Monoclonal antibodies data analysis

Figure 10.3 Whole-mass analysis of a monoclonal antibody. (A) Direct infusion of the antibody generates an envelope of high m/z ions ranging from 2000 to 3500. Deconvolution of the ion current signal gives the mass of the complete native molecule (147, 100.97 Da) and resolves some heterogeneity linked to the A-glycan structures. The major forms are consistent with molecules carrying biantennary structures capped with 0, 1, or 2 hexose (G = galactose) residues. (Data generated on an ESI-Q-Star instrument, Sciex-Applied Biosystems.)...

The effect of CVS is T-cell mediated, since no effect was observed in athymic nu/nu mice of BALB/c background which were hereditarily T cell deficient (data not shown). Therefore, we examined the participation of T-cell subsets in the antitumor effect of CVS following their in vivo depletion with monoclonal antibodies in the Meth A and BALB/c system, Fig. (6)-Protocol B. CVS was injected i.t. 3 times from day 2, and tumor i.v. rechallenging was performed on day 9. Anti-CD3 treatment completely inhibited the antitumor effect of CVS (data not shown). Anti-CD4 and anti-CD8 treatment partially inhibited the effect of CVS. Less than 1.0% of the CD3-, CD4-, or CD8-positive cells were detected in the spleen or lymph nodes following in vivo treatment with the corresponding antibodies by flow-cytometric analysis using FACS (Becton Dickinson, USA). 

Expression levels of the mutant thrombin receptors were determined in transiently transfected COS cells by assaying the level of surface expressed thrombin receptors by FACS analysis, usually for mutant receptors that showed a significant diminution of function. COS cells were transfected by calcium phosphate-DNA co-precipitation30 with 10 ng of DNA. The transfected cells were analyzed by FACS 48 h after transfection. A monoclonal antibody specific for the amino terminal region was used as the primary antibody to detect thrombin receptors present on the surface of the cells.31 A FITC conjugated goat anti-mouse IgG was used as a secondary antibody. Expression levels of the mutant receptors were determined relative to expression levels of wild-type controls. Expression-level data (% of control) for some of the mutant receptors are listed in Table 2 (wild-type thrombin... 

Mass cytometry, however, has important limitations. First of all, the cells employed in the analysis cannot be recovered. Second, the potential huge number of labels complicates the analysis and data interpretation. Finally, the prize of the stable isotope conjugated monoclonal antibodies is quite high, also necessary to test their specificity, affinity, stability, and resistance (43). 

Interpretation of the results is again a problem. Tests using home made monoclonal antibodies are suspect until the antisera is made available to other investigators for conformation. The use of well characterized antisera from companies which supply to others is better at this stage. Since the tissue section is examined and scored by an observer, the data from these studies are not really available for analysis by the reader. Computerized image analysis techniques are still not widely used. Thus, in evaluating the results, possible bias of the investigator must be taken into account. 

Langley RG, Papp K, Gottlieb AB, Krueger GG, Gordon KB, WUliams D, et al. Safety results from a pooled analysis of randomized, controlled phase II and III clinical trials and interim data from an open-label extension trial of the interleukin-12/23 monoclonal antibody, briakinumab, in moderate to severe psoriasis. J Eur Acad Dermatol Venereol 2013 27(10) 1252-61. 

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