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Keywords:

  • CD4;
  • Quantibrite™ PE quantification kits;
  • ABC value;
  • MESF value;
  • FITC-labeled microspheres;
  • CYTO-TROL Control Cells;
  • pH effect;
  • antibody binding affinity;
  • effective F/P ratio

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Background:

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO-TROL™ Control Cells and T-lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate.

Methods:

Calibration with Quantibrite PE-labeled microspheres and the use of unimolar CD4-PE conjugates provided direct measurement of the antibody bound per cell value (ABC) for CD4 expression on normal T-lymphocytes. Calibration for CD4-FITC monoclonal antibody (Mab) labeled CYTO-TROL Control Cells and normal T-lymphocytes was based on molecules of equivalent soluble fluorochrome (MESF) as determined by FITC-labeled microspheres traceable to NIST RM 8640. The MESF value for CD4-FITC Mab was determined that enabled the conversion of the MESF values obtained for CYTO-TROL cells to ABC. We investigated the likely pH change in the fluorescein microenvironments within FITC-labeled Mab and cells stained with FITC-labeled Mab using a pH sensitive indicator.

Results:

The mean ABC value for T-lymphocytes prepared from fresh whole blood using CD4-PE conjugate (48,321) was consistent with previous results, and it was much higher than the mean ABC using CD4-FITC Mab (22,156). The mean ABC value for CYTO-TROL cells using CD4-PE conjugate (43,090) was also higher than that using CD4-FITC conjugate (34,734), although the discrepancy was not as great. Further studies suggested the discrepancy in CYTO-TROL results may be accounted for by the low pH of the membrane microenvironment, but the greater discrepancy in T-lymphocytes could not be fully explained.

Conclusion:

CD4 expression on fresh normal whole blood samples and CYTO-TROL cells can be consistently quantified in ABC units using Quantibrite PE quantification beads and unimolar CD4-PE conjugates. Quantification with CD4-FITC conjugate is not as consistent, but may be improved by the use of CD4 T-cells as biological calibrators. This approximation is valid only for surface receptors with consensus ABC values measured by different QFC methods serving as biological standards. Published 2007 Wiley-Liss, Inc.

The use of quantitative immunofluorescence measurements has increased significantly since the mid-1990s. Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on a cell. Though the ultimate goal of quantification is to measure the number of antigens or ligand binding sites on a cell, this task is carried out by measuring the number of antibodies bound per cell (ABC). In 1998, a special issue of the journal Cytometry was dedicated to the topic of “Quantitative Fluorescence Cytometry: an Emerging Consensus” (1) which provided a comprehensive status report on the quantification effort. There was a consensus that fluorescence signal should be quantified in terms of molecules of equivalent soluble fluorochrome (MESF). It was also generally agreed in the community that the current goal is to develop practical, reliable, and yet inter-laboratory comparable methods for quantifying the number of fluorescent antibodies or other biomolecules bound on cells.

Five QFC methods have been developed and used for quantification including MESF FITC microsphere calibration, the use of Quantibrite calibration beads, quantitative indirect immunofluorescence assay (QIFI), quantum simply cellular (QSC) bead method, and the use of CD4 on normal lymphocytes as a biological calibrator (2, 3). The first two methods are based upon the MESF concept. The assignment of MESF values relies on the equality of fluorescence yields from two solutions defined as the product of fluorochrome concentration and the molecular quantum yield (4). With the use of a set of microbeads, each with a known MESF value, the MESF value of an analyte of interest can be determined on the basis of a flow cytometric calibration curve generated by using the reference bead set (5). To obtain the ABC value of the monoclonal antibody (Mab) specific for the analyte, the MESF value or the effective fluorophore per antibody (F/P) ratio of the Mab is required. The ABC value is the ratio between the MESF value of the cells stained with labeled Mab and the MESF value of the labeled antibody. If the manufacturer could provide the MESF value or the effective F/P ratio for the conjugated Mab, it would be straightforward for users to calculate the ABC value for the conjugated Mab. This value is not available for most Mab, such as fluorescein-labeled Mab. Hence, it is not yet practical for the determination of ABC values with the use of fluorescein as the labeling fluorochrome. Quantibrite calibration beads (BD Bioscience), on the other hand, are a set of microbeads with different amount of immobilized phycoerythrin (PE) molecules. By using unimolar conjugates of PE to Mab (effective F/P ratio = 1), ABC values of PE-labeled Mab can be determined directly according to the calibration curves generated by utilizing Quantibrite beads.

Quantitative indirect immunofluorescence assay, QIFI, was first developed by Poncelet and Carayon (5). This method employs a series of microbeads coated with different known amounts of mouse immunoglobulin in an attempt to mimic cells coated with Mabs at saturating concentration. A second layer, composed of labeled goat anti-mouse IgG under saturating conditions, is then used to calibrate flow cytometers in terms of antibody binding capacity. By measuring fluorescence signals from biological cells incubated with saturating amounts of Mabs, followed by incubation with labeled secondary antibodies, ABC values can be determined from the calibration curves. Using the QIFI method, however, Bikoue et al. observed that different Mabs recognizing the same antigen could give different ABC values on the same cells (6). Keep in mind that different Mabs might recognize different epitopes of the same receptor and Mab binding could be monovalent or bivalent depending on the epitope (7).

The QSC bead method utilizes a series of four microsphere populations labeled with different amounts of goat anti-mouse or goat anti-human IgG. These microspheres are stained in parallel with the same fluorescently labeled Mab as cells. Each microsphere population will bind a known amount of Mab and hence, four populations produce a cytometric calibration curve for measuring the ABC value of cells. Similar to the QIFI method described earlier, different antibody clones directed against the same antigen molecule and even the same Mab conjugated with different fluorochromes could give different ABC values by using this method (8). In addition, an apparent endless avidity of QSC beads that seems unable to saturate may introduce further variability in quantitative fluorescence measurements (9).

In 1991, Poncelet et al. reported that CD4+ T cells from HIV-infected individuals bound consistently about 46,000 CD4 Mab molecules measured according to the calibration curve generated with cell lines expressing known amounts of CD5 molecules detected via radio-labeled CD5 Mab (5, 10). Since then, CD4+ expression on T cells has been suggested and implemented as a biological calibrator for quantification of other surface antigens (11). A similar CD4 expression level on fresh normal whole blood was also demonstrated by Davis et al. using unimolar Leu 3a-PE conjugate and Quantibrite PE beads (7). Nonetheless, measurements of CD4 expression depend on variables such as fixation conditions, antibody clones, fluorochrome and conjugation chemistries, and quantitation methods used. Because of these variables, CD4 expression levels in terms of ABC values reported in literature vary significantly (2, 6–8, 12–15). Resolution of these discrepancies would be useful.

In this study, we focus on CD4 ABC quantification by employing MESF-based methods including MESF FITC microspheres and Quantibrite PE beads. The use of Quantibrite PE beads and unimolar CD4-PE conjugates is a straightforward method for the determination of ABC values. Different sample preparation procedures are implemented for evaluating their effects on the ABC values. CYTO-TROL Control Cells, lyophilized lymphocytes, are used to determine the ABC values by the ratio of the MESF values of control cells stained with CD4-FITC Mab and the MESF value of CD4-FITC Mab itself. We compare the ABC value obtained for CYTO-TROL cells using MESF FITC microspheres calibration method with the value determined by using Quantibrite PE beads and unimolar CD4-PE conjugates. The ABC values for CD4 expression on normal T lymphocytes determined by using two MESF-based methodologies are also compared.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Heparinized normal donor samples were obtained from NIH Department of Transfusion Medicine. This sample source is exemption approved by the institutional review board. Monoclonal antibodies, CD4-PE unimolar conjugate (clone Leu-3a) (Catalog No: 340586, Lot: 16533) and Quantibrite PE Quantitation kits (Catalog No: 340495, Lots: 50351) were purchased from BD Biosciences (San Jose, CA).1 CYTO-TROL Control Cells (Catalog No: 6604248) and CD4-FITC Mab (T4 FITC, Catalog No: 6603862) are products from Beckman Coulter (Fullerton, CA). A pH-sensitive fluorophore, SNARF-1, succinimidyl ester, was obtained from Molecular Probes/Invitrogen (Carlsbad, CA). A set of microspheres labeled with different amounts of fluorescein molecules that is traceable to the NIST certified reference material, RM 8640 (16), was used for quantifying fluorescein fluorescence signals in terms of MESF values.

For labeling carrier protein-free unlabeled CD4 Mab with SNARF-1, succinimidyl ester, Mab in PBS went through buffer exchange to 0.1 M sodium carbonate buffer, pH 8.3, by using Pro Spin columns from Princeton Separations. (Adelphia, NJ). Conjugation of SNARF-1 to Mab was carried out according to the manufacturer-recommended procedure. After the labeling reaction, the spin column separation was performed twice to ensure the separation of free fluorophores from the labeled Mab and at the same time exchange the buffer to PBS, pH 7.2, 0.05% sodium azide.

The procedure for whole blood staining was described previously (17). Briefly, the whole blood washed with 1× PBS was stained with labeled antibodies for 30 min at RT. The cell suspensions were subsequently lysed with 1× FACS™ Lysing Solution (BD Biosciences). After washing twice with 1× PBS, the obtained leukocytes were resuspended in 0.5–1 mL of PBS either with 1% fixative (Formaldehyde, Electron Microscopy Sciences, Fort Washington, PA) or without the fixative serving as fresh whole blood samples. Mononuclear cells were obtained from the whole blood (30 mL) using the Ficoll Hypaque density gradient separation procedure [Lymphocyte separation medium (ICN Biomedical, Aurora, OH)] (18). A small amount of remaining red cells were removed by lysing with ammonium chloride (ACK Lysing Solution, Biowittaker). Mononuclear cells were stained with monoclonal antibodies for 30 min at RT. After washing twice with PBS, the stained cells were resuspended in 0.5–1 mL of PBS either with or without 1% fixative. For obtaining enriched cells, 100 μL of CD19 microbeads from Miltenyi Biotec. (Auburn, CA) was added to a 2 mL of whole blood sample and incubated for 15 min at 4°C followed by addition of a 15 mL of 1X PBS/0.5% BSA (w/w) and centrifugation at 450g for 10 min. The supernatant was carefully pipetted off and cell pellet was resuspended in 1× PBS. The cell suspension was subsequently applied to magnetic separation with the autoMACS™ Separator (Miltenyi Biotec, Auburn, CA). The staining procedure for the enriched cells via negative selection with labeled Mab is the same as that for the whole blood samples described earlier.

Labeling CYTO-TROL Control Cells with CD4-FITC Mab followed the manufacturer-recommended procedure. A 100 μL volume of reconstituted CYTO-TROL cells (from an original volume of 1 mL of cells plus reconstitution buffer) was stained with a 10 μL of CD4-FITC Mab. After incubation for 30 min at room temperature, 1 mL of PBS was added, and cells were analyzed using two different flow cytometers. For labeling CYTO-TROL cells with unimolar CD4-PE conjugates, a titration curve was first generated to ensure cell staining under saturation conditions. A 30-μL aliquot of unimolar CD4-PE conjugate was used for staining a 100 μL volume of reconstituted CYTO-TROL cells at RT for 30 min. After washing with PBS, the stained cells were resuspended in PBS. For the pH titration measurements, the control cells in PBS were spun at 600g for 5 min, and cells were then reconstituted in 0.5 mL of one of four buffer media: borate buffer (pH 9.0), PBS (pH 7.3), (2-[N-morpholino]ethane sulfonic acid) buffer solution (MES, pH 6.2), and sodium acetate buffer (pH 5.2).

The flow cytometric measurements were carried out using a FACSCalibur (BD BioSciences, San Jose, CA) and a custom-made research cytometer. “CellQuest” software and “QuantiCalc” software (BD BioSciences) were utilized in the BDB cytometer for data acquisition and analysis, respectively. For the measurements, lymphocyte populations were gated by using 2D side and forward scatter plots, and median log channel numbers obtained from fluorescence histograms were used for the determination of MESF values or number of PE molecules bound per cell. Back gating with CD3 versus CD4 was also used to ensure the inclusion of the total T-lymphocyte population.

The emission spectra and the MESF assignment of CD4-FITC Mab were performed by using a custom-made, calibrated spectrofluorimeter described previously (19). The sample cell was a semi-micro cuvette (Starna, Type 9F). The laser beam was focused in the middle of the cuvette using a lens with a focal length of 60 mm. Approximately, 0.5 mL of a solution (or suspension) was stirred using a small magnetic bar and used for the measurements. One potential problem associated with this sample cell is the change of illuminating intensity due to scattering by the microspheres with respect to the capillary sampling method reported previously (19). A few percent systematic reductions of measured MESF values are expected.

The assignment of MESF value of CD4-FITC Mab was carried out according to the published procedures (19) and based on the equality of the measured fluorescence yields of labeled antibody solution and fluorescein standard reference solution [dilutions of the standard reference material, SRM 1932, from NIST (20)]. With the assumption that the molecular absorptivity is the same for the two solutions, the equality of fluorescence yields is expressed as

  • equation image(1)

where Nfl and NMab refer to the molar concentrations of soluble fluorescein and labeled Mab, respectively, and ϕ denotes the fluorescence quantum yield for the two entities given in the subscript. Using the reference solution as the unit of fluorescence yield, a MESF value can be assigned to fluorescein-labeled Mab as

  • equation image(2)

The relationship between a MESF value and an ABC value per cell is given by

  • equation image(3)

In the case of unimolar PE- Mab conjugate, an ABC value is calculated as a ratio of the number of PE molecules bound per cell and the number of PE molecules per Mab which is one. The fluorescence properties of the PE molecule in PE-Mab conjugate were measured to be the same as the properties of cells stained with Mab through direct fluorometer comparison of cells and CD4-PE solution (7). With the known ABC value obtained through the use of unimolar CD4-PE conjugate, an effective F/P ratio of the CD4-FITC conjugate can then be determined by the ratio of the MESF value measured and the ABC value obtained [Eq. (4)]. This allows the comparison of the MESF value measured for CD4-FITC Mab [Eq. (2)] and the effective F/P ratio calculated [Eq. (4)].

  • equation image(4)

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Figure 1 shows the number of PE molecules bound per T-cell determined for numerous samples according to the calibration curves generated by using Quantibrite PE Quantification kits and with the use of unimolar CD4-PE conjugate. Three different sample preparation procedures were implemented including whole blood staining, Ficoll-Hypaque density gradient protocol producing mononuclear cells, and enriched cells via negative selection using CD19 Mab labeled with magnetic beads. In addition, the effect of sample fixation was explored. Fresh samples without fixation, in general, display higher ABC values than those with fixation when using the same sample preparation protocol. For instance, the mean ABC value of 48,321 (CV, 2.6%) for the fresh whole blood samples is higher than the value of 43,432 (CV, 3.6%) for the fixed whole blood samples. Under the same fixation condition, whole blood samples give higher values than mononuclear cells. The mean ABC value of 43,432 for the fixed whole blood samples is higher than the value of 36,596 (CV, 7.1%) for the fixed mononuclear cells. The latter went through more purification steps that likely affect the nature of ligand-receptor binding processes. The mean ABC values for fresh and fixed enriched cells are 45,480 (CV, 4.5%) and 43,336 (CV, 3.9%), respectively; both are very close to the values for the respective whole blood samples as expected. Importantly, the number of PE molecules bound per cell determined for fresh whole blood samples, 48,321, is consistent with the value, ∼48,000, reported by Davis et al., with the use of the same quantification method (7).

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Figure 1. The number of PE molecules per cell determined on the basis of calibration curves generated by using QuantiBrite PE Quantification kits and the use of unimolar CD4-PE conjugate. Three different sample preparation procedures were implemented including whole blood, mononuclear cells, and enriched cells by negative selection using magnetic CD19 Mab. Additionally, fixation effect was investigated through comparison of fresh and fixed sample measurements.

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CYTO-TROL Control Cells were stained with CD4-FITC Mab following the manufacturer-recommended procedure. On the basis of the calibration curves generated by using FITC-labeled microspheres traceable to NIST RM 8640, the mean MESF value of stained CYTO-TROL cells was determined to be 40,292 (CV, 4.0%) for six separate experiments. Moreover, by implementing the calibration curve attained with a series dilution of the fluorescein SRM 1932 (Fig. 2), we assigned a MESF value to CD4-FITC Mab. The equation shown in Figure 2 allows the determination of the equivalent fluorescein concentration, Nfl, in Eq. (1) given in the ‘Materials and Methods’ section using the measured fluorescence signal from CD4-FITC Mab solution (FS). With a known concentration of CD4 Mab provided by Beckman Coulter, the MESF value of CD4-FITC antibody was calculated to be 1.16 according to Eq. (2). This MESF value could inherit as much as 10–15% uncertainties because of variation in the concentration of CD4-FITC Mab. An accurate concentration could in principle, be determined by amino acid analysis (21). The ABC value for CD4-FITC Mab was then calculated to be 34,734 on the basis of Eq. (3) (Table 1). To make sure that CD4 receptor folding on CYTO-TROL cell surface is in a similar fashion as that in whole blood samples, we determined the ABC value of CYTO-TROL cells by using Quantibrite PE calibration method and unimolar CD4 PE conjugate. The ABC value is obtained to be 43,090 (CV, 2.6%) for five separate experiments (Table 1).

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Figure 2. A calibration curve attained on the calibrated spectrofluorimeter using a series dilution of the NIST fluorescein standard reference material, SRM 1932. The solid line is the linear fitting curve of the experimental data points (solid circles) via the expressed equation that relates measured fluorescence signal, FS, to soluble fluorescein concentration (C). This equation allows the determination of the equivalent fluorescein concentration for CD4-FITC Mab, which is pointed by the arrow intersected with y-axis.

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Table 1. ABC Values Obtained for CYTO-TROL Cells and Different Sample Preparations by Using Both CD4-PE and CD4-FITC Conjugates
SamplesABC
CD4-PECD4-FITCCD4-FITC revised 15% for pH effect
  1. ND means “not done.”

CYTO-TROL43,09034,73440,864
Fresh whole blood48,32122,15626,066
Fixed whole blood43,43217,87921,034
Fresh mononuclear cells42,00119,29222,697
Fixed mononuclear cells36,596NDND
Fresh enriched cells45,48022,05125,942
Fixed enriched cells43,33620,18723,749

It is well known that fluorescein displays pH-sensitive fluorescence (22, 23). Therefore, we speculate that as a first approximation, fluorescein molecules on the outer surfaces of lymphocytes experience the same pH microenvironment as fluorescein attached to the antibodies or in bulk solution. To verify this approximation, SNARF-1, a pH sensitive fluorophore (24, 25), was conjugated to the carrier protein-free unlabeled CD4 Mab in the same fashion as the FITC conjugation to the Mab. Figure 3 exhibits emission spectra of SNARF-conjugated CD4 Mab and whole blood stained with the labeled antibodies, both in 1× PBS, pH 7.3. On the basis of the ratios of the two fluorescence peaks at 582 nm and 645 nm, the pH values were estimated to be ∼8.0 for SNARF-labeled Mab and 7.1 for the antibody-stained whole blood, respectively. The pH value that fluorescein molecules are experiencing when attached to CD4 Mab is higher than the value of the buffer medium. On the other hand, the pH value that fluorophores are sensing on cell surfaces is slightly lower than that of the medium.

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Figure 3. The normalized solution emission spectrum of SNARF-1 conjugated unlabeled CD4 Mab and smoothed emission spectrum of a whole blood sample stained with SNARF-1 conjugated unlabeled CD4 Mab; both were measured in 1× PBS, pH 7.3.

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To obtain estimation as to the percentage decrease of the fluorescence signal due to the pH change at the cell interface, we performed these titration measurements using identical number of CYTO-TROL cells in the four different buffer media. Figure 4 shows the titration curve with respect to fluorescein solution. The titration curves for three brightly fluorescent FITC-labeled bead populations are also included. The signals at highest pH value, ∼9.0, are normalized to be 1. All curves trail a similar trend except that the signals from stained CYTO-TROL cells drop more severely at low pH. Based on these measurements, a ∼15% decrease of fluorescence signal is expected from single FITC-labeled CD4 Mab to CYTO-TROL cell stained with single CD4 antibody.

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Figure 4. The fluorescence signal relative to the signal at pH 9.0 as a function of the medium pH value: fluorescein solution (open square), No. 5 FITC-labeled microsphere population (solid circle), No. 4 microsphere population (solid square), No. 3 microsphere population (solid triangle), and CYTO-TROL cells stained with CD4-FITC Mab (diamond). The sigmoidal fitting curves are shown for fluorescein, No. 4 microsphere population, and stained CYTO-TROL cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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We further determined MESF values for numerous normal blood samples stained with CD4-FITC Mab following various sample preparation procedures. These values are given in Figure 5, showing trends similar to CD4 expression levels measured by using unimolar CD4-PE conjugate displayed in Figure 1. Fresh prepared whole blood gives the highest mean MESF value 25,701 (CV, 2.2%). The mean values for fixed whole blood and fresh prepared mononuclear cells are 20,740 (CV, 1.6%) and 22,379 (CV, 7.2%), respectively. As expected, the mean MESF value for fresh enriched cells, 25,579 (CV, 4.8%), is about the same as that of fresh whole blood samples. However, the mean value for the fixed enriched cells, 23,417 (CV, 3.6%), is slightly higher than the value for the fixed whole blood. Note that the mean MESF value of fresh prepared whole blood, 25,701, is lower than the mean value determined for stained CYTO-TROL cells (40,292).

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Figure 5. MESF values determined according to the calibration curves generated by using FITC-labeled microspheres traceable to RM 8460 and with the use of CD4-FITC Mab. Three different sample preparation procedures were implemented including whole blood, mononuclear cells, and enriched cells by negative selection using CD19 Mab labeled with magnetic beads. Fixation effect was also investigated through comparison of fresh and fixed sample measurements.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The mean number of PE molecules bound per cell determined by using unimolar CD4 PE conjugate is 48,321 for fresh prepared whole blood samples (Fig. 1). This value is about the same as the value reported by Davis et al. (48,000) who employed the same calibration methodology (7). In addition, this value is consistent with the value, 46,000, published by Poncelet et al. according to the calibration curve generated with cell lines expressing known amounts of CD5 molecules detected by radio-labeled CD5 Mab (9). The use of two different QFC methods for obtaining similar ABC values enhances the measurement confidence in quantification of CD4 expression. Considering that the use of unimolar CD4-PE conjugate is critical in the present method, the reliability of the nominal 1:1 molar ratio of the conjugate must be ensured by the manufacturer. This study also demonstrated that fixation and additional sample preparation steps clearly resulted in lower ABC values (Fig. 1). This suggests that fresh and fixed cell preparations should be compared when attempting antigen quantification. It also implies that if the CD4 lymphocyte is to be used as a biological control to normalize a given calibration curve, a fresh cell preparation is needed. In fact, fresh preparations can serve as references and may be used to assign values to the fixed preparations. The authors realize that it may not always be possible to use fresh clinical samples due to concerns about the use of hazardous materials. However, it should be possible to at least explore the relationship between fresh and fixed values using normal blood bank samples.

We have used CYTO-TROL Control Cells stained with CD4-FITC Mab for quantifying CD4 expression level on T cells by means of a FITC MESF microsphere calibration curve. The mean MESF value determined is 40,292 according to the calibration curves generated using FITC microbeads traceable to RM 8640. By dividing the MESF value of 1.16 assigned to CD4-FITC Mab, the ABC value is calculated to be 34,734 (Table 1). This value is smaller than the ABC value, 43,090, determined for CYTO-TROL cells stained with unimolar CD4 PE conjugate by using Quantibrite PE calibration method.

One of the likely reasons for the lower value could be related to the pH effect since fluorescein molecules display pH-sensitive fluorescence (22, 23). To verify this speculation, the pH-sensitive SNARF-1 fluorophore was conjugated to carrier protein-free unlabeled CD4 Mab and used to stain CYTO-TROL cells. The solution emission spectra of SNARF-conjugated CD4 Mab and the whole blood stained with the labeled antibodies (Fig. 3) show the pH difference between the two microenvironments surrounding the SNARF fluorophore. Their pH values differ by ∼1 unit, i.e., ∼pH 8.0 for SNARF-labeled Mab and pH 7.1 for the antibody-stained whole blood, respectively. To estimate the percentage drop in fluorescence signals due to this pH change, we performed titration measurements using CYTO-TROL Control Cells stained with CD4-FITC Mab. As shown in Figure 4, the fluorescence signal of stained CYTO-TROL cells decreases by about 15% from pH 8.0 to pH 7.1. If the MESF value of CD4-FITC Mab were lowered by 15%, the ABC value of CD4-stained CYTO-TROL cells would be 40,864 by using Eq. (3) instead of 34,734 (Table 1). This value is reasonably close to 43,090 determined by using Quantibrite PE quantification method. The results show that the ABC values are fairly independent of the type of fluorophore used for this cell preparation.

An equally important consideration is that FITC conjugation generally reduces antibody affinity more than unimolar PE conjugation. This is due to the higher number of FITC fluorochromes per antibody molecule (∼5 fluorophores per antibody), which alters the binding kinetics. Lower binding affinity of FITC-labeled antibodies could result in lower ABC values relative to the unimolar CD4-PE conjugate. Additionally, it's also likely that fluorescein fluorescence quenching occurs because of their vicinity to the cell surface (26) and proximity to each other (27). The PE molecule, on the other hand, is composed of 30 fluorophores (phycoerythrobilin and phycourobilin) that interact closely among themselves and preserve a high fluorescence quantum yield (28). This fluorophore has a molecular weight of 243 kD and is about twice as big as an antibody molecule (150 kD). Therefore, fluorescence quenching due to the vicinity of cell surface and amino acids of the antibody is much less likely; nevertheless, the spatial effect may exist because of the large size of the PE molecule. Ideally, radio-labeled Mab would have a minimal perturbation on the affinity binding interaction between surface antigen and Mab (5).

An additional reason for the lower ABC value determined by means of the FITC microbead calibration method with respect to Quantibrite PE Quantification methodology (Table 1) could be due to the refraction index difference in Quantibrite PE beads and FITC MESF beads. These two types of microbeads are composed of different polymers and both are different from blood lymphocytes. Because of the difference in the index of refraction, we performed MESF assignments on one Calibrite™ FITC bead population relative to the No. 4 FITC-labeled microsphere population (RM 8640) by means of the calibrated spectro-fluorimeter and flow cytometer measurements. The polymer composition for Calibrite beads is similar to that of Quantibrite beads. The two MESF values obtained for this Calibrite bead population differ by less than 10%, suggesting that the refraction index parameter in this case plays a minor role in the two measurement methodologies. It is well known that the fluorescence signal depends on the electric field intensity at the surface of the microsphere. We calculated the electric field intensity at the distance of 10 nm from the surface of a sphere of radius equal to 7 × 10−6 m using the application “Near Field IE 2.00” from Valley Scientific, Inc. The intensity was integrated over the surface of the microsphere and compared to the integrated intensity of a sphere with the same index of refraction as the medium (this corresponds to the case of a homogeneous material). For spheres of index of refraction n= 1.45 and n = 1.55, the difference in integrated intensity was less than 1%. This relatively small difference in integrated intensity would not yield perceptible results in the resulting fluorescence signal from microspheres made with poly(methyl methacrylate) and latex.

Because of the facts discussed earlier, it seems more reasonable to use an effective F/P ratio than the assigned MESF value of the antibody for the determination of the ABC value according to Eq. (3). In principle, with a known ABC value for a specific surface receptor that is established by different quantification methods, one could determine the effective F/P ratios of labeled Mabs from different vendors by using Eq. (4). In the present study, the effective F/P ratio for CD4-FITC Mab is calculated to be 0.54 when we divide the mean MESF value of 25,701 measured for the normal blood samples (Fig. 5) by the generally agreed ABC value of 48,000 for CD4 expression. This ratio value (0.54) is only half of the MESF value (1.16) determined by implementing rigorously MESF assignment procedure. This mathematically assigned MESF value of the Mab is referred as the effective F/P ratio obtained by cuvette fluorimetry in the CLSI guideline document (29). By binding to a microbead for the determination of the effective F/P ratio of CD4-FITC conjugate used, nonetheless, Vogt et al. have obtained results consistent with the ∼48,000 ABC consensus value (30). It is therefore, more appropriate to measure the fluorescence of a conjugate bound to a biological calibrator than the solution fluorescence of the conjugate for the determination of an effective F/P ratio of the conjugate.

The ABC value of 43,090 obtained for unimolar CD4 PE-stained CYTO-TROL cells is reasonably close to the value of 48,321 obtained for fresh whole blood. This suggests that the effects of lyophilization are negligible. However, a revised ABC value of 40,864 for CD4-FITC Mab labeled CYTO-TROL cells versus 26,066 for fresh whole blood (Table 1) was unexpected and more difficult to explain. In other words, why are the PE-labeled CYTO-TROL cells and fresh whole blood in reasonable agreement, whereas the FITC-labeled CYTO-TROL cells and fresh whole blood are so different. Although FITC conjugation as discussed earlier undoubtedly changes antibody affinity, the most likely explanation for this close to 2-fold difference seems to be attributable to the surface interaction of the CD4-FITC Mab and CD4 antigen receptor on fresh whole blood cells. The result of this surface interaction is thought to cause fluorescence quenching as the major explanation for this observation. We are aware that the CD4 PE and CD4 FITC clones used in this study are not identical. However, the MESF values obtained for the BD CD4-FITC clone was even lower than the values for the Coulter Beckman clone (data not shown) further supporting our observations. Studies are underway to address this issue of quenching, valency, effects of lipid rafts, and the acidic glycocalyx loss on CYTO-TROL cells.

  • 1

    Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED
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