Detection and quantification of the human IgG4 half-molecule, HL, from unpurified cell-culture supernatants.

The presence and absence of inter-heavy-chain disulphide linkages contribute to the existence of the tetrameric (H 2 L 2 ) and half (HL) human IgG molecules, respectively [Schuurman, Perdok, Gorter and Aalberse (2001) Mol. Immunol. 38, 1-8]. Reduced effector response in the human IgG4 subclass presents an alternative therapeutic platform in a monoclonal-antibody (mAb) development program. During the initial cell-selection stage, titres of the recombinant human antibody present in crude cell-culture supernatants are determined by ELISA, a technique requiring nano-gram quantities of mAb. In the case of an IgG4 antibody, this material is represented mainly by the combination of the tetrameric (H 2 L 2 ) and dimeric (HL) forms of the antibody. The determination of concentrations or ratios of tetramer and dimer usually requires at least one chromatographic purification step, and thus frequently this is evaluated later in the mAb development process when the number of potential clones has been reduced. In the present paper we describe a Western-blot-based method that detects and quantifies IgG4 half-molecules, HL, from crude cell-culture supernatants without purification so that H 2 L 2 /HL ratios can be included as a part of early clonal evaluation along with the screening of mAb titres. This method was demonstrated (I) to have a linear HL detection range of 0.5-10 ng, (2) to require microlitre volumes of culture and (3) to react specifically with human IgG4 produced from hybridoma and Chinese-hamster ovary cell cultures. Moreover, this protocol is applicable to evaluate and monitor potential H 2 L 2 /HL variations as a result of changes during the process-development stage of a mAb development program.