G2 (M), MM62, Camp, MM98, Gale, Hay, Pol, Goe, Ren, Mcw, IgG3 polyclonal, MM147, Rea, Jan, Will, Cart, J. Wil, Ze

Figure 3

Reactivity of IgG3 specific MAbs with enzymatic fragments of MM98 (IgG3). Control represents a mouse MAb (HP6050) with anti-IgG3 specificity

Figure 4A

Immunoblot analysis of 1F18G7 MAb reactivity with IgG subclasses. Lanes 1 to 5 represent reduced forms of MM1 (IgG1), MM12 (IgG2), Hay-ward (Heavy chain of IgG3), MM98 (IgG3) and MM147 (IgG4), respectively. Lanes 6 to 13 represent non reduced forms of MM1, MM12, Hay-ward, MM98 (whole molecule), MM98 (Fab), MM98 (Fc), IgG3 polyclonal and MM147, respectively.

Figure 4B

Immunoblot analysis of 1F18A8 reactivity with IgG subclasses. See footnote to figure 4A.

Figure 5A

Representative binding curves employed for extrapolation of affinity constant of 1F18G7 MAb.

Figure 5B

Representative binding curves employed for extrapolation of affinity constant of 1F18A8 MAb.