Fab Portion Research Articles

Degradation of humoral host defense by Candida albicans proteinase.

The effect of an extracellular proteinase from the pathogenic yeast Candida albicans on the bactericidal and opsonizing activities of human serum was studied. The ability of human polymorphonuclear leukocytes to kill Staphylococcus aureus was greatly reduced when the bacteria were opsonized with human serum treated with the proteinase. The reduction in the opsonizing activity of human serum was attributed to degradation of the Fc portion of immunoglobulin G by the action of C. albicans proteinase as determined by immunoprecipitation reaction. However, the Fab portion of immunoglobulin G was resistant to proteolysis by the proteinase. A clear reduction in the bactericidal activity of human serum against Escherichia coli was observed when the serum was treated with C. albicans proteinase. The reduction of serum bactericidal activity was attributed to the degradation of complement C3 by proteolysis by the proteinase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while C5 resisted the action of the proteinase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteinase also degrades endogenous proteinase inhibitors, such as alpha 2 macroglobulin and alpha 1 proteinase inhibitor, which are involved in regulating inflammation. These results suggest that destruction of a host's defense-oriented or regulatory proteins facilitates debilitation of the infected host.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

The Third IgG-Binding Domain from Streptococcal Protein G: An Analysis by X-ray Crystallography of the Structure Alone and in a Complex with Fab

Protein G is a cell surface-associated protein from Streptococcus that binds to IgG with high affinity. We have determined the X-ray crystal structures of the third IgG,-binding domain (domain III) alone to a resolution of 1.1 Å (final R-factor of 19.3%), and in complex with an Fab fragment to 2.6 Å (final R-factor of 16.8%). The structure of domain III is similar to the lower-resolution crystal structures of protein G domais determined previously by other investigators, but shows some minor diffrence when compared with the equivalent NMR structures. Domain III binds to the immunoglobulin by formation of an antiparallel interaction between the second β-strand in domain III and the last bet2a;-strand in the CH1 domain. There is also a minor site of interaction between the C-terminal end of the α-helix in protein G and the first β-strand in the CH1 domain. Previous studies by NMR on the interactions between protein G and IgG have concluded that different portions of the protein G domain are involved in binding to the Fab and Fc portions. The results presented here support these findings and permit a detailed analysis of the recognition of Fab by protein G; formation of the complex buries a large water-accessible area, of a magnitude comparable with that found in antibody/antigen interactions. The majority of hydrogen bonds between the two protons involve main-chain atoms from the CH1 domain. The CH1 domain residues that are in contact with protein G are shown to be highly conserved in alignments of mouse and human γ chain amino acid sequences. We conclude that the binding site for protein G on Fab is relatively invariant across different species and γ chain subclasses, providing an explanation for the widespread recognition of Fab fragments from mouse and human antibodies by protein G. The solution of the crystal structures of domain III alone and bound to Fab has demonstrated that there is no major structural change apparent in either protein on formation of the complex.

Characterization of biosynthetic IgG oligomers resulting from light chain variable domain duplication

We have characterized a human IgG1 monoclonal antibody composed of altered light chains. Each light chain consists of two identical variable domains and a kappa constant domain, in association with a normal gamma chain. This antibody assembled biosynthetically into a mixture of stable oligomers and monomers. Employing gel filtration, PAGE, and electron microscopy, we examined the antibody and the nature of the associations involved in oligomer formation. By engineering a protease factor Xa site between the duplicated light chain variable domains and examining the fragments produced following factor Xa cleavage, we demonstrated the association of the IgG monomers occurred through their duplicated VL domains. Electron microscopy showed the oligomeric antibody to be predominantly dimers and trimers in which the monomeric units were associated through the tips of the Fab portion of the antibody, presumably through the protuding N-terminal VL domains. Similar examination of monomers demonstrated several molecular forms, including individual molecules with self-crosslinked Fab arms and others displaying the open Y and T shapes typically observed for IgG antibodies. The monomers also displayed distally protruding domain-like structures. The oligomers produced by this cell line therefore occurred through the nonconvalent interaction between the extra light chain variable domains.

Crystallization of two monoclonal Fab fragments of similar amino-acid sequence bound to the same area of horse cytochrome c and interacting by potentially distinct mechanisms.

The mouse monoclonal antibodies (mAb), 2E5.G10 and 1F5.D1, are specific for horse cytochrome c and appear to bind the same epitope, since their heavy (H) and light (L) chains are functionally interchangeable. Comparison of the amino-acid sequences suggests that slightly different interactions may be involved in antigen recognition. In addition, the H chains differ at only a few amino-acid residues from the H chain of a rat cytochrome c-specific mAb suggesting that specificity for one protein over another may be determined by these amino-acid differences. To address these possibilities, the three-dimensional structures of the Fab portions of the mAb bound to cytochrome c are being determined by X-ray diffraction analysis. Here we describe the preparation and crystallization of the two complexes with horse cytochrome c. The complex of the Fab fragment of 2E5.G10 with horse cytochrome c yielded crystals of X-ray diffraction quality under two sets of conditions; in both the space group was P2(1). The corresponding complex of 1F5.D1 under one of these conditions crystallized in the P2(1)2(1)2(1) space group. Three-dimensional X-ray data for these two complexes have been collected with nominal resolutions of 2.86 and 2.48 A, respectively.

Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency.

In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by the HER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amont of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185HER2 receptor.

Identification of PECAM-1 in solid tumor cells and its potential involvement in tumor cell adhesion to endothelium.

PECAM-1 (CD31/EndoCAM) is an adhesion molecule in the immunoglobulin supergene family that is expressed on endothelial cells, platelets, and some hematopoietic lineage cells. In this paper, using several polyclonal and monoclonal antibodies against PECAM-1, we identified PECAM-1 molecules on human, rat, and murine solid tumor cell lines. Immunocytochemical labeling and flow cytometric analysis using either polyclonal, monoclonal, or Fab portion of the antibodies against PECAM-1 detected a distinct distribution on tumor cell surface. Immunoblotting revealed proteins ranging from 120 to 130 kDa in tumor cells derived from different species. Immunoprecipitation and subcellular fractionation studies indicated that PECAM-1 is constitutively expressed on the surface of human tumor cells (i.e. colon adenocarcinoma). The specificity of a major polyclonal anti-PECAM-1 used in the current study (i.e. SEW-3) was confirmed by the preabsorption studies. PECAM-1 molecules on tumor cells appear to bear terminal carbohydrate moieties (i.e. sialic acid residues) different from those on platelets, since neuraminidase treatment of tumor cells, unlike platelets, did not result in a mobility shift. Polymerase chain reaction (PCR) analysis of genomic DNA derived from tumor cell lines of different species revealed the presence of PECAM-1 gene in the genome. The mRNAs of PECAM-1 in tumor cells were detected by reverse transcription-PCR followed by Southern hybridization. Screening of more than 20 human, rat, and murine solid tumor cell lines indicated that PECAM-1 is widely expressed, although the level of expression varies considerably among different cell lines. The expression of PECAM-1 message in tumor cells was confirmed by Northern blotting. DNA sequencing of the PCR fragment revealed that human tumor cell PECAM-1 matches 100% to the human endothelial cell counterpart. Finally, it was demonstrated that tumor cell PECAM-1 is involved in mediating tumor cell adhesion to endothelium, as evidenced by the ability of anti-PECAM-1 antibodies to decrease the adhesion of unstimulated tumor cells to microvascular endothelial cells.

Purification, structure and partial characterization of the major sperm activating protein complex in human serum

Serum has long been known to enhance sperm motility, and the major factor that mediates this activation has been shown to be a macromolecule complex with a MW of about 250 kD. This macromolecule, named Sperm Activating Protein (SPAP) has now been purified by a four step protocol. Starting with 100 mL of serum 20 to 200 micrograms of purified SPAP was recovered (N = 40). Studies on the identification and structure of SPAP revealed that the macromolecule is formed by an IgG molecule to which an apolipoprotein A-I molecule is bound. Further studies on the structure of the complex was performed by partial proteolytic cleavage of SPAP by pepsin and papain and interaction with poly- and monoclonal antibodies. Based on these results the most plausible structure of SPAP places one molecule of apo A-I between the two arms of the Fab portion of an IgG4 molecule. Human spermatozoa incubated with polyclonal anti-SPAP serum and a second FITC-labelled antiserum showed fluorescence at a distinct band at the lower part of the sperm head, indicating that SPAP acts by direct interaction with the spermatozoa. The SPAP molecule shows a new mechanism mediated by the immunoglobulins, that may be very important for male fecundity.

Spontaneous and antibody directed cytotoxicity of double-negative T cells from autoimmune mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice develop a syndrome similar to systemic lupus erythematosus in humans. This strain of mice is characterized by the progressive accumulation of CD4-CD8- (double-negative; DN) T cells which express increased levels of cell adhesion molecules such as CD44 and heat stable antigen (HSA). The DN T cells exhibited a higher level of spontaneous cytolytic activity and contained a higher level of serine esterase as compared with T cells of MRL/Mp-+/+ (MRL/+) mice. We also found that mAbs against CD44, Mel-14, CD45R, and HSA could augment the cytolytic activity of DN T cells of MRL/lpr mice. Antibody-mediated augmentation of cytolytic activity of DN T cells was due to conjugate formation in which the Fc portion of mAb bound to the Fc gamma receptor on target cells and the Fab portion of mAb bound to corresponding cell surface antigens on DN T cells. The antibody-mediated augmentation of cytolytic activity was not detected in T cells of MRL/+ mice and lymphokine activated killer (LAK) cells of C57BL/6 mice. In contrast, anti-CD3 mAbs could augment the cytolytic activity of DN T cells, T cells as well as LAK cells. mAbs against LFA-1 and VLA-4 failed to augment the cytolytic activity of three different effector cells. It should be noted that anti-CD3 mAb-mediated cytolytic activity of DN T cells was substantially reduced by anti-LFA-1 mAb. However, CD44, Mel-14, CD45R as well as HSA-mediated cytolytic activity of DN T cells was not inhibited by anti-LFA-1 mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and binding properties of a monoclonal anti-idiotypic autoantibody to anti-DNA with epibody activity.

We report the isolation and characterization of a mouse autoanti-idiotypic mAb (D7.4 IgG2a), which is directed against a major public Id (A52 IgG2b) in the murine and human autoimmune response to DNA. The natural anti-Id mAb has been produced in the course of the SLE-like disease in a female NZB/NZW F1 mouse and showed a dual specificity (epibody activity) for the public Id (A52) and for the autoantigen (DNA). The two binding activities were shown to reside in the Fab portion of the epibody and were highly specific for their respective Ag. A complete nucleotide sequence analysis of the D7.4 H and L chain V-region genes combined with computer comparisons to available Ig sequences may suggest a charge interaction between the H chain CDR3 segments of the Id and anti-Id antibodies. The D7.4 epibody may be a component of the self-binding, idiotypically connected network of natural antibodies. Alternatively, it could be elicited against the potentially pathogenic, DNA-containing immune complexes in order to facilitate their removal from the circulation of diseased individuals.

Crystal structure of a streptococcal protein G domain bound to an Fab fragment.

Protein G is a cell-surface protein from Streptococcus which binds to IgG molecules from a wide range of species with an affinity comparable to that of antigen. The high affinity of protein G for the Fab portion of IgG poses a particular challenge in molecular recognition, given the variability of heavy chain subclass, light chain type and complementarity-determining regions. Here we report the crystal structure of a complex between a protein G domain and an immunoglobulin Fab fragment. An outer beta-strand in the protein G domain forms an antiparallel interaction with the last beta-strand in the constant heavy chain domain of the immunoglobulin, thus extending the beta-sheet into the protein G. The interaction between secondary structural elements in Fab and protein G provides an ingenious solution to the problem of maintaining a high affinity for many different IgG molecules. The structure also contrasts with Fab-antigen complexes, in which all contacts with antigen are mediated by the variable regions of the antibody, and to our knowledge provides the first details of interaction of the constant regions of Fab with another protein.

Crystallization and preliminary X-ray analysis of the complex between a mouse Fab fragment and a single IgG-binding domain from streptococcal protein G.

The Fab fragment of a mouse immunoglobulin G1, complexed with a single IgG-binding domain from streptococcal protein G, has been crystallized in a form suitable for analysis by X-ray diffraction. The needle-shaped crystals were grown from polyethylene glycol 4000 using vapour diffusion methods and diffract to 2.3 A resolution. The space group is P2(1)2(1)2(1) (a = 64.5 A, b = 70.5 A and c = 120.1 A), with one Fab-protein G domain complex in the asymmetric unit. Solution of the three-dimensional structure of the complex will permit a detailed analysis of the molecular interactions between protein G and the Fab portion of IgG.

Localization of a sequence motif complementary to the nuclear localization signal in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy

A sequence motif complementary to the nuclear localization signal (NLS) has been localized in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy using sequence-specific antibodies. The antibodies were generated in two different ways: by immunization with a carrier-coupled peptide and by isolation of the sequence-specific antibody from an immune serum against native proteasomes using a peptide-affinity column. The sequence specificity of the isolated antibody was confirmed by a PEPSCAN-ELISA performed on overlapping nonapeptides deduced from the sequence of the α-subunit of the Thermoplasma proteasome. Compared to the antibody induced by the carrier-coupled peptide this antibody fraction showed a much higher affinity for native proteasomes. The attachment site of the Fab portion of the antibody to the proteasome was mapped by electron microscopy in conjunction with image processing. The antibody was found to bind to the periphery of the two outer “disks” of the proteasome complex formed by the α-subunits.

Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen

A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se 155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 μg/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.

Preliminary studies for an immunotherapeutic approach to the treatment of human myeloma using chimeric anti-CD38 antibody

Multiple myeloma is a disease in which conventional chemotherapy has only limited value, but which may be ideal for treatment with passive antibody against a suitable cell surface antigen on the neoplastic plasma cell. The CD38 antigen is known to be present on the majority of neoplastic plasma cells, and this was confirmed by detailed examination of bone marrow aspirates from three patients. Strong expression of CD38 was confined to cells which, by the criteria of light-scattering profiles and possession of cytoplasmic Ig, were plasma cells. The vast majority of neoplastic plasma cells appeared to be involved. Using a cell line as a model, it was found that the CD38 antigen acts as a target for a chimeric antibody prepared from the antibody OKT10. The chimeric antibody consists of the Fab portion of the mouse monoclonal antibody linked by a stable thioether bond to an Fc molecule derived from human IgG1, thereby forming mouse Fab-human Fc. In contrast to the parent antibody, the chimeric molecule mediates antibody-dependent cellular cytotoxicity (ADCC) very efficiently with human blood mononuclear effector cells, and is effective at low concentration. Also, even though the CD38 antigen is present on natural killer cells, there appears to be little deleterious action of the antibody on effector cell function. The antibody also failed to affect the growth of progenitor cells of the granulocyte/macrophage or erythroid lineages present in normal bone marrows, despite the suspicion that these cells express the antigen. Other advantages of the CD38 molecule are that it is not found in the serum of patients with myeloma, and it does not appear to modulate in vitro. Fourteen patients with florid myeloma and on various chemotherapeutic regimes had an undiminished capacity to mediate ADCC with the chimeric antibody, when compared with normal individuals. The maintenance of ADCC activity, coupled with the known suppression of the antibody response in these patients, augers well for treatment with chimeric antibody.

Preliminary Studies for an Immunotherapeutic Approach to the Treatment of Human Myeloma Using Chimeric Anti-CD38 Antibody

Multiple myeloma is a disease in which conventional chemotherapy has only limited value, but which may be ideal for treatment with passive antibody against a suitable cell surface antigen on the neoplastic plasma cell. The CD38 antigen is known to be present on the majority of neoplastic plasma cells, and this was confirmed by detailed examination of bone marrow aspirates from three patients. Strong expression of CD38 was confined to cells which, by the criteria of light-scattering profiles and possession of cytoplasmic Ig, were plasma cells. The vast majority of neoplastic plasma cells appeared to be involved. Using a cell line as a model, it was found that the CD38 antigen acts as a target for a chimeric antibody prepared from the antibody OKT10. The chimeric antibody consists of the Fab portion of the mouse monoclonal antibody linked by a stable thioether bond to an Fc molecule derived from human IgG1, thereby forming mouse Fab-human Fc. In contrast to the parent antibody, the chimeric molecule mediates antibody-dependent cellular cytotoxicity (ADCC) very efficiently with human blood mononuclear effector cells, and is effective at low concentration. Also, even though the CD38 antigen is present on natural killer cells, there appears to be little deleterious action of the antibody on effector cell function. The antibody also failed to affect the growth of progenitor cells of the granulocyte/macrophage or erythroid lineages present in normal bone marrows, despite the suspicion that these cells express the antigen. Other advantages of the CD38 molecule are that it is not found in the serum of patients with myeloma, and it does not appear to modulate in vitro. Fourteen patients with florid myeloma and on various chemotherapeutic regimes had an undiminished capacity to mediate ADCC with the chimeric antibody, when compared with normal individuals. The maintenance of ADCC activity, coupled with the known suppression of the antibody response in these patients, augers well for treatment with chimeric antibody.

Shared idiotypic determinant in mono- and polyclonal anti-phospholipid antibodies with lupus anticoagulant activity

Of 42 purified human myeloma proteins tested, two (IgG3 Her and IgM Mag) were found to possess strong lupus anticoagulant (LA) and anti-cephalin activity, as assessed by a dilute activated partial thromboplastin time (dAPTT) and ELISA test, respectively. For these proteins, we confirmed the observation reported by others that LA activity is present in the antigen-binding (Fab) portion of the immunoglobulin molecule. Rabbit anti-idiotype antibodies against IgG3 Her inhibited the anti-cephalin activity of this protein, suggesting that the anti-cephalin activity of IgG3 Her depends on the hypervariable part of the immunoglobulin and thus most probably is a true antigen-antibody reaction. The anti-Her idiotype antibodies were also able to bind to and inhibit the anti-cephalin activity of IgM Mag. ELISA binding and inhibition experiments showed that the anti-idiotype antiserum contained at least two sets of anti-idiotypes; one set that recognizes a cross-reactive idiotype shared by IgG3 Her and IgM Mag, and another set that seems to be unique to the immunizing protein IgG3 Her. Both sets of anti-idiotype antibodies also bound weakly to polyclonal (patient) IgG, indicating an idiotypic cross reaction.

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable plate-like crystals which belong to the space group P2 12 12 with unit cell constants of a = 66.5 A ̊ , b = 74.3 A ̊ and c = 105.3 A ̊ . There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 Å.

The specificity of antiglobulin autoantibodies in patients with primary Sjögren's syndrome

Antiglobulin autoantibodies have already been demonstrated in the sera of patients with primary Sjögren's syndrome (primary SS). In our study, the specificity of primary SS antiglobulins for different regions of IgG molecules was examined by employing both direct binding and competitive inhibition enzyme-linked immunosorbent assay. We found that a considerable amount of total antiglobulins in primary SS was specific for the Fab portion, although the remainder was specific for the Fc portion, namely rheumatoid factor (RF). In contrast, most of the antiglobulins in RA were specific for the Fc portion of IgG. These results indicate that in primary SS, antiglobulins directed against epitopes different from those of RF are produced. These antiglobulins may prove to have a different role in primary SS than that ascribed to RF in RA.

Antigenic studies on flocculating brewer's yeast, Saccharomyces cerevisiae NCYC 227

Batch cultures of a brewer’s strain of Saccharomyces cerevisiae, NCYC 227, in a defined medium exhibited characteristic flocculation during the late exponential phase of growth. However, early exponential cells were non-flocculent and flocculation of such cells could not be induced even in the presence of Ca2+. A specific glycoprotein, absent from the cell walls of non-flocculating exponentially growing cells of this yeast and those of non-flocculating strains of S. cerevisiae, was identified on the cell wall surface of flocculating exponentially growing cells of S. cerevisiae NCYC 227. Flocculent cells of S. cerevisiae NCYC 227 dispersed with EDTA and coated with monovalent Fab portions of the antibody showed reduced flocculation. Removal of the monovalent antibody portions from the cell surfaces induced cell flocculation in the presence of Ca2+. These results suggest a role for this glycoprotein in yeast cell flocculation.