Determination of CD4 antigen density on cells: Role of antibody valency, avidity, clones, and conjugation

Davis, Kenneth A.; Abrams, Barnaby; Iyer, Sujata B.; Hoffman, R. A.; Bishop, James E.

doi:10.1002/(sici)1097-0320(19981001)33:2<197::aid-cyto14>3.0.co;2-p

Cited by 145 publications

(152 citation statements)

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“…The ABC values obtained using unimolar conjugates are given in Table 1. The mean value determined for unimolar CD4-PE conjugate (36,800) is slightly larger than that for the standard grade CD4 PE (34,400); both are consistent with the reported values (8). Though our values are MESF values deter mined based on calibration curves generated by using NIST RM 8640, the number of PE molecules per cell obtained based on calibration curves generated by using QuantiBRITE TM PE Quantification kits and unimolar PE conjugates, and calculated antibody bound per cell values for CD20 FITC using CD4 expression on T cells as the biological calibrator.…”

Section: Discussionsupporting

confidence: 89%

“…1) determined using standard grade anti-CD4 PE and based on calibration curves generated by QuantiBRITE PE Quantification kits is 34,400, with a CV of 5.5%. This value is comparable to the value reported by Davis et al, *38,000 PE molecules, using standard grade CD4 PE from BD Biosciences and freshly prepared blood samples without fixation step (8). The authors showed that the number of PE molecules determined depended on fixation condition, and varied with CD4-PE conjugates obtained from different sources.…”

Section: Discussionsupporting

confidence: 85%

“…We utilized CD4 PE as a positive control and gating reagents that define major lymphocyte as subsets: T cells, B cells, and NK cells. CD4 was chosen because it has been studied extensively (6)(7)(8)(9), shown little variation between normal subjects, and demonstrated excellent baseline separation. The validation efforts on CD4 quantification should ensure the confidence of quantifying the B-cell marker, CD20.…”

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Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Wang

Abbasi

Gaigalas

et al. 2006

Cytometry Part B Clinical

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Methods: We measured CD20 expression on normal and B-CLL B-cells, using FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, we also measured CD4 expression on T-cells with FITC and PE Mab. Calibration curves were constructed using the CLSI (formerly NCCLS) consensus guidelines for QFC. Calibration with QuantiBRITE TM PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurement of antibody bound per cell (ABC) for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF), as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC, using the CD4 T-cell as a biologic calibrator, to normalize FITC and PE results for CD20 expression.Results: On normal B cells, the mean ABC value for unimolar CD20-PE conjugate was 143,500 (CV 6 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV 6 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20-FITC on normal B-cells was 199,300. CD20-FITC staining on B-CLL cells was generally too weak for accurate quantification. On normal T-cells, the mean ABC value for CD4 unimolar PE conjugate was (36,800 6 10.4)%, and it did not differ significantly in CLL samples.Conclusion: The expression of CD20 on normal and B-CLL lymphocytes can be quantified in ABC units using unimolar CD20-PE conjugates. In addition, CD4 expression on T-cells can be used as a biological calibrator to quantify CD20-FITC ABC, with reasonable agreement between the two conjugates with different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective F/P ratios for FITC conjugates will require additional laboratory studies. q

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 85%

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Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Wang

Abbasi

Gaigalas

et al. 2006

Cytometry Part B Clinical

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“…In some cases custom conjugates were used. Cells were fixed prior to analysis, which causes a reduction in CD4 binding, and the number of CD4 antibodies bound per cell was estimated to be 40,000 (18). CS & T beads were analyzed under the same conditions as the CD4 cells, and the fluorescence scale in ABD units was Figure 9.…”

Section: Cytometer Biological Signal Calibrationmentioning

confidence: 99%

Toward quantitative fluorescence measurements with multicolor flow cytometry

et al. 2007

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A procedure is presented for calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure involves two steps. First, each of the fluorescence channels of the flow cytometer is calibrated using Ultra Rainbow beads with assigned values of equivalent number of reference fluorophores (ERF). The objective of this step is to establish a linear relation between the fluorescence signal in a given fluorescence channel of multicolor flow cytometers and the value of ERF. The second step involves a biological standard such as a lymphocyte with a known number of antibody binding sites (e.g., CD4 binding sites). The biological standard is incubated with antibodies labeled with one type of fluorophores for a particular fluorescence channel and serves to translate the ERF scale to an ABC scale. A significant part of the two-step calibration procedure involves the assignment of ERF values to the different populations of Ultra Rainbow beads. The assignment of ERF values quantifies the relative amount of embedded fluorophore mixture in each bead population. It is crucial to insure that the fluorescence signal in a given range of fluorescence emission wavelengths is related linearly to the assigned values of ERF. The biological standard has to posses a known number of binding sites for a given antibody. In addition, this antibody has to be amenable to labeling with different types of fluorophores associated with various fluorescence channels. The present work suggests that all of the requirements for a successful calibration of a multicolor flow cytometer in terms of ABC values can be fulfilled. The calibration procedure is based on firm scientific foundations so that it is easy to envision future improvements in accuracy and ease of implementation. Published 2007 Wiley-Liss, Inc. y Key terms multicolor cytometry; calibration; quantitation; biological standard IN a flow cytometer measurement, cells are carried by a flowing stream across focused laser beams (1). Prior to aspiration of the cells into the sheath fluid stream of the flow cytometer, the cells are incubated with a mixture of labeled antibodies specific to various receptors on the cell surface or intracellular protein antigens. Nuclear antigens can also be detected and are often useful as proliferation markers. After the incubation, the labeled antibodies are attached to specific receptors on the surface of the cell or intracellular protein antigens. As the cells flow past the laser beams, the fluorescence of fluorophores is detected in discreet fluorescence channels (FC) via photomultipler tubes (PMT). Fluorescence signal detected in a specific fluorescence channel is representative of the expression level of a given protein antigen. Because immune cells work in a cooperative manner (2), disease diagnostics and immunotherapies are mostly carried out by monitoring expression of multiple cell receptors, such as HIV-1 infection (3,4) and chronic lymphocytic leukemia (5,6). A CLSI guideline (7) exists for fluorescen...

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“…When QuantiBRITE PE beads are used in conjunction with PE conjugates with a PE-to-monoclonal antibody (mAb) ratio of 1:1, PE molecules can be converted to ABC (28). Finally, if the antibody-to-antigen binding stoichiometry is known, the number of antigens per cell can be determined (29). PE is an ideal fluorochrome for use with fluorescence quantitation because of its high sensitivity and lack of self-quenching.…”

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confidence: 99%