Protein G-gold complex: comparative evaluation with protein A-gold for high-resolution immunocytochemistry

We combined the protein G-gold complex with several polyclonal and monoclonal antibodies for localization of various antigenic sites. The labelings were compared with those obtained using the protein A-gold complex. The results from either the immunodot experiment or immunoelectron microscopy have demonstrated that, for rabbit and guinea pig antibodies, both protein G-gold and protein A-gold complexes label several different specific antibodies with similar efficiency. However, with antibodies raised in goats or in mice, and particularly with mouse monoclonal antibodies, protein G-gold yielded intense and specific labeling, whereas protein A-gold yielded intense and specific labeling, whereas protein A-gold was very variable; it either gave weaker signals or failed to reveal any specific site or, as with one monoclonal, both protein G and protein A gave similar results. The higher affinity and versatility of protein G over protein A, established by the immunochemical approach, was confirmed by immunocytochemistry. Because of its enhanced reactivity with monoclonal antibodies and its broader affinity for polyclonal antibodies, protein G-gold complex appears to be a better and more versatile probe for high-resolution immunocytochemistry.     

Protein AG-gold complex: an alternative probe in immunocytochemistry.

The purpose of this study was to evaluate the use of protein AG tagged with colloidal gold as a reliable immunocytochemical reagent. Protein AG is a recombinant of 47.3 KD molecular weight and pI = 4.3, which displays immunoglobulin Fc binding sites for both staphylococcal protein A and streptococcal protein G. It adsorbs to 10-nm colloidal gold particles with a lower affinity than does protein A, and is saturable. A maximal number of 12 protein AG molecules could be accommodated on the gold particle surface. Protein AG-gold conjugates yielded positive signals in post-embedding immunocytochemical assays when used as a secondary reagent in conjunction with several species and classes of polyclonal (rabbit, goat, sheep, guinea pig) and mouse monoclonal immunoglobulins (IgG1, IgG2, and IgG3). In addition, protein AG-gold was found to be a useful reagent in immunoblot analysis because of its ability to bind and identify nitrocellulose-immobilized IgGs (rabbit, mouse, goat, sheep, rat, and cow). Its spectrum of specificity towards various types of antibodies combines those of the parental protein A and protein G molecules. The protein AG-gold complex therefore appears to be a highly versatile and convenient alternative probe for immunochemical and immunocytochemical studies.     

Immune complex formation enhances the binding of staphylococcal protein A to immunoglobulin G

Staphylococcal protein A binds efficiently to the Fc region of goat immunoglobulin G antibodies only after they are immune complexed to immobilized, but not fluid-phase, polyvalent antigen (human myeloma immunoglobulin E protein) or monovalent hapten (methotrexate). Compared to fluid-phase or immobilized free immunoglobulin G, the reactivity of anti-immunoglobulin antibodies bound to solid-phase antigen was enhanced at least 300-fold. Results with immobilized methotrexate indicated that two molecules of immunoglobulin G must be bound in proximity to bind one molecule of protein A. Thus, aggregation appears to be a necessary condition for protein A binding.     

Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.     

T cell unresponsiveness to the mitogenic activity of OKT3 antibody results from a deficiency of monocyte Fc gamma receptors for murine IgG2a and inability to cross-link the T3-Ti complex

We recently identified defective monocyte accessory function as the cause of T cell unresponsiveness to the mitogenic activity of OKT3 antibody in cultures of peripheral blood mononuclear cells (PBMC) from five healthy subjects, members of one family. We now report that the underlying abnormality in nonresponders is at the level of monocyte Fc gamma receptors for murine IgG2a. T cell unresponsiveness was not restricted to the signal provided by OKT3 but occurred also for two other anti-T3 antibodies of the IgG2a subclass, in contrast to a normal proliferative response to IgG1 anti-T3 antibodies in one of the OKT3 nonresponders. By using cytofluorography, we found that monocytes from responders but not from nonresponders bound OKT3-FITC to their membrane. The binding could be blocked by mouse IgG2a and by human IgG, but not by mouse IgG1 nor by serum albumin. The data suggest that, through specific Fc gamma receptors for murine IgG2a, monocytes bind the Fc portion of OKT3 during T cell activation. The function of this Fc gamma receptor binding was further studied by culturing PBMC from nonresponders on plates coated with affinity-purified goat anti-mouse IgG antibodies as a substitute for monocyte Fc gamma receptors. The addition of OKT3 to nonresponder PBMC, cultured on such plates, resulted in T cell activation, as evidenced by thymidine incorporation, IL 2 production, and expression of IL 2 receptors. Soluble anti-mouse IgG was not able to substitute for monocyte Fc gamma receptors. The results demonstrate the existence of polymorphism in monocyte Fc gamma receptors for murine IgG2a. They also substantiate that an essential helper function of monocytes in T cell activation by anti-T3 is to provide a matrix for multimeric binding of the Fc portion of the anti-T3 antibodies in order to cross-link T3 molecules.     

A novel methodology for double protein A-gold immunolabeling utilizing the monovalent fragment of protein A.

A method for sequential protein A-gold immunolabeling is described whereby the binding of second gold probe to the first antibody-protein A-gold complex is reduced to acceptably minimal levels. Immunolabeling of thin sections of embedded pituitary tissue was used as a model system. After an initial immunolabeling for prolactin, sections were incubated in normal serum (rabbit) followed by a monovalent fragment of protein A. These latter two incubations reduced artifactual second gold probe label over prolactin-labeled secretory granules to minimal levels (much less than 1 particle per granule) when sections were subsequently immunolabeled with normal serum. The combination of normal serum and protein A fragment incubations saturates IgG and protein A binding sites on the first antibody-gold probe complex. The latter is thereafter unable to bind further IgG (and thus gold probe) because of the monovalent nature of the protein A fragment. It is suggested that this methodology may be extended to multiple immunolabeling procedures for electron microscopy. In addition, when used before single labeling this method may be an effective way to minimize nonspecific IgG binding in cases where the tissue or antibody under study may be a problem.     

Streptococcal Fc receptors. II. Comparison of the reactivity of a receptor from a group C streptococcus with staphylococcal protein A

The reactivity of a soluble Fc receptor from a group C streptococcus ( FcRc ) was compared antigenically and functionally with the staphylococcal Fc receptor, protein A. Protein A and FcRc were found to inhibit each others' binding to the Fc region of human IgG, indicating that they bind to sites that are in close proximity on the Fc region of human IgG. The two bacterial Fc receptors were antigenically unrelated. Differences were observed in the species and subclass reactivity of the two receptors. The patterns of binding of protein A and FcRc under various conditions suggested that these receptors reacted with distinct regions on the Fc region of immunoglobulins. FcRc bound more efficiently to goat, sheep, and cow IgG, protein A bound more efficiently to dog IgG, and neither receptor bound to rat IgG. Differences were also observed in the reactivity towards human IgG subclasses. The FcRc bound to all samples of the four human IgG subclass standards. Protein A bound to IgG1, IgG2, and IgG4, and to one of two IgG3 myeloma proteins tested. The reactivity of our soluble FcRc corresponds to a type III streptococcal Fc receptor classified by the reactivity of intact bacteria.     

The mitogenic activity of type III bacterial Ig binding proteins (protein G) for human peripheral blood lymphocytes is not related to their ability to react with human serum albumin or IgG

The mitogenic potential of bacterial IgG Fc binding proteins for human PBL is controversial. Wild type and recombinant type III IgG Fc binding proteins induce a wide spectrum of proliferative responses ranging from non-mitogenic to potent responses. To understand the reason for these differences, three recombinant forms of a type III IgG Fc binding protein derived from a single human group C streptococcal strain, 26RP66, were generated. Form I bound human IgG and human serum albumin, form II bound IgG alone and form III bound human serum albumin alone. These functionally distinct forms were compared with the corresponding wild type preparation from the same strain for mitogenic potential. A mitogenic response was induced only with the form I recombinant or the native wild type protein. These proteins shared the functional characteristics of binding human serum albumin and IgG. Mixtures of the IgG binding (form II) and human serum albumin binding fragments (form III) failed to reconstitute the mitogenic potential of the full length proteins. These results demonstrate that the type III IgG Fc binding protein has mitogenic potential for human PBL that is not related to its ability to react with human serum albumin or IgG.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Purification and some properties of streptococcal protein G, a novel IgG-binding reagent

Protein G, a bacterial cell wall protein with affinity for immunoglobulin G (IgG), has been isolated from a human group G streptococcal strain (G148). Bacterial surface proteins were solubilized by enzymatic digestion with papain. Protein G was isolated by sequential use of ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and affinity chromatography on Sepharose 4B-coupled IgG. The presence of protein G in various pools and fractions during the isolation was followed by their ability to inhibit the binding of radio-labeled IgG to G148 bacteria. A highly purified protein G was obtained. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the apparent m.w. was 30,000, and on agarose gel electrophoresis the purified protein gave rise to a single band in the alpha 1-region. Protein G was found to bind all human IgG subclasses and also rabbit, mouse, and goat IgG. On the IgG molecule, the Fc part appears mainly responsible for the interaction with protein G, although a low degree interaction was also recorded for Fab fragments. IgM, IgA, and IgD, however, showed no binding to protein G. This novel IgG-binding reagent promises to be of theoretical and practical interest in immunologic research.     

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.     

The gene sequence and some properties of protein H. A novel IgG-binding protein

The gene for protein H, a novel bacterial cell wall protein with specific affinity for human IgG Fc, was cloned from a group A Streptococcus and expressed in Escherichia coli. Recombinant E. coli cells produced two forms of a human IgG Fc-binding protein, one with an apparent Mr of 42 kDa in a periplasmic fraction and the other with an apparent Mr of 45 kDa in a mixed fraction of cytoplasms and membranes. Both 42-kDa and 45-kDa protein preparations similarly bound to human IgG1 to IgG4, human IgG Fc, and rabbit IgG, but not to IgG of mouse, rat, bovine, sheep, goat, and human IgA, IgD, IgE, and IgM. The complete nucleotide sequence of the cloned 1.8-kb DNA fragment was determined. An open reading frame encoded a hypothetical protein of 376 amino acid residues (Mr = 42,498). The N-terminal amino acid sequence, consisting of 41 residues, which was removed post-translationally had typical characteristics of Gram-positive bacterial signal peptides. Thus, the mature form of protein H was suggested to consist of 335 residues (Mr = 38,162). There were 3 repeated sequences consisting of 42 residues that were highly homologous to those of protein Arp, an IgA-binding streptococcal cell wall protein, and streptococcal M6 and M24 proteins. The C-terminal amino acid sequence consisting of 93 residues, directly following the repeated sequences, was also highly homologous to that of M6 and M24 proteins. No sequence homology was found between protein H and protein A or protein G, two other IgG-binding bacterial cell wall proteins.     

Determinant analysis and interaction studies on monoclonal antibodies to bovine IgG1 and IgG2.

Two mouse monoclonal antibodies SKb1 and SKb6 were prepared by fusion of myeloma cells with spleen cells of female Balb/c mouse immunized with a mixture of bovine IgG1 and IgG2. In radioimmunoassay, SKb1 bound specifically to IgG2 but SKb6 reacted with both IgG1 and IgG2 molecules. In the competition experiments, heavy chain isolated from bovine IgG could inhibit the binding of 125I-IgG1 and 125I-IgG2 to SKb6, while it failed to inhibit the binding of 125I-IgG2 to SKb1. The epitope reacting with SKb1 was found to be present not only on bovine IgG2 but also on goat IgG and was not present on IgG molecules isolated from the serum of rabbit, rat, sheep, horse, human and monkey. Similarly, the epitope reacting to SKb6 was found to be present on bovine IgG1 and IgG2 and also on IgG molecules isolated from goat and sheep serum but was absent in the IgG molecules isolated from the serum of rabbit, rat, horse, human and monkey. The association constants of the interactions of SKb1 with 125I-IgG2 and of SKb6 with 125I-IgG1 and 125I-IgG2, determined by Scatchard analysis, Steward-Petty plot and Sips plot, were found to be in the order of 10(8)-10(10) L/M. The association constants were determined at varying temperatures to obtain the thermodynamic parameters. The enthalpy (delta H0) and entropy (delta S0) values for the above antigen-antibody interactions were in the range of 9.15-15.96 kcal/mole and 36.96-41.15 eu/mole respectively. The heterogeneity indices for similar interactions determined by Sips equation were consistent with the expected values for binding of monoclonal antibodies with homogeneous protein determinants.     

A simple preparative procedure to extract and purify protein G from group G streptococci.

A rapid method for the solubilization of the bacterial type III Fc binding protein, protein G, from a group G streptococcus is described. Treatment of intact bacteria with cyanogen bromide results in the solubilization of a homogeneous Mr approximately 50,000 protein which retains IgG and human serum albumin binding properties. The solubilized protein could be purified to homogeneity by molecular sieving chromatography and retained all of the functional properties of the native protein.     

Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fc gamma RI).

The high affinity receptor that binds the Fc domain of immunoglobulin G (IgG) subclasses 1 and 3 (Fc gamma RI) mediates important immune defense functions by inducing cell surface changes on human leukocytes. In this article, we document direct high affinity binding of Fc gamma RI to the actin filament cross-linking protein, actin-binding protein (ABP). In the absence of IgG, all Fc gamma RI molecules in undifferentiated cells of myeloid line U937 bound to ABP over a 9-fold range of Fc gamma RI expression induced by human IFN-gamma. Binding of IgG to U937 cells constitutively expressing Fc gamma RI or to COS cells genetically transfected to express Fc gamma RI rapidly decreased the avidity of Fc gamma RI for ABP. This finding suggests the existence of a pathway communicating a signal between a functional IgG receptor and intracellular components involved in the effector responses to Fc gamma RI-ligand interaction.     

Gene for an immunoglobulin-binding protein from a group G streptococcus.

The gene (spg) for an immunoglobulin G (IgG)-binding protein from a Streptococcus clinical isolate of Lancefield group G was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and 5'-flanking sequences was determined. The DNA sequence includes an open reading frame which encodes a hypothetical protein of 448 amino acid residues (Mr = 47,595). The 5' end of this open reading frame encodes a sequence resembling a typical secretion signal sequence, and the remainder of the encoded protein has features reminiscent of staphylococcal protein A and of streptococcal M6 protein, including repeated sequences and a similar C-terminal structure. Aside from this C-terminal structure, the encoded protein has little direct amino acid sequence homology to either protein A or M6 protein. In E. coli, the cloned gene directs the synthesis of a protein which binds to immunoglobulins, including rabbit immunoglobulin, goat IgG, and human IgG3(lambda). Its binding properties are similar to those of the protein G described by Bj?rck and Kronvall (L. Bj?rck and G. Kronvall, J. Immunol. 133:969-974, 1984), a type III Fc receptor from a group G streptococcus.     

Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies

Protein G is an immunoglobulin (IgG)-binding bacterial cell wall protein recently isolated from group G streptococci. We have investigated the avidity of protein G for various monoclonal and polyclonal Ig of the IgG class, and compared it with the binding properties of protein A, the staphylococcal Fc-binding protein. Radiolabeled Ig were mixed with Sepharose-coupled protein G or protein A, and the amounts of radioactivity bound to the matrix-coupled bacterial proteins were determined. The avidity was found to be greater for protein G than for protein A for all examined Ig. Protein G bound all tested monoclonal IgG from mouse IgG1, IgG2a, and IgG3, and rat IgG2a, IgG2b, and IgG2c. In addition, polyclonal IgG from man, cow, rabbit, goat, rat, and mouse bound to protein G, whereas chicken IgG did not. The binding property of protein G was additionally exploited in the Western blot assay, in which iodine-labeled protein G was used successfully for the detection of a rat monoclonal antibody against ovalbumin, and for the detection of rabbit and goat polyclonal whole antisera against human urinary proteins. In these experimental situations, protein G was found to be a powerful reagent for the detection of IgG, and consequently the antigen against which these antibodies are directed.     

Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG

Various Gram-positive bacteria express different types of IgG-binding receptors, each of which displaying certain unique binding properties. To evaluate specificity and avidity aspects of the differential binding pattern, a set of competitive binding assays was employed, by using staphylococcal protein A (SPA), streptococcal protein G (SPG), and a chimeric protein AG. These receptors were analyzed, in a reciprocal fashion, for binding and inhibition of binding to a selected panel of polyclonal and monoclonal Ig. Results of the study reveal that a majority of the determinants on human and bovine IgG, recognized by SPA and SPG, are either coextensive or closely overlapping. Accordingly, a minor portion of the determinants appear to be unique in the sense that a particular determinant(s) is selectively identified by one of the two receptors. Binding assays involving purified Fc fragments from human IgG, suggest that SPG shows exclusive specificity for an Fab region determinant(s) not recognized by SPA, whereas the Fc determinants for SPA and SPG are identical or overlapping. Furthermore, one of the IgG subclasses of bovine origin appears to be seen by the SPG receptor only. The competition study also demonstrates that the novel chimeric protein AG receptor shows higher or equal avidity for variants of human IgG molecules compared to the best of its parental constituents. It can thus be deduced that chimeric receptors might be useful as optimized tools for immunologic applications.     

Characterization of the 2:1 complex between the class I MHC-related Fc receptor and its Fc ligand in solution.

The neonatal Fc receptor (FcRn) facilitates the transfer of maternal immunoglobulin G (IgG) to offspring and prolongs the half-life of serum IgG. FcRn binds IgG in acidic intracellular vesicles and releases IgG upon exposure to the basic pH of the bloodstream. The crystal structure of an FcRn/Fc complex revealed FcRn dimers bridged by homodimeric Fc molecules to create an oligomeric array with two receptors per Fc [Burmeister et al. (1994) Nature 372, 379-383], consistent with the 2:1 FcRn:Fc stoichiometry observed in solution [Huber et al. (1993) J. Mol. Biol. 230, 1077-1083; Sánchez et al. (1999) Biochemistry 38, 9471-9476]. Two distinct 2:1 FcRn/Fc complexes were present in the cocrystal structure: a complex containing an FcRn dimer interacting with an Fc and a complex in which single FcRn molecules are bound to both sides of the Fc homodimer. To determine which of the two possible 2:1 FcRn/Fc complexes exists in solution, we generated recombinant Fc molecules with zero, one, and two FcRn binding sites and studied their interactions with a soluble form of rat FcRn. The wild-type Fc with two FcRn binding sites binds two FcRn molecules under all assay conditions, and the nonbinding Fc with no FcRn binding sites shows no specific binding. The heterodimeric Fc with one FcRn binding site binds one FcRn molecule, suggesting that the 2:1 FcRn/wild-type Fc complex formed in solution consists of single FcRn molecules binding to both sides of Fc rather than an FcRn dimer binding to a single site on Fc.     

Rheumatoid factors may bear the internal image of the Fc gamma-binding protein of herpes simplex virus type 1

Affinity-purified rheumatoid factors (RF) from 20 patients with rheumatoid arthritis were tested for their reactivity with the mAb II-481 against glycoprotein E (gE), the Fc gamma-binding protein of HSV-1, as well as with a panel of mAb against human Fc gamma R. All RF bound to mAb II-481 in preference to mAb IV.3 (anti-human Fc gamma RII) or MOPC 141 (control mAb) which belong to the same IgG2b subclass. Five RF showed strong reactivity with II-481. No significant reactivity was observed between RF and mAb against human Fc gamma R. Non-RF human IgM did not react with any of the mAb. Clear-cut binding to II-481 was also seen with monoclonal IgM-RF derived from MRL/1 mice (mRF-2). The reaction between RF and II-481 was completely inhibited by human IgG. It was also inhibited by BHK cell extract infected with HSV-1, and with purified gE. II-481 inhibited the binding of human IgG Fc to the infected cell extract, confirming that II-481 recognizes the Fc-binding site on gE. II-481 did not react directly with human IgG or Fc of IgG. mAb to human IgG2 showed stronger binding to II-481 than to MOPC 141, suggesting II-481 has conformational similarity to human IgG H chain. These results suggest that at least some RF bear the "internal image" of HSV-1 Fc gamma-binding protein and support the hypothesis that some RF may be generated as anti-idiotype antibodies against antiviral antibodies.