Dear Philippe (Poncelet) I read your message with interest and would like to receive Part B. >From the viewpoint of a binding study, I think the generalised analysis we did in Ron Chatelier's paper in the EMBO J still raises the question of what happens when there are several different cell-types which occur within a mixture such as whole blood, each of which has (different?) binding specificity for the target antigen, as applies with all of the non-lineage specific antibodies. This is why the initial process needs to be done with "pure"(?) cell lines, which express just a single class of specific binding sites; of course, this needs to be demonstrated via proper binding curves (best done by the isoparametric method I would say). I don't accept your enthronement of the radio method as the gold standard, because of its bulk reporting response and because of the influence of dead-cell uptake. The gold standard will be fluorescent ligands with defined univalent antigen-specific binding, used initially via an isoparametric study and calibrated against a series of bi-valent Mabs, eg those offered by Dako, BC and BD. I am intersted to hear more on QIFI though. Bob -----Original Message----- From: Michel Canton [SMTP:mcanton@wcube.fr] Sent: Wednesday, September 10, 1997 3:37 PM To: Cytometry Mailing List Subject: Re: Cell surface receptor Dear group, I fully agree with Bob Ashcroft's answer to this very interesting question regarding the use of antibodies and the concern of monovalent/bivalent attachment as well as the influence of affinity. The most recognized and popular method of measuring cell-associated receptor numbers is radio-binding. This method may be considered as a gold standard for any other method. Flow cytometry (FCM) methods presents many advantages over radio-binding (and in addition, we all like it so much!). This is Titus et al. ((1981), PNAS, 78, p519) measured Fc receptors on human cells with FCM after calibrating their assay using radio-labeled aggregated IgG. They demonstrated a linear relationship between MFI (corrected from background) and the number of cell bound IgG molecules as determined by radio-binding. By simply applying the same principle with anti-CD5 MAbs, we designed the QIFI technology (quantitative indirect fluorescence immuno assay) that proved to be valid for any mouse IgG Mab (J.Immunol.Methods, 85: 65-74, 1985). This method is now being fully exploited in application kits. Initially CEM sub-clones with known CD5 Mab binding capacity were used as reference calibration standards for quantitative FCM (Leucocytes Typing II, vol.2, 329-343). Later on non fluorescent latex beads covered with mouse IgG were calibrated towards our cellular standards. Such beads still serve as pseudo-cells covered with known numbers of IgG Mab to relate MFI to Mab binding, i.e., number of bound Mab molecules per cell. Infering antigen density from Mab binding is a more critical approach as underlined by Bob Ashcroft. We experienced various situations: 1- CD5 T101 Mab had proved in our hands to monovalently bind to CEM cells under saturating conditions as checked by radio-binding using both IgG and Fab fragments. 2- During the 2nd International Workshop we found that a majority of different CD8 Mabs revealed about 130,000 sites per CD8+ lymphocytes whereas few others revealed only half this number , i.e., about 65,000 sites. In fact each CD8 complex consists in one alpha-beta heterodimer that could express an epitope present on both chains. Each CD8 complex binds either 2 Mab molecules or only one depending on the specificity of the Mab. 3- A recent study from Wagner et al. (Blood, 88:907, 1996) showed that even when the target antigen is expressed at a very high density and inter-molecule distance is shorter than the size of a F(ab')2 fragment, divalent binding occurs even under conditions of entibody excess. This is demonstrated for the GpIIa-IIIa complex on the platelet surface. Although monovalent binding could be taken as a rule when saturating concentration is used there may be exceptions that one needs to know to be able to measure antigenic sites more appropriately (use of Fab fragment). Under the monovalent binding assumption we published some numbers for major CDs on human leucocytes which could (?) serve as a basis for further studies (Cytometry , CCC, 26: 137, 1996). Some of these numbers have been, at least, rather consistent over time (years), when using various cytometers and also between different laboratories. There are for instance about 50,000 CD4 sites/CD4+T lymphocyte (Res. Immunol., 142: 291, 1991) or about 200,000 CD45 sites on normal PBL (Cytometry, 12 suppl. 5, 418A, 1991). To summarize I just wanted to illustrate that besides fluorescent beads there are other means (non fluorescent beads covered with mouse IgG or whatever) that can also be useful and at least very flexible and that in any of these different quantitation strategies radio-binding must still remain the referecence when designing such quantitative flow assays. If you allow me I shall send you the part B of my answer in few days focusing on the specific question of quantifying receptors and measuring receptor occupancy. Hope this long part A (too long ?) can already help! Philippe Poncelet, PhD Scientific Director BioCytex 140, chemin de l'armee d'afrique 13010 Marseille France Tel +33 (0) 4 91 94 29 39 Fax: +33 (0) 4 91 47 24 71 E-mail: use my boss's E-mail: mcanton@wcube.fr
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