Purification and Characterization of Membrane Protein (90 kDa) from Mycoplasma salivarium,Which Binds Immunoglobulin (Ig) G Fc Fragment

A 90 kDa protein of Mycoplasma salivarium was released from cell membranes of the organism with Triton X-100 and purified by ion-exchange chromatography and chromatofocusing. The protein was eluted at pH 5.5 by chromatofocusing. The protein was shown to react with the Fc fragments of IgG from human and nine different animal species and did not distinguish between IgG from different species, while protein A, tested for comparative purposes, displayed a strong specificity for human and swine IgG. Furthermore, the protein reacted with antigen specific goat IgG (specific for gamma chains of human IgG), sheep red blood cells (SRBC) sensitized with rabbit antiserum to SRBC, that is, the Fc part of rabbit IgG, and concanavalin A as well. These findings may suggest that the protein is a lectin which binds the carbohydrate moiety of the Fc part of IgG.     

Development of fluorescence change-based, reagent-less optic immunosensor

A reagent-less, regenerable and portable optic immunosensor was developed. A model sample, immunoglobulin G (IgG), was detected with this system based on changes in fluorescent intensity of fluorescent labeled protein A with specific reactivity to IgG depending on a reaction between the proteins. A glass plate immobilized with Qdot-labeled protein A was placed on the top of optic fibers designed for both excitation and fluorescence emission. The optic fibers with the Qdot-labeled protein A-immobilized glass plate were inserted into a solution of pH 7.4 phosphate buffered saline. After stabilization of the fluorescence intensity, IgG was added and the time-course of the fluorescence intensity was measured on a fluorometer connected with the optic fibers. Furthermore, the fluorescence response of a transient state was evaluated with the same system. When the Qdot-labeled protein A bound to IgG, fluorescence intensity decreased because of the inhibition by IgG. The degree of fluorescence decrease depends on the IgG concentration at a steady state and also in a transient state.     

A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties

Protein G, an IgG-binding molecule, was prepared from the cell walls of a group G streptococcal strain, G-148. The protein could be extracted from the cells by papain digestion and purified by the sequential use of ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Sepharose-coupled human IgG, and gel chromatography on Sephadex G-200. Two protein bands with similar molecular weight, 34,000 and 36,000, were obtained when analyzing the pure protein G on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield using this purification scheme was 27% of the protein G solubilized from the cells or 70 micrograms/ml packed bacteria. The Stokes radius and frictional ratio of protein G were determined to 3.53 nm and 1.64, respectively, suggesting an elongated fibrous molecule. The protein did not contain any intrachain disulfide bonds. The amino acid composition of protein G was determined and was found to be different from that of protein A, the well known staphylococcal IgG-binding protein. The equilibrium constants of the reactions between protein G and human, rabbit, mouse, and goat polyclonal IgG, determined by Scatchard plots, ranged between 1 X 10(10) and 7 X 10(10), for rat polyclonal IgG 1.4 X 10(9), and human monoclonal IgG1, IgG2, IgG3, and IgG4 between 2 X 10(9) and 6 X 10(9). These affinity constants were always greater than the corresponding values for protein A. The binding between protein G and various polyclonal and monoclonal IgG was pH dependent between 2.8 and 10, strongest at pH 4 and 5, and weakest at pH 10.     

Rabbits immunized with mixtures of staphylococcal protein A and autologous IgG produce anti-human IgG antibodies

The results of this study provide evidence that protein A may render IgG immunogenic in the autologous host. Antibodies to human but not rabbit IgG were detected in sera of rabbits immunized with a mixture of autologous serum and protein A. Anti-human IgG antibodies appeared within 2 weeks at which time the antibodies were of the IgM class. Upon further immunization, both IgM and IgG antibodies were produced with the IgG class predominating. The antibodies elicited by a mixture of protein A with autologous IgG resembled those which arise in response to autologous IgG that has been denatured by physicochemical means, in that they react mainly with foreign species IgG and weakly, if at all, with IgG of rabbit origin.     

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.     

Evidence for functional heterogeneity in IgG Fc-binding proteins associated with group A streptococci

A number of group A streptococcal isolates have been compared for their nonimmune reactivity with each human IgG subclass, and rabbit, pig, or horse IgG. The results obtained demonstrate considerable heterogeneity in the expression of type II IgG-binding proteins among and within group A isolates. Extraction and analysis of type II IgG-binding proteins from selected strains demonstrate the existence of five functionally distinct IgG-binding proteins. The type IIo IgG binding protein displayed the greatest range of reactivities, binding to all four human IgG subclasses, and rabbit, pig, and horse IgG. A variant of this protein, designated type II'o, bound all four human subclasses and rabbit IgG, but failed to react with pig or horse IgG. A type IIa protein was recovered from certain group A strains which bound human IgG1, IgG2, IgG4, as well as reacting with rabbit, pig, and horse IgG. A functionally related type IIc activity that displayed all of the reactivities of the type IIa protein but did not bind with human IgG2 was also identified. The final functional form of group A IgG-binding protein, the type IIb protein, bound exclusively to human IgG3. Comparison of these functionally different type II IgG-binding proteins demonstrated no simple structure-function relationship. These studies underscore the heterogeneity of type II Ig-binding proteins expressed by different group A streptococci and document that a single strain can change its pattern of expression of type II IgG-binding protein both quantitatively and qualitatively.     

S-layer-mediated display of the immunoglobulin G-binding domain of streptococcal protein G on the surface of Caulobacter crescentus: development of an immunoactive reagent

The immunoglobulin G (IgG)-binding streptococcal protein G is often used for immunoprecipitation or immunoadsorption-based assays, as it exhibits binding to a broader spectrum of host species IgG and IgG subclasses than the alternative, Staphylococcus aureus protein A. Caulobacter crescentus produces a hexagonally arranged paracrystalline protein surface layer (S-layer) composed of a single secreted protein, RsaA, that is notably tolerant of heterologous peptide insertions while maintaining the surface-attached crystalline character. Here, a protein G IgG-binding domain, GB1, was expressed as an insertion into full-length RsaA on the cell surface to produce densely packed immunoreactive particles. GB1 insertions at five separate sites were expressed, and all bound rabbit and goat IgG, but expression levels were reduced compared to those of wild-type RsaA and poor binding to mouse IgG was noted. To remedy this, we used the 20-amino-acid Muc1 peptide derived from human mucins as a spacer, since insertions of multiple tandem repeats were well tolerated for RsaA secretion and assembly. This strategy worked remarkably well, and recombinant RsaA proteins, containing up to three GB1 domains, surrounded by Muc1 peptides, not only were secreted and assembled but did so at wild-type levels. The ability to bind IgG (including mouse IgG) increased as GB1 units were added, and those with three GB1 domains bound twice as much rabbit IgG per cell as S. aureus cells (Pansorbin). The ability of recombinant protein G-Caulobacter cells to function as immunoactive reagents was assessed in an immunoprecipitation assay using a FLAG-tagged protein and anti-FLAG mouse monoclonal antibody; their performance was comparable to that of protein G-Sepharose beads. This work demonstrates the potential for using cells expressing recombinant RsaA/GB1 in immunoassays, especially considering that protein G-Caulobacter cells are more cost-effective than protein G beads and exhibit a broader species and IgG isotype binding range than protein A.     

Isolated DNA repeat region from fcrA76, the Fc-binding protein gene from an M-type 76 strain of group A streptococci, encodes a protein with Fc-binding activity

The DNA repeat region of fcrA76, the gene encoding a group A streptococcal Fc-binding protein, was subcloned in-frame into an Escherichia coli plasmid expression vector. The expressed protein product displayed the same Fc-binding properties as the full-length Fc-binding protein expressed from fcrA76. The affinity-purified, full-length Fc-binding protein was found to compete with staphylococcal protein A or streptococcal protein G for binding to beads coated with human IgG. These results are consistent with earlier studies suggesting that the binding sites on human IgG for protein A, protein G and the type II Fc-binding protein from group A streptococci are located at the interface of the CH2 and CH3 domains of the Fc region.     

Detection of IgG bound at the erythrocyte membrane by means of an immunohistochemical gold-silver technique

The binding of IgG at the erythrocyte membrane during aging plays an important role in the elimination process of the cells by the reticulohistiocytic system. By means of an indirect protein A gold- and protein A gold-silver-method bound IgG was detected immunocytochemically on physiologically aged, pronase and neuraminidase treated red blood cells (RBC). At the light and electron microscopic level an increased binding of IgG at "old" as well as enzymatic treated cells in comparison to "young" and normal RBC was noted. The silver enhancement of gold particles visualized the immunostaining and permitted the semiquantitative analysis of the RBC by a scanning microdensitometer.     

Study of rabbit IgG, modified with carboxycarbonic anhydride of diethylenetriaminopentacetic acid

The affinity-purified by chromatography on immobilized antigen rabbit IgG was modified with mixed carboxycarbonic anhydride of DTPA which markedly alters the interaction of charged residues in the protein molecule. To study the correlation between the antigen binding activity and the conformational mobility of IgG, the reactivity of modified IgG towards conformational probes targeted at variable and constant IgG domains, was investigated. The antibody against CH2 domains of IgG, staphylococcal protein A and protein antigen ferritin were used as conformational probes. It was found that modification of IgG amino groups entails the global increase in conformational mobility involving the Fab fragments, CH2 and, probably, the CH3 domains of the Fc portion of IgG. Taking advantage of Fab fragments modification it was shown that two processes contribute to the global increase in the conformational mobility of IgG. These processes are: i) stimulation of segmental flexibility and, ii) increase in the mobility within the Fv domains of the Fab fragments.     

Increasing immunoglobulin G adsorption in dextran‐grafted protein A gels

The formation of a stable spatial arrangement of protein A ligands is a great challenge for the development of high‐capacity polymer‐grafted protein A adsorbents due to the complexity in interplay between coupled ligands and polymer chain. In this work, carboxymethyl dextrans (CMDs) with different molecular weight were introduced to provide stable spatial ligand arrangement in CMD‐grafted protein A gels to improve IgG adsorption. The result showed that coupling of protein A ligand in CMD‐grafted layer had no marked influence on pore size and dextran layers coupled with the ligands were stable in experimental range of salt concentrations. The result of IgG adsorption revealed that carboxymethyl dextran T10, a short CMD, was more suitable as a scaffold for the synthesis of high‐capacity protein A gels. Moreover, the maximal adsorption capacity for IgG was obtained to be 96.4 mg/g gel at ionic capacities of 300–350 mmol/L and a ligand density of 15.2 mg/g gel. Dynamic binding capacity for IgG exhibited a higher capacity utilization in CMD‐grafted protein A gels than non‐grafted protein A gel. The research presented a tactics to establish a stable dextran layer coupled with protein A ligands and demonstrated its importance to improve binding capacity for IgG.     

IgG subclass responses to a transmembrane protein (gp41) peptide in HIV infection

The IgG subclass distribution to the E34/E32 peptides, derived from the HIV-1 glycoprotein 41 transmembrane protein, was analyzed in ELISA. Sera from individuals at different stages of the disease were assayed. A restricted subclass response of mainly IgG1 and IgG2 was found. The subclass response was of a different type than the one observed to HIV whole Ag and to a synthetic peptide from the C'-terminal part of the HIV-1 p24 core protein. An increased subclass restriction was observed in progressed stages of the disease.     

Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel

Immunoglobulin G (IgG) purification from human plasma with protein A attached supermacroporous poly(hydroxyethyl methacrylate) [PHEMA] cryogel has been studied. PHEMA cryogel was prepared by bulk polymerization which proceeds in aqueous solution of monomer frozen inside a plastic syringe (cryo-polymerization). After thawing, the PHEMA cryogel contains a continuous matrix having interconnected pores of 10–200 μm size. Protein was covalently attached onto the PHEMA cryogel via cyanogen bromide (CNBr) activation. The maximum IgG adsorption on the PHEMA/protein A cryogel was found to be 83.2 mg/g at pH 7.4 from aqueous solutions. The non-specific IgG adsorption onto the PHEMA cryogel was about 0.38 mg/g. The macropore size of the cryogel makes it possible to process blood cells without blocking the column. Higher adsorption capacity was observed from human plasma (up to 88.1 mg/g). Adsorbed IgG was eluted using 0.1 M glycine–HCl buffer (pH 3.5) with a purity of 85%. PHEMA–protein A cryogel was used for repetitive adsorption/desorption of IgG without noticeable loss in IgG adsorption capacity after 10 cycles. PHEMA–protein A cryogel showed several advantages such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–desorption cycles.     

Chemiluminescence response of human phagocytes against IgG-opsonized staphylococci with and without protein A activities

The effects of immunological IgG binding to Staphylococcus aureus and IgG binding via protein A on the chemiluminescence (CL) response of human phagocytes were examined. The results obtained by enzyme immunoassay showed a clear correlation between the magnitude of the CL response and amount of IgG on protein A-deficient HL-87 strain. Despite no difference in protein A activity between 209P and Cowan I strains, the CL response to IgG-opsonized 209P cells was lower than that to Cowan I cells similarly opsonized. Moreover, the CL response to opsonized HL-87 cells was identical with that of opsonized Cowan I cells, which was a protein A-rich parent strain of the HL-87. The protein A activity of Cowan I cells was significantly decreased when the cells were treated with the Fc fraction of IgG before opsonization, but such a treatment did not change the phagocytic CL response. These results strongly suggest that IgG bound to protein A via its Fc portion has no effect on the phagocytic CL response and that IgG immunologically bound to S. aureus is responsible for the opsonization of the bacteria.     

Binding of human IgM from a rheumatoid factor to IgG of 12 animal species

The binding of IgM from a rheumatoid factor (RF-IgM) to IgG from 12 animal species was analyzed by an ELISA system. The RF-IgM bound various animal IgG with dissimilar affinities. The binding of RF-IgM to animal IgG was inhibited by addition of protein A, which binds some animal IgG by recognizing the junctional site on CH2-CH3 domains in the Fc region. As previously reported, no significant correlation was observed between the binding of RF-IgM to IgG and the content of galactose-free oligosaccharides, which is increased in IgG of rheumatoid arthritis patients or autoimmune mice. We suggest that the crucial epitope of IgG for RF-IgM binding is not the oligosaccharide structure generated specifically in IgG of autoimmune diseases but that RF-IgM may recognize a certain protein conformation of a region in IgG near the binding site of protein A.     

Comparative analysis of the interaction of protein A from Staphylococcus aureus with native, reduced and aggregated IgG]

In a comparative study the affinity of rabbit and human IgG, native, reduced and aggregated by various methods, to protein A obtained from the cell wall of Staphylococcus aureus was determined. For this purpose the method of the passive hemagglutination inhibition test was developed. The affinity to protein A was found to grow considerably after specific or non-specific IgG aggregation and to decrease by 60% after local damage affecting the structure of the IgG molecule waist as a result of the dissolution of the disulfide bond between its heavy chains. The problem of similarity between such effector properties of IgG as its ability to activate the complement system and to combine with protein A is considered, as well as the problem of the pathogenetic role of immune complexes bound with protein A.     

Thermodynamic Stability and Functional Activity of Tumor-Associated Antibodies

Tumor-associated antibodies of human IgG1 subclass were eluted from cell-surface antigens of human carcinoma cells and studied by differential scanning calorimetry and binding to local conformational probes, protein A from Staphylococcus aureus and a monoclonal antibody targeted to the CH2 domain of the Fc fragment. At pH 2.0-7.0, we observed virtually identical enthalpies of thermal unfolding for IgG1 from normal human sera and tumor-associated IgG1. The exact values of calorimetric enthalpy (h) at pH 7.0 were 6.1 and 6.2-6.3 cal/g for IgG1 from normal serum and IgG1 from carcinoma cells, respectively. The affinity constants of protein A binding to the CH2–CH3 domain interface demonstrated differences between serum IgG1 and tumor associated IgG1 that did not exceed 3-8-fold. The binding affinity toward the anti-CH2 monoclonal antibody determined for serum IgG1 and IgG1 from carcinoma cells differed not more than 2.5-fold. The thermodynamic parameters of IgG1 from carcinoma cells strongly suggest that protein conformational stability was essentially unaltered and that the Fc fragment of the tumor-derived IgG1 preserved its structural integrity.     

Purification of immunoglobulins G by protein A/G affinity membrane chromatography

An affinity membrane grafted with protein A/G or protein A was characterized for human and mouse immunoglobulins G purification. Breakthrough curves up to ligand saturation were measured and used to study the effects of flow velocities, feed solution concentrations and protein A/G versus protein A membranes. Increased flow-rate did not decrease the amount of IgG bound to the membranes. Increased feed solution concentration allowed more IgG to bind prior to breakthrough. Kinetic parameters for immunoglobulins G sorption to immobilized protein A were measured in batch experiments. The static binding capacity was determined to be 6.6 mg ml⁻¹ membrane volume. Finally, this affinity membrane was used to purify IgG from cell culture supernatant. The electrophoresis of the purified IgG fractions did not show any contaminant.     

Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography-mass spectrometry

A method including protein A purification, limited Lys-C digestion, and mass spectrometry analysis was used in the study to quantify a recombinant monoclonal antibody in cynomolgus monkey serum. The same antibody that was isotopically labeled was used as an internal standard. Interferences from serum proteins were first significantly reduced by protein A purification and then by limited Lys-C digestion of protein A bound IgG, including both monkey and the recombinant IgG. Fab fragment of the recombinant human IgG was analyzed directly by LC–MS, while monkey IgG and the Fc fragment of the recombinant human IgG remained bound to protein A resin. Quantitation was achieved by measuring the peak intensity of the Fab from the recombinant human IgG and comparing it to that of the Fab from the stable isotope-labeled internal standard. The results were in good agreement with the values from ELISA. LC–MS can therefore be used as a complementary approach to ELISA to quantify recombinant monoclonal antibodies in serum for pharmacokinetics studies and it can also be used where specific reagents such as antigens are not readily available for ELISA.     

Certain characteristics of isolation of human myelomic IgG of different subclasses and their distribution according to the allotypes, subclasses and types of light chain]

A combination of the methods of preparative electrophoresis in agar gel and of the ion-exchange chromatography on DE-32 cellulose permitted to obtain 32 immunochemically pure human myelomic IgG. The proteins of the first three subclasses were obtained by elution in the 0.01 phosphate buffer at pH 7.6. IgG4 was eluted with the increase of the gradient to 1 M NaCl in the phosphate buffer. Of the 32 human myelomic IgG 26 represented IgG1,4--IgG2, 1--IgG3, and 1--IgG4. Among the 26 IgG1 11 were of the Gm(a) allotype, and 15 proteins had the Gm(f) determinant; one IgG2 protein was Gm(n+) and 3--Gm(n-). One IgG3 protein was referred to the Gm(b) variant. The majority of the IgG proteins of the subclass I had chi-type of the L-chains, and the chi: lambda ratio constituted 2.71.