Antibody Binding Capacity Research Articles

Comparison of IgE reactivity of recombinant and natural pen a 1 by RAST and mediator release

Rationale Pen a 1 is the major shrimp allergen of Penaceus aztecus and has been identified as the muscle protein tropomyosin. It is responsible for the majority of IgE-mediated hypersensitivity to shrimp. Since recombinant proteins may be useful in diagnosis and treatment of food allergic individuals, recombinant Pen a 1 (rPen a 1) was cloned and expressed in E. coli. This study was performed to compare the immunological activity of rPen a 1 and natural Pen a 1 (nPen a 1) by RAST and mediator release. Methods Sera from six shrimp-allergic individuals with positive skin tests to shrimp extract were tested. RAST was performed using discs coated with whole shrimp extract, nPen a 1 (purified by preparative SDS-PAGE), and rPen a 1 at different concentrations (10.0 - 0.01 μg per disc). Peanut-coated discs were used as controls. Mediator release was performed by sensitizing humanized rat basophilic leukemia cells with human IgE from shrimp-allergic individuals. Results All six of the shrimp-allergic sera had RAST reactivity at all concentrations to both nPen a 1 and rPen a 1. Non-allergic control sera did not show reactivity. Linear regression analysis demonstrated identical IgE antibody binding capacity of rPen a 1 and nPen a 1. This was confirmed by the mediator release assay. Conclusion The use of rPen a 1 may have a role in determining shrimp-allergic individuals. Since this study is small, further serological testing should be conducted to confirm these results and skin testing with rPen a 1 should be performed.

Group-selective immunoassay for the detection of morphine in urine.

A new competitive inhibition immunoassay (group-selective immunoassay; GSI) has been developed to detect free morphine in urine with the Fab' fragments of monoclonal antibodies (MAbs) (1B(12)F(9)B(4), IgG(1), kappa, K(aff) = 9.66 x 10(10)M(-1)). At the first assay step, microtiter plates were coated with morphine-ovalbumin (M-6-S-OVA), in which free amino acids were protected by a glutaraldehyde cross-linking modification. The modification did not essentially influence the antibody-binding capacity of the immunosorbent. At the second assay step, anti-morphine MAbs' Fab' fragments, in which free amino groups were biotinylated by N-hydrosuccinimide-biotin ester, were bound to chemically modified immunosorbent. The biotin residues were then detected by the streptavidin-peroxide conjugate. This method has a sensitivity of 3.50 x 10(-15) mol/L using very little volume of sample, covering up to almost 1.20 x 10(-11) mol/L of standard concentration of morphine with good reproducibility. Standard curve prepared in urine indicated a good correlation between the concentration of morphine and the value of OD (y = 1/ax + b; r = 0.99939257, S = 0.01138127). Coefficients of variation for this immunoassay were 1.41 approximately 6.61% within-a-day assay and 2.31 approximately 8.99% between days assay. The recoveries were 94 approximately 101.4% from negative urine and 95.2 approximately 107.5% from positive urine samples, respectively. This method has application as a specific screen for morphine in drug abusers, to study the metabolism of the drug in the body, or to screen the monoclonal antibodies (MAbs) against morphine.

Magnetic cell separation: characterization of magnetophoretic mobility.

Magnetic cell separation has become a popular technique to enrich or deplete cells of interest from a heterogeneous cell population. One important aspect of magnetic cell separation is the degree to which a cell binds paramagnetic material. It is this paramagnetic material that imparts a positive magnetophoretic mobility to the target cell, thus allowing effective cell separation. A mathematical relationship has been developed to correlate magnetic labeling to the magnetophoretic mobility of an immunomagnetically labeled cell. Four parameters have been identified that significantly affect magnetophoretic mobility of an immunomagnetically labeled cell: the antibody binding capacity (ABC) of a cell population, the secondary antibody amplification (psi), the particle-magnetic field interaction parameter (DeltachiV(m)), and the cell diameter (D(c)). The ranges of these parameters are calculated and presented along with how the parameters affect the minimum and maximum range of magnetophoretic mobility. A detailed understanding of these parameters allows predictions of cellular magnetophoretic mobilities and provides control of cell mobility through selection of antibodies and magnetic particle conjugates.

Humoral immune response in aspergillosis: an immunodominant glycoprotein of 35 kDa from Aspergillus flavus.

A glycoprotein preparation containing 70% carbohydrates and 30% proteins was isolated from the mycelium of two strains of Aspergillus flavus and fractionated by ConA-Sepharose affinity chromatography. An immunodominant 35-kDa antigen was detected in a ConA-bound fraction (B fraction). It contained mannose and galactose in a 1.4:1.0 ratio. This antigen seems to be able to elicit an antibody response in patients with aspergillosis and in rabbits immunized with A. flavus whole cells. The carbohydrate units of the BF fraction appeared to be responsible for the antigenicity, since treatment with periodate removed most of the antibody binding capacity.

Tissue-dependent effects of immunization with a nicotine conjugate vaccine on the distribution of nicotine in rats

Vaccination of rats against nicotine reduces nicotine distribution to brain even at nicotine doses greatly exceeding the estimated binding capacity of the available antibody. This observation suggests a differential effect by which vaccination reduces nicotine distribution to brain to a greater extent than to other tissues. To test this hypothesis, vaccinated rats received a single intravenous nicotine dose equal to twice the estimated binding capacity of nicotine-specific antibody in vaccinated rats. The total and bound serum nicotine concentrations were higher in the vaccinated rats compared to controls, while the unbound serum nicotine concentration was lower. Distribution of nicotine to brain was reduced in vaccinated rats in a time-dependent manner, with a greater reduction at 1 min (64%) than at 25 min (45%). Vaccination reduced nicotine distribution to muscle, testis, spleen, liver, heart, and kidney, but to a lesser extent than to brain, while nicotine distribution to fat was increased. Chronically infused nicotine showed a similarly altered pattern of tissue distribution in vaccinated rats, but differences were in general smaller than after a single nicotine dose; brain nicotine concentration was 24% lower in vaccinated rats, while lung nicotine concentration was higher. The presence of nicotine-specific antibody in tissues may have contributed to the increased nicotine concentrations in fat and lung. These data suggest that vaccination reduces nicotine distribution to brain not only by sequestering nicotine in serum but also by redirecting tissue distribution disproportionately away from brain, such that nicotine concentrations are reduced to a greater extent in brain than in other tissues.

Combination hepatitis B immune globulin and lamivudine versus hepatitis B immune globulin monotherapy in preventing recurrent hepatitis B virus infection in liver transplant recipients

UNTIL RECENTLY, liver transplantation in patients infected with the hepatitis B virus (HBV) was associated with a high rate of graft loss and poor survival because of viral recurrence. With the use of hepatitis B immune globulin (HBIG), allograft rejection rates have decreased (15% to 50%). However, recurrences still occur due to the saturation of the antibody-binding capacity of HBIG by the high viral load or by mutations in the hepatitis B surface antigen (HBsAg) molecule that render HBIG ineffective. Lamivudine, a purine nucleoside analog that serves as a reverse transcriptase inhibitor, is known to be a potent inhibitor of HBV replication. Lamivudine, administered before and after liver transplantation to prevent graft reinfection, was found to produce a complete and sustained suppression of viral replication. However, lamivudine escape mutations in the YMDD locus of the HBV DNA polymerase have been reported more frequently. Following 52 weeks of therapy post–liver transplantation, Perrillo et al documented a mutation rate of 27%. Our long-term study showed that 62.5% of patients developed lamivudine resistance. The aim of the present study was to determine the efficacy of the combination of HBIG and lamivudine compared with that of HBIG monotherapy to prevent recurrent HBV infection in patients undergoing orthotopic liver transplantation (OLT).

Modification of ISFETs with a monolayer of latex beads for specific detection of proteins

The so-called ion-step method is a novel potentiometric approach that can detect protein adsorbed onto the gate area of modified ion-sensitive field-effect transistors (ISFETs). In this report, a generic technology is described for immobilization of peptides and proteins to the ISFET gate in order to confer specific binding properties to the ISFET. For this, the surface of the ISFET was covered with a monolayer of Amino beads (diameter, 0.9 μm) followed by immobilization of protein ligands onto these beads. Amino beads are latex spheres that contain primary amino groups at the outer surface. Preactivation of the latex-bound amino groups with glutaraldehyde, and consecutive incubation with polylysine resulted in covalent immobilization of this polyamine, as revealed by ion stepping measurements. For ImmunoFET applications, human serum albumin (HSA) was immobilized onto the Amino bead-covered ISFETs, by passive adsorption but also by covalent coupling. Resulting devices were used for qualitative detection of α-HSA antibodies by means of the ion step method. The binding of antibody was very specific and fast (most of the binding was accomplished in 15 min) with signal yields up to 17 mV. Efforts to increase the antibody-binding capacity of the solid phase on the ISFET exploiting amino group activation (with glutaraldehyde or other homobifunctional cross linkers) before HSA coupling, did not improve signal yield. The bead technology described in this report is an easy, generic method for coating the ISFET with a solid phase that, using the ion-step method, can be applied to immunosensing.

Quantification of CD38 Expression in B-cell Chronic Lymphocytic Leukemia (B-CLL): a Comparison Between Antibody Binding Capacity (ABC) and Relative Median Fluorescence (RMF)

Quantification of CD38 Expression in B-cell Chronic Lymphocytic Leukemia (B-CLL): a Comparison Between Antibody Binding Capacity (ABC) and Relative Median Fluorescence (RMF)

Quantification improves the prognostic value of CD38 expression in B-cell chronic lymphocytic leukaemia.

Recent studies have shown that CD38 expressed as a percentage of the antigen positivity can predict prognosis and disease progression in patients with B-cell chronic lymphocytic leukaemia (B-CLL). The present study showed that quantification of CD38 expressed as antibody-binding capacity (ABC) improves the prognostic value of the percentage of CD38 positivity in B-CLL. In a cohort of 81 patients with B-CLL, a level of CD38 expression of > or = 30% and an ABC value of 250 proved statistically valid cut-off points to predict disease progression (% CD38: P=0.0027; ABC: P < 0.0001). There was a positive and significant correlation between the percentage of CD38 expression and ABC (r=0.7; P < 0.0001). There was a better discrimination of survival using ABC rather than percentage CD38 positivity (P < 0.0001 compared with P=0.0027). Only ABC predicted for survival in patients under 60 years of age (P=0.0076) or with stage A disease (P=0.0195). Both percentage CD38 and ABC discriminated between time to first treatment for all patients but only ABC predicted time to treatment for stage A patients (P=0.0004). In conclusion, CD38 positivity is an important prognostic factor in B-CLL. However, quantification of CD38 is superior to the percentage positivity and should be used clinically in conjunction with other variables of predictive value to identify B-CLL patients that are likely to progress.

Stabilised cellular immuno-fluorescence assay: CD45 expression as a calibration standard for human leukocytes

We first confirmed the precision of the quantitative indirect immunofluorescence (QIFI) test by demonstrating that blood lymphocytes from different individuals expressed CD45 leucocyte antigens at a very similar level (mean: 201×10 3 antibody binding capacity (ABC)/lymphocyte) with only minimal variation (CV% 2.5%). These values were maintained for 4 days in blood samples when kept at 20 °C, and for up to 14 days in samples fixed with TransFix. Using long-term stabilisation, after an initial drop of 10–15% the CD45 ABC/lymphocyte values remained stable at an 85–90% level for >1 year. These biological standards were used to check other quantitative IF techniques. The quantum simply cellular (QSC) method showed variable results (85–240%), and the QuantiBRITE method gave values as low as 30–40% of the expected values, indicating that biological standards such as CD45 ABC/lymphocytes are absolutely essential to check the performance of methods that claim to quantify immunofluorescence (IF). Next, these standards were used to establish the stabilised cellular immuno-fluorescence assay (SCIFA) as follows. The ABC×10 3/cell values established by QIFI on leucocyte populations such as lympho-, mono- and granulocytes were used to create calibration curves for the CD45 antigen and its isoforms CD45RA, -R0 and -RB. The same cell populations were then stained with monoclonal antibodies (MAbs) directly conjugated to different fluorochromes in order to translate the mean fluorescence intensity (MFI) values seen with the directly labelled reagents to values of ABC×10 3/cells. Using SCIFA with a triple-colour direct IF, the display of CD45 and its isoforms were quantitated on the ‘virgin’ or ‘unprimed’ (CD45RA +) and ‘primed’ (CD45R0 +) subsets in both the CD4 + and CD8 + T cell lineages. We also observed that the CD4 + and CD8 + T cells in transit between the ‘virgin’ and ‘primed’ subsets frequently displayed different levels of the CD45RA and -R0 molecules, pointing to the physiological variability of the CD45RA-R0 switching process. In conclusion, internal biological standards, with known stable expression of ABC/cell, should be used to evaluate IF staining patterns in a quantitative manner during routine investigations.

Evidence of an affinity threshold for IgE-allergen binding in the percutaneous skin test reaction.

Atopy is an aberrant immune response involving allergen-specific IgE production, though serum IgE concentration is not an entirely reliable diagnostic tool, particularly for epidemiological and genetic studies. There is no clear correlation between IgE and other indicators of atopy such as skin prick tests (SPT)s, and physiological associations are difficult to justify in cases with detectable IgE but negative SPT results. IgE reflects the number of molecules available to produce an atopic response, but the degree of the response is determined by the binding strength (affinity) between receptor-bound IgE and the allergen. We sought to determine if there was an association between binding affinity and SPT results in people with histories of atopy. Standard SPTs (whole allergen extracts) were administered to people with histories of sensitivities to ragweed and house dust mite. The concentrations and affinities of serum allergen-specific IgEs were determined using the purified allergens Amb a 1 and Der p 1. There was a positive correlation between weal area and allergen-specific IgE among SPT-positive donors. However, for those individuals with detectable amounts of allergen-specific IgE, there was considerable overlap of IgE values between SPT-positive and -negative groups. Among sensitized donors, IgE-allergen interactions were characterized by two or three specific reactions of very high affinity (K(A) range 10(8) -10(11) M). Negative SPT reactions were associated with lowered IgE binding affinities to major allergens. This delimited two groups with atopic disorders: specific IgE(+)/ SPT(+) and specific IgE(+)/SPT(-). The product of antibody affinity and concentration, which we define as antibody capacity (CAP = K(A) x IgE), is more informative with regard to describing allergen sensitivity than antibody concentration alone. Antibody binding capacity provides physiological evidence of atopy in some subjects who do not test positively by common methods and suggests an affinity threshold to produce a positive SPT reaction.

Analysis of Epstein-Barr virus (EBV) receptor CD21 on peripheral B lymphocytes of long-term EBV- adults.

Primary infections with EBV are rarely observed after the age of 20. Some individuals even remain seronegative all their lives. Previously, a lack of EBV receptors on B cells of persistently EBV- adults was described as a reason for long-term EBV-seronegativity. The present study examined the CD21 receptor status of 20 repeatedly EBV- healthy adults and 32 EBV+ volunteers by means of flow cytometry. CD21 molecules on the surface of CD19+ B cells were quantified using anti-IgG-coated microbeads. The percentage of CD19+/CD21+ B lymphocytes was slightly lower in the peripheral blood of EBV- donors, but the CD21 antibody binding capacity on CD19+ B cells showed no significant differences between EBV- and EBV+ adults. In vitro studies showed an equally good EBV transformability of peripheral B lymphocytes of EBV- and EBV+ donors. Since HLA-DR was recently described as a co-receptor for EBV infection of B cells, we also determined HLA-DRB1 alleles in the EBV- group. We found a significant negative association of EBV-seronegativity with HLA-DR13 in comparison with 111 healthy blood donors. In summary, a biologically significant lack of the EBV receptor CD21 on peripheral B lymphocytes of persistently EBV- adults was excluded as a reason for long-term EBV-seronegativity.

CD34 and CD117 are overexpressed in AML and may be valuable to detect minimal residual disease

We estimated by quantitative flow cytometry (FC) the expression of CD13, CD33, CD34 and CD117 antigens in cells from 64 patients with acute myeloid leukaemia (AML) and 22 normal bone marrows (BMs). The method converts fluorescence intensity into number of antigen molecules per cell, measured by antibody binding capacity (ABC). The number of molecules per cell in normal BM was 9.5±5.7 for CD13, 7±2.3 for CD33, 6±0.7 for CD34, and 6.3±1.5×10 3 for CD117. AML blasts expressed 11.4±12.4 molecules per cell for CD13, 9.5±9.7 for CD33, 74±2328.5 for CD34 and 12.5±33×10 3 for CD117. The number of CD34 and CD117 molecules were significantly higher in AML than in normals ( P<0.0001 and P<0.05, respectively) while only in a few cases, CD13 and CD33 were abnormally expressed in myeloblasts. Our results indicate that quantitative analysis of CD34 and CD117 may be useful to detect minimal residual disease (MRD) and could be tested in a future to monitor therapy in AML.

Skin Test Results but not Serology Reflect Immediate Type Respiratory Sensitivity: A Study Performed with Recombinant Allergen Molecules

The diagnosis of type I allergy, an IgE-antibody-mediated hypersensitivity disease affecting more than 25% of the population, is based on the measurement of allergen-specific serum IgE levels and provocation testing. Whether the determination of allergen- specific serum IgE levels can replace in vivo provocation testing for allergy diagnosis is a controversial issue. We used purified recombinant timothy grass and birch pollen allergens to compare by skin prick and nasal provocation testing as well as by serology in vivo sensitivity with antibody-binding capacity in 24 pollen allergic patients and eight control individuals. Results from biologic tests were correlated with each other and with allergen-specific IgE and IgG1-4 levels. IgE-reactive allergens induced immediate skin and nasal reactions, but the intensity of the allergic tissue reactions was not correlated with either the levels of allergen-specific IgE or the levels of allergen-specific IgG antibodies. Less frequently detected allergens with low IgE-binding capacity were able to induce strong allergic reactions comparable to those caused by major allergens with high IgE-binding capacity. In contrast, skin test and nasal provocation results were significantly correlated (r = 0.63, p < 0.01). Our study thus demonstrates on a molecular level that skin testing provides a better reflection of immediate type respiratory sensitivity than serologic measurements. These results have implications for allergy diagnosis and, in particular, for the selection of relevant allergen components for specific immunotherapy.

Flow cytometric analysis of the association between blood group-related proteins and the detergent-insoluble material of K562 cells and erythroid precursors.

The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100-treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM-associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.

Measurement of CD2 expression levels of IFN-?-treated fibrosarcomas using cell tracking velocimetry

A methodology and a mathematical relationship have been developed that allow quantitation of the expression levels of cellular surface antigens, in terms of antibody binding capacities (ABC). This methodology uses immunomagnetically labeled cells and calibration microbeads combined with cell tracking velocimetry (CTV) technology to measure magnetophoretic mobilities corresponding to cellular ABC. The mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads and cells in a nearly constant magnetic energy gradient. Transformed fibrosarcoma cells were given controlled treatments of interferon-alpha in order to manipulate CD2 antigen expression levels. These cells were then immunomagnetically labeled with anti-CD2 FITC antibodies and anti-FITC MACS paramagnetic nanoparticles. Measured magnetophoretic mobilities were used to calculate ABC for these cells, corresponding to CD2 expression levels. The results from CTV and flow cytometry (FCM) qualitatively verify that these fibrosarcoma cells express elevated levels of CD2 molecules with increasing interferon-alpha treatment from 0 to 24 h. The mean basal CD2 expression level, in terms of ABC, was calculated to be 27,000 from CTV analysis, whereas FCM indicates a comparable ABC value of 33,000.

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Quantitative analysis of XG blood group and CD99 antigens on human red cells.

CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantitative polymorphism co-regulated with the Xga blood group polymorphism. The co-expression of X-linked MIC2 and XG genes is presumably controlled at the transcriptional level by a single XGR locus in the pseudoautosomal region of sexual chromosomes. This locus is composed of two alleles, XGR(low) and XGR(high), which determine low or high CD99 levels (CD99-L, CD99-H) and the Xg(a-)/ Xg(a+) status. To test this hypothesis, the phenotypic relationship between Xga and CD99 antigens on human RBCs was investigated by quantitative flow cytometry using NBL-1 (anti-Xga) and 12E7 (anti-CD99) monoclonal antibodies and semi-quantitative estimate of membrane proteins and RNA by Western blot and Northern blot, respectively. The antibody binding capacity of RBCs, which is an estimation of the antigen density, was determined for 118 blood donors including 60 males and 58 females. Xg(a+) RBCs, which all belong to the group of CD99-H expressors, carry 159+/-13 and 960+/-50 copies of Xga and CD99 molecules/cell, respectively. Xg(a-) RBCs have no Xga antigen, but are subdivided into CD99-H (all male) and CD99-L expressors carrying 747+/-28 and 200+/-22 CD99 copies/cell, respectively, with identical CD99 levels between CD99-L males and females. However, among males, the CD99 expression was higher in Xg(a+) than in Xg(a-)/CD99-H individuals (P<0.01). In addition, CD99-H expressors in Xg(a+) males could be clearly subdivided into two categories, high and super high expressors, which are presumably heterozygous and homozygous for the XGR(high) allele, which fits the above hypothesis. This was not the case for Xg(a+) females where CD99-H subcategories were not found. Quantitative differences were confirmed by Western blot analysis of red cell membrane preparations from individuals of different Xga and CD99 phenotypes and by Northern blot analysis showing that the reticulocytes from CD99-L individuals expressed a reduced level of MIC2 transcripts compared to CD99-H donors. These findings further support the hypothesis of a single genetic control of CD99 and Xga expression by the XGR locus.

Effect of cryopreservation on adhesion molecule expression in peripheral blood derived CD34+ cells

Homing of stem cells to the bone marrow after grafting is essential for hematopoietic recovery and supposedly depends on cellular adhesion molecules (CAMs). Since cryopreservation may affect expression of CAMs, members from integrin-(CD11a, CD49d), immunoglobulin-(CD54, CD58), selectin-(CD62L), and CD44 families were analyzed on CD34+ cells in fresh leukapheresis products (LKP) and after their cryopreservation. Two parameters were compared: the percentage of CD34+ cells with CAM expression, and the degree of CAM antigen expression; the latter can be estimated by quantitating the number of molecules of monoclonal antibody (MAb) that bind to the cell surface, and is called antibody-binding capacity (ABC). In fresh LKP 100% of CD34+ cells were positive for CD44 and CD58; the majority of CD34+ cells coexpressed CD11a, and CD49d; 73% coexpressed CD62L (L-selectin). CD54 was expressed with remarkable inter-individual variability. In thawed LKP the percentage of CD34+ cells coexpressing adhesion molecules CD54, CD11a was slightly (but not significantly) lower than in fresh samples. When the ABC is compared, generally a slightly lower expression of CAMs was observed. A marked difference was found for expression of CD62L; in the thawed LKP the surface antigen density and fraction of CD62+ cells was significantly reduced. Our results show that freezing-thawing process influences on qualitative change of the graft reflected by the decrease of CAMs on CD34+ cells, especially L-selectin. This might hamper homing, and possibly lead to suboptimal duration of aplasia.

Fluorescence Intensity Calibration for Immunophenotyping by Flow Cytometry

Fluorescence intensity (FI) is the basis for classifying phenotypes by fluorescence-label flow cytometry. FI is customarily recorded as an arbitrary relative value, but with proper calibration it can be expressed in stoichiometric units called molecules of equivalent soluble fluorochrome (MESF) that reflect the concentrations of the fluorescent conjugates and the receptors they stain. Forthcoming availability of authoritative standards and consensus methods will alleviate many of the difficulties encountered in making valid MESF measurements. FI calibration establishes the true values for the critical parameters of the fluorescence measurement, a useful feature for quality control. It further allows the establishment of a comparable window of analysis across different times and laboratories, and it permits numeric assessment of antibody-binding capacity (ABC) values in selected cell populations. The relation between ABC values and receptor expression is complicated by several factors, but careful assessment of the binding chemistry can establish the actual number of receptors on cells stained by fluorescent conjugates.