Quantification of Monocional Human1gm

Pontet et al. (1) recently argued that, in contrast to polyclonal 1gM, accurate data on concentrations of monoclonal 1gM in different patients is difficult to obtain because of the dependence of the results on the technique. This statement is at variance with previously published work of De Bruijn and Klein (2, 3) who came to different conclusions. According to our findings, confirmed by Out et al. (4), the facts are as follows: #{149} Monoclonal as well as polyclonal human 1gM from different individuals may both show large discrepancies in the results by radial iminunodiffusion (RID), in the sense that the same quantity of different 19S 1gM may give precipitation circles of widely different diameter. #{149} These discrepancies are caused by interaction with the diffusion medium (5) and therefore do not show up in nephelometric and turbidimetric methods, where polyethylene glycol does not seem to interfere (5). #{149} Reduction to 7S monomers (2) or the use of agarose (5) as diffusion medium abolishes the discrepancies and permits a quantification by RID of monoclonal was well as of polyclonal 1gM masses. Such figures compare well with those obtained by electrophoresis (monoclonal 1gM only) and by nephelometry or turbidimetry without previous reduction. #{149} Determinations carried out in this way yield results (3, 6) well below those based on WHO estimates (7). These properties of the RID technique are independent of the batch of antiserum. Their correctness has been proven by comparison with the results of quantifications by analytical ultracentrifugation or electrophoresis of sera containing monoclonal 1gM (2, 3). Thus there can be no doubt that monoclonal 1gM can be correctly determined mass units by RID, electrophoresis, nephelometry, and turbidimetry. Contrary to current expectations (e.g., 8) the discrepancies found when determining different monoclonal 1gM by RID without previous reduction are not greater than when different polyclonal 1gM are investigated. The significant differences between techniques found by Pontet et al. for monoclonal 1gM may arise from comparison of results by electrophoresis with those by RID without reduction and partly also by use of different standards for each technique. Most commercial standards are directly or indirectly based on the data supplied by the wHO for their reference preparations (7). Such standards are therefore incorrect as far as mass units are concerned (3) and cannot be compared for use in RiD without previous reduction. Correct primary standards may be prepared by adding known quantities of any punfled human 1gM preparation to sera previously freed of 1gM by immunoabsorption. The correctness of such standards has been demonstrated by recovery experiments (3, 5). Different standards must be used for high and low concentrations of 1gM, because the RID results are affected by the ratio of 1gM to other serum proteins (5). Secondary standards can then be derived from primary ones by RID with previous reduction or by nephelometry. The nephelometric dose-response curves published by Pontet et al. (1) clearly show that use of that technique will produce comparable results if measurements are carried out in sufficient antibody excess, as most manufacturers prescribe. The shape of the doseresponse curves around the equivalence points for various 19S 1gM cannot be expected to be related to the diameter of precipitation circles of reduced 1gM in RID. After reduction, antigenic or possibly other differences between the IgMs are no longer expressed in the latter technique. In addition, interference by native monomers is obviated because all 1gM is quantified in the monomeric form. Determination of monoclonal 1gM should pose no problem if based on properly calibrated standards. If RID is involved, the 1gM should be reduced first. Mass units or relative (international) units can be used, as desired.