IgG Binding Affinity Research Articles

Isotype modulates epitope specificity, affinity, and antiviral activities of anti–HIV-1 human broadly neutralizing 2F5 antibody

The constant heavy chain (CH1) domain affects antibody affinity and fine specificity, challenging the paradigm that only variable regions contribute to antigen binding. To investigate the role of the CH1 domain, we constructed IgA2 from the broadly neutralizing anti-HIV-1 2F5 IgG1, and compared 2F5 IgA2 and IgG binding affinity and functional activities. We found that 2F5 IgA2 bound to the gp41 membrane proximal external region with higher affinity than IgG1. Functionally, compared with IgG1, 2F5 IgA2 more efficiently blocked HIV-1 transcytosis across epithelial cells and CD4(+) cell infection by R5 HIV-1. The 2F5 IgG1 and IgA2 acted synergistically to fully block HIV-1 transfer from Langerhans to autologous CD4(+) T cells and to inhibit CD4(+) T-cell infection. Epitope mapping performed by screening a random peptide library and in silico docking modeling suggested that along with the 2F5 IgG canonical ELDKWA epitope on gp41, the IgG1 recognized an additional 3D-conformational epitope on the gp41 C-helix. In contrast, the IgA2 epitope included a unique conformational motif on the gp41 N-helix. Overall, the CH1 region of 2F5 contributes to shape its epitope specificity, antibody affinity, and functional activities. In the context of sexually transmitted infections such as HIV-1/AIDS, raising a mucosal IgA-based vaccine response should complement an IgG-based vaccine response in blocking HIV-1 transmission.

Chemical synthesis and evaluation of a backbone‐cyclized minimized 2‐helix Z‐domain

The Z-molecule is a small, engineered IgG-binding affinity protein derived from the immunoglobulin-binding domain B of Staphylococcus aureus protein A. The Z-domain consists of 58 amino acids forming a well-defined antiparallel three-helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three-helix bundle. The small size and the stable three-helix structure are two attractive properties comprised in the Z-domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein-based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z-domain and joining the N- and C-termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two-helix Z-domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N- and C-termini.

Megalin/Cubilin-mediated Uptake of FITC-labeled IgG by OK Kidney Epithelial Cells

In this paper, we characterize the uptake mechanism of fluorescein isothiocyanate-labeled human immunoglobulin G (FITC-hIgG) in opossum kidney (OK) epithelial cells, which have been shown to express megalin and cubilin. Confocal immunofluorescence microscopy showed the punctate expression of the neonatal Fc receptor FcRn in the cytoplasm, but not on the cell surface membrane. Temperature- and energy-dependent uptake of FITC-hIgG was observed at pH 7.4 but not at pH 6.0, indicating that the internalization of FITC-hIgG might not be due to FcRn, which has a binding affinity for IgG under acidic conditions. Under physiological pH conditions, human and bovine serum γ-globulin decreased FITC-hIgG uptake in a concentration-dependent manner. In addition, FITC-hIgG uptake was inhibited by various megalin and/or cubilin ligands including albumin, cytochrome c, transferrin and gentamicin. Endosomal acidification inhibitors (bafilomycin A(1) and chloroquine) significantly decreased the uptake of FITC-hIgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) decreased FITC-hIgG uptake. Potassium depletion and hypertonicity, conditions known to inhibit clathrin-dependent endocytosis, also decreased FITC-hIgG uptake. In contrast, caveolin-dependent endocytosis inhibitors (nystatin and methyl-β-cyclodextrin) did not decrease, but rather increased the uptake of FITC-hIgG. These observations suggest that the internalization of FITC-hIgG in OK cells might be, at least in part, due to megalin/cubilin-mediated, clathrin-dependent endocytosis.

A genome-wide association study identifies three new susceptibility loci for ulcerative colitis in the Japanese population

Ulcerative colitis is one of the principal forms of inflammatory bowel disease with complex manifestations. Although previous studies have indicated that there is a genetic contribution to the pathogenesis of ulcerative colitis, the genes influencing susceptibility to the disease have not been fully determined. To identify genetic factors conferring risk of ulcerative colitis, here we conducted a two-stage genome-wide association study and subsequent replication study using 1,384 Japanese individuals with ulcerative colitis and 3,057 control subjects. In addition to the expected strong association with the major histocompatibility complex (MHC) region, we identified three new susceptibility loci: the immunoglobulin receptor gene FCGR2A (rs1801274, P = 1.56 x 10(-12)), a locus on chromosome 13q12 (rs17085007, P = 6.64 x 10(-8)) and the glycoprotein gene SLC26A3 (rs2108225, P = 9.50 x 10(-8)). rs1801274 is a nonsynonymous SNP of FCGR2A that is reported to have a critical effect on receptor binding affinity for IgG and to be associated with other autoimmune diseases. Our findings provide insight into the molecular pathogenesis of ulcerative colitis.

IgG Subclass and the FcG Receptor Iia Polymorphism Associate to Dengue Fever, Dengue Hemorrhagic Fever and Asymptomatic Dengue Infection in Cuba

Background: FcG Receptors II (FcGRII) mediate a variety of biological response, including Antibodies Dependent Enhancement (ADE). The ADE theory shows that the presence of circulating dengue-specific IgG antibodies can enhance the viral entry trough FcGR and constitutes the largest risk factor for the individuals to develop of Dengue Hemorrhagic Fiver (DHF). An Arginine (R) to Histidine (H) substitution at position 131 of the FcGRIIa gene change the IgG binding affinity of the receptor with reduced opsonization of IgG2 associated with the arginine variant. The exceptional epidemiological circumstances in Cuba allow us to maintain a homogeneous sample with a similar history of natural dengue virus infections. In this work we investigate whether homozygosity for the arginine variant might be associated with a reduced risk of DHF caused by ADE and the subclass of IgG in the acute serum samples from individuals who suffered secondary infection to dengue 2 virus in the epidemic of Santiago de Cuba in 1997 with different clinical pictures. Methods: The Subclass of IgG were detected using Dot Blot in the soluble fraction and the immunocomplex fraction of the dengue acute serum samples kinetically collected. The DNA samples were studied to determine the polymorphism associated to Fc gamma receptor IIa. The FcGRIIa genotypes (R/R131, H/H131 and R/H131) and allelic frequencies were compared. Results: IgG1 are the predominant IgG subclass during the dengue acute infection even as a part of the immunocomplex or free in the serum. On the other hand IgG3 levels, only detected in the immunocomplex fraction, were observed in DF cases. The distribution of FcGRIIa genotype frequencies in Santiago de Cuba population showed the predominant RH genotype and the homozygocity for arginine variant did not found any protective effect associate to dengue infection in our population. Conclusion: The IgG subclass involved in dengue infection are IgG1 and IgG3 and the ADE phenomenon probably take place by the FcGRIIIa and FcGRIIIb associate with these IgG.

Ferrous Ions and Reactive Oxygen Species Increase Antigen-binding and Anti-inflammatory Activities of Immunoglobulin G

Polyspecific antibodies represent a first line of defense against infection and regulate inflammation, properties hypothesized to rely on their ability to interact with multiple antigens. We demonstrated that IgG exposure to pro-oxidative ferrous ions or to reactive oxygen species enhances paratope flexibility and hydrophobicity, leading to expansion of the spectrum of recognized antigens, regulation of cell proliferation, and protection in experimental sepsis. We propose that ferrous ions, released from transferrin and ferritin at sites of inflammation, synergize with reactive oxygen species to modify the immunoglobulins present in the surrounding microenvironment, thus quenching pro-inflammatory signals, while facilitating neutralization of pathogens.

Engineered Human IgG Antibodies with Longer Serum Half-lives in Primates

The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.

In vitro assay of biological and chemical toxins using antibodies against lethal toxin neutralizing factor

Lethal Toxin Neutralizing Factor (N-LTNF), MW 63.0 kDa, was isolated from opossum serum. After trypsin digestion, the active domain of N-LTNF was isolated and sequenced. The synthetic peptide consisting of ten amino acids was designated as LT-10. N-LTNF and LT-10 inhibited the lethality of animal, plant and bacteria toxins when tested on mice non-immunologically. The antibodies against N-LTNF and LT-10 only reacted immunologically with toxins and not with non-toxic substances. Anti-LTNF and anti-LT-10 reacted immunologically by ELISA test with toxins that were not detected by mouse test, such as cholera toxin and digoxin. Anti-LTNF and anti-LT-10 failed to react immunologically with non-toxic substances, such as nerve growth factor and collagen. Currently, mouse bioassay is in use for toxin detection and assay. Binding affinity of IgG from anti-LT-10 showed a linear relationship with mouse bioassay by ELISA detection limit to some toxins only. This may be due to the fact that anti-LTNF and anti-LT-10 detected the toxins that were not lethal to mouse. Thus, anti-LTNF and anti-LT-10 can be useful in assaying toxins as an alternative to mouse bioassay.

‘Morphs’ (MRFs): metal-reversible folding domains for differential IgG binding

Background: Selective recognition and binding of IgG molecules is the basis for a host of immunological and affinity purification techniques. Capture of an IgG in these procedures relies chiefly on its interaction with one of a variety of reengineered bacterial receptors which bind to the Fc region of IgG molecules with very high affinity. While this interaction is extremely efficient in trapping IgG molecules, the tight interaction between the binding partners often requires denaturing conditions for disruption of the complex, which can adversely affect the yield of purified IgG and also limit the lifetime of the receptor matrix. An effective receptor/IgG binding system which could be modulated by less extreme conditions is of considerable general interest. Results: We describe the properties of a series of modified Fc receptor domains which are competent to bind IgG with high affinity but which can be reversibly unfolded upon addition of modest amounts of transition metal ions. Data are presented demonstrating loss of the secondary structural content of the domains as a function of increasing metal concentration, with a concomitant decrease in IgG binding affinity. Variants of the Fc receptor differing at a single amino acid position display increased sensitivity to metal-induced unfolding, while retaining comparable IgG binding ability in the absence of metal. Conclusions: The interaction of this series of Fc receptors with metal ions abolishes IgG binding, but removal of metal ions allows refolding of the domains with restoration of the IgG binding function. Examples of the utility and potential applications of these metal-modulated IgG binding domains are discussed.

The FcgammaRIIIA-158V allele is a risk factor for ITP

In those patients with idiopatic thrombocytopenic purpura (ITP) that do not respond to standard therapy with corticosteroids and/or splenectomy, high-dose intravenous IgG or low dose anti-D is given to achieve transient increases in circulating platelet counts. The effect of these treatments is thought to be blockade of macrophage Fc receptors. Macrophages express FcγRIa, FcγRIIa and FcγRIIIa, all three classes may be involved in binding and phagocytosis of sensitized platelets. The FcγRIIa-131R/H and FcγRIIIa-158V/F polymorphisms influence the IgG-binding affinity of the receptors. In literature, skewing of distribution of the FcγRIIA alleles towards the low-affinity-genotype: FcγRIIA-131R/R has been described in ITP patients. Others did not confirm these findings. We genotyped a group of 83 patients with chronic ITP and 87 ethnically comparable hospital controls for the FcγRIIA, FcγRIIIA and FcγRIIIB polymorphisms, the latter Fc receptor is only expressed by neutrophils. In the ITP patient population, the gene frequency of the FcγRIIIA-158V allele was 0·55, vs. 0·43 in the control population (P = 0·001). The genotype distribution of the FcγRIIIA-V/F polymorphism was also significantly different from that of a control population (P = 0·006). The allele and genotype distributions of the FcγRIIA-131R/H or the FcγRIIIB-NA1,2 polymorphisms was similar in patients and controls. Our observations show that macrophage FcaRIIIa may be the most important receptor in clearance of sensitized platelets, or that FcγRIIIa-bearing cells (NK cells or macrophages) are implicated in the pathogenesis of this autoimmune disease.

Spectroscopic investigation of tertiary fold of staphylococcal protein A to explore its engineering application

Staphylococcal protein A is a cell wall constituent of most strains of Staphylococcus aureus, and it is characterized by its binding affinity to some immunological classes. A mutated low molecular weight type protein A (LPA; Mwt=27 kDa) which consists of the domains, E, D, A, B and 13 residues of the C-domain was prepared in this study. Since LPA does not possess a cell wall-bound region in contrast to wild-type protein A (WPA; Mwt=42 kDa), we have established a methodology of large scale purification of LPA without using any extracellular expression systems such as Escherichia coli. Using this relatively abundant protein, the immobilization of the LPA with silk fibroin of Bombyx mori was performed. Thermal stability of LPA immobilized with silk fibroin is higher than that of free LPA at high temperature judging from the immunoglobulin G (IgG)-binding affinity. However, the apparent value of its affinity decreased relative to that of immobilized WPA. These results indicate that structural information is essential to explore improvement of IgG-binding affinity of immobilized LPA. Therefore, secondary structure of free LPA was detected by its characteristic helical pattern in circular dichroism (CD) in aqueous solution. In addition to this, tertiary fold of four IgG-binding domains were investigated by two-dimensional 1H-NMR spectra. Four significantly high-field shifted cross-peaks attributed to methyl signals of alanine residues suggest that all four domains pack into a three helix bundle motif in solution. These structural data and properties of IgG-binding affinity suggest that spatial arrangement of four IgG-binding domains are packed into a compact globular molecular shape. This causes a certain active site of immobilized LPA to be buried in the silk fibroin fiber.

Molecular Modeling and Experimental Approaches toward Designing a Minimalist Protein Having Fc-binding Activity ofStaphylococcalProtein A

Protein A (PA), a cell wall constituent ofStaphylococcus aureus,has got the unique property of binding with the Fc fragment of IgG from various species. The sequence data indicate five highly homologous Fc-binding regions in protein A. Computer sequence analysis provided the tryptic and chymotryptic fragments of IgG-binding domains of protein A. Molecular modeling in conjunction with molecular mechanical calculation has been used to search for the smallest possible proteolytic fragments of PA, still retaining Fc-binding activity. A 20-residue peptide (typtic fragment) and a 16-residue peptide (chymotryptic fragment) have been indicated, by molecular modeling studies, to possess IgG-binding affinity comparable to that of the B domain of Protein A. Binding of a 20-residue peptide has been substantiated experimentally by immunoprecipitation, capillary electrophoresis, and circular dichroism spectroscopic analyses.

T FcRIIIA158F allele is a risk factor for systemic lupus erythematosus

Objective To study whether the FcγRIIIA-158V/F polymorphism, which affects IgG binding affinity, is a risk factor for systemic lupus erythematosus (SLE). Methods We genotyped a group of 70 Caucasian SLE patients for all known FcγR polymorphisms. Of this group, 45 patients (64%) had nephritis. In 35 patients, this diagnosis was confirmed by renal biopsy. Results In the total group of 70 SLE patients, the frequency of the FcγRIIIA-158F allele was 0.74, versus 0.57 in healthy controls (P = 0.003). The genotype distribution of the FcγRIIIA-158V/F polymorphism was also significantly different from that of the control population (P = 0.004). The distribution of the other FcγR polymorphisms-FcγRIIA-131R/H, FcγRIIIBNA(1,2), and FcγRIIIA-48L/R/H-was similar in SLE patients and controls. Conclusion In our group of SLE patients, only the distribution of the alleles of the FcγRIIIA-158V/F polymorphism was significantly different from that in the control group. This might indicate that macrophage expression of the FcγRIIIA-158F isoform is involved in the disturbed clearance of immune complexes in patients with SLE.

The Fc gammaRIIIA-158F allele is a risk factor for systemic lupus erythematosus.

To study whether the Fc gammaRIIIA-158V/F polymorphism, which affects IgG binding affinity, is a risk factor for systemic lupus erythematosus (SLE). We genotyped a group of 70 Caucasian SLE patients for all known Fc gammaR polymorphisms. Of this group, 45 patients (64%) had nephritis. In 35 patients, this diagnosis was confirmed by renal biopsy. In the total group of 70 SLE patients, the frequency of the Fc gammaRIIIA-158F allele was 0.74, versus 0.57 in healthy controls (P = 0.003). The genotype distribution of the Fc gammaRIIIA-158V/F polymorphism was also significantly different from that of the control population (P = 0.004). The distribution of the other Fc gammaR polymorphisms--Fc gammaRIIA-131R/H, Fc gammaRIIIB-NA(1,2), and Fc gammaRIIIA-48L/R/H--was similar in SLE patients and controls. In our group of SLE patients, only the distribution of the alleles of the Fc gammaRIIIA-158V/F polymorphism was significantly different from that in the control group. This might indicate that macrophage expression of the Fc gammaRIIIA-158F isoform is involved in the disturbed clearance of immune complexes in patients with SLE.

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein A and rabbit IgG.

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbent assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.

Information technology law: What does the future hold?

IgG responses are crucial in antiviral defence and instrumental for the serodiagnosis of infections. Fcγ receptors (FcγRs), which recognize the Fc-part of IgG, differ regarding their IgG binding affinity, IgG subclass preference, cellular expression profile and pathogen elimination mechanisms elicited upon activation. Assessing their activation in vitro is of fundamental importance, but technically difficult. Therefore, a novel assay for measuring antiviral IgG antibodies triggering activation of individual host Fcγ receptors was established. The assay comprises the co-cultivation of virus-infected target cells with immune IgG antibodies and mouse BW5147 hybridoma cells stably expressing chimeric FcγR–CD3ζ chain molecules consisting of the extracellular domain of human FcγRIIIA, FcγRIIA or FcγRI, respectively, fused to the transmembrane and intracellular domains of the mouse CD3ζ chain. Triggering of the chimeric FcγR receptors by immune complexes formed on the surface of IgG-opsonized virus-infected target cells resulted in FcγR activation leading to IL-2 secretion by BW5147 cells, which was quantified as a surrogate marker in an ELISA. Target cells infected with various human pathogenic viruses including herpes simplex virus type 1 (HSV-1), Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), measles virus (MV), and respiratory syncytial virus (RSV) or displaying human immunodeficiency virus-1 (HIV-1) gp120 evoke dose-dependent IgG responses demonstrating the universal applicability of the assay. Taken together, a new reliable and simple tool for measuring antibodies triggering activation of Fcγ receptors was established. This assay will be instrumental for defining novel correlates of IgG immunity and the design of new therapeutic IgGs.

The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region.

A family of chimeric immunoglobulins (Igs) bearing the murine variable region directed against the hapten dansyl linked to human IgG1, -2, -3, and -4 has been characterized with respect to binding to the human high affinity Fc gamma receptor, Fc gamma RI. Chimeric IgG1 and -3 have the highest affinity association (Ka = 10(9) M-1), IgG4 is 10-fold reduced from this level, and IgG2 displays no detectable binding. A series of genetic manipulations was undertaken in which domains from the strongly binding subclass IgG3 were exchanged with domains from the nonbinding subclass IgG2. The subclass of the CH2 domain was found to be critical for determining IgG receptor affinity. In addition, the hinge region was found to modulate the affinity of the IgG for Fc gamma RI, possibly by determining accessibility of Fc gamma RI to the binding site on Fc. A series of amino acid substitutions were engineered into the CH2 domain of IgG3 and IgG4 at sites considered potentially important to Fc receptor binding based on homology comparisons of binding and nonbinding IgG subclasses. Characterization of these mutants has revealed the importance for Fc gamma RI association of two regions of the genetic CH2 domain separated in primary structure by nearly 100 residues. The first of these is the hinge-link or lower hinge regions, in which two residues, Leu (234) and Leu(235) in IgG1 and -3, are critical to high affinity binding. Substitution at either of these sites reduces the IgG association constant by 10-100-fold. The second region that appears to contribute to receptor binding is in a hinge-proximal bend between two beta strands within the CH2 domain, specifically, Pro(331) in IgG1 and -3. As a result of beta sheet formation within this domain, this residue lies within 11 A of the hinge-link region. Substitution at this site reduces the Fc receptor association constant by 10-fold.

Immunoaffinity for IgG antibodies of protein A labelled with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid

Protein A was covalently labelled with 4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid in several molar ratios. The binding affinity for IgG and the delayed fluorescence of the corresponding complexes with Eu(III) were determined for each labelled compound. The immunoaffinity decreased slowly with increase in ratio of the label. A better immunoaffinity/labelling ratio was obtained by coupling protein A to the chelate via bovine serum albumin. A suitable ratio of chelating agent to protein A for producing a universal marker to be used in time-resolved fluoroimmunoassays can be determined.

Isolation of cDNAs for two distinct human Fc receptors by ligand affinity cloning.

Two cDNA clones encoding different but related receptors for immunoglobulin G constant domains were isolated from cDNA expression libraries by a ligand-mediated selection procedure ('affinity cloning'). Because both of the receptors encoded by the cDNAs react with CDw32 monoclonal antibodies, and both show the appropriate IgG binding affinity, both appear to be forms of the receptor formerly thought to be a single species called FcRII. The extracellular domains encoded by the isolated clones are closely related to the murine IgG2b/1 beta receptor extracellular domains, but the intracellular domains are unrelated. The receptors expressed in COS cells show a preference for IgG1 among IgG subtypes and no affinity for IgM, IgA or IgE. Abundant expression of the RNAs was detected in myeloid cell lines and placenta.

Measurement of platelet surface-bound IgG by a monoclonal 125I-anti-IgG assay.

We have described the use of a monoclonal 125I-labeled anti-IgG (125I-MA) to assay IgG antibody displayed on the surface of platelets from normal and immune thrombocytopenic patients and reported levels of IgG 10-100-fold lower than previous studies. This report describes the immunologic characteristics of the 125I-MA and the assay for surface IgG. The 125I-MA has a high binding affinity for surface-displayed IgG (2.22 X 10(9) M-1), reacts equally well with all four subclasses of IgG and not at all with IgM or IgA. In our assay, the binding of 125I-MA was found to be greater than or equal to 99% specific for IgG (no nonspecific association of 125I-MA with platelets) and the binding ratio of 125I-MA to IgG displayed on the cell surface was 0.91 (close to unity). Finally, platelet lysates were found to contain large amounts of IgG protein (39,597 +/- 27,418 molecules/platelet) as compared to surface-displayed IgG (124 +/- 86 molecules/platelet). This assay has excellent characteristics for quantitation of IgG on platelets and the discrepancy with other techniques may, in part, be due to intentional or inadvertent lysis of platelets during assay conditions.