Extraction and characterization of IgG Fc receptors from group C and group G streptococci.

Immunoglobulin G (IgG) Fc receptors (FcRs) were extracted by proteolytic digestion of four strains each of group C and group G streptococci. The solubilized proteins were analyzed in Western blots and multiple IgG-binding bands were obtained. The banding patterns of some of the strains were very similar, but this property was independent of which streptococcal group the strains belonged to. Highly purified FcRs were prepared from one group C and one group G strain. The 13 N-terminal amino acids were determined, and found to be identical, whereas comparison with the sequence of staphylococcal protein A did not reveal any homology. The isolated streptococcal FcRs also appeared closely related antigenically and functionally. Thus, both molecules were capable of inhibiting each others binding to immobilized IgG, and the radiolabelled group G FcR was completely inhibited from binding to IgG by an antibody to the group C FcR. Finally, in a direct binding assay both proteins were capable of reacting to a similar degree with a wide variety of IgGs, thereby demonstrating the great potential of streptococcal FcRs as tools for binding and detection of IgG antibodies.

Interaction of bacterial Fc receptors with goat immunoglobulins.

The interaction of type I (staphylococcal protein A) and type III (streptococcal FcRc) bacterial Fc receptors with goat immunoglobulins has been studied. Staphylococcal protein A bound poorly to the majority of goat immunoglobulins at all pHs tested. There was some evidence that protein A bound IgG2 better than IgG1, particularly at pH 8 and above. One of 10 sera tested demonstrated a high level of reactivity with protein A and this was shown to correlate with the presence of a natural antibody to protein A. The streptococcal Fc receptors, FcRc, bound efficiently to all goat IgG and goat sera tested. Both goat IgG subclasses reacted efficiently with the FcRc between pH 6 and 8. Inhibition of binding of 125I-FcRc to immobilized goat IgG enabled levels of IgG in goat serum to be estimated. These results suggest the streptococcal FcRc will be of value as an immunochemical reagent in studies involving isolation and quantitation of goat immunoglobulins.

Detection of receptors for the Fc region of IgG on streptococci.

A semi-quantitative absorption test to measure Fc-reactive proteins on the surface of streptococci is described. The ability of bacteria to remove intact IgG in the presence of an equimolar amount of F(ab')2 fragments was used to identify streptococci with Fc-reactive proteins on their surface. This method was found to be more objective, reproducible and quantitative than the hemagglutination and 125I-labeled IgG binding methods currently in use. Methods for detection of secreted Fc-reactive materials with protein A-like reactivity are also described.

Enzyme-labeled type III bacterial Fc receptors. A versatile tracer for immunoassay.

The type III bacterial Fc receptor isolated form a group C streptococcus has been conjugated to alkaline phosphatase and used as a tracer in a variety of direct and indirect immunoassays. These immunoassays have utilized specific antibodies prepared in species whose immunoglobulins are poorly reactive with the type I Fc receptor, staphylococcal protein A. The value of the type III Fc receptor as a tracer for immunoassays utilizing antibodies produced in sheep and goats is documented.

Selective colony blotting to expand bacterial surface receptors: applications to receptors for rat immunoglobulins.

Many bacterial surface receptors demonstrate a heterogeneous expression pattern among individual colonies. Methods have been developed to select bacteria expressing high levels of a stable surface receptor. This process is illustrated using a Streptococcus zooepidemicus isolate demonstrating a high level of Fc receptors for rat immunoglobulins. This strain was selected and expanded to obtain a bacterial isolate demonstrating approximately 100 fold greater reactivity with rat immunoglobulins than protein A positive Staphylococcus aureus or 30-40 fold higher reactivity for rat IgG than type III Fc receptor positive streptococcal group G strains. The optimal pH for rat IgG binding and the reactivity with rat IgG subclasses and certain rat monoclonal antibodies is described. The potential application and limitations of the selected rat Fc receptor positive bacterial strain to immunoassays based on the specificity of rat monoclonal and polyclonal antibodies is discussed.

A hemolytic assay for the measurement of equine complement.

A hemolytic assay was developed for the measurement of functional equine complement activity. The assay utilizes antibody sensitized chicken erythrocytes as the target cell and was specific for classical pathway (antibody dependent) complement activity. The assay was found to be reproducible and more sensitive than previous reports using other species of target cells. Total serum complement (CH50) values were determined for five mares and their foals and followed over a period of 3 months.

Solid-phase radioimmunoassay for the detection of immunoglobulins against bovine Brucella abortus.

A sensitive radioimmunoassay for the detection of Brucella abortus antibody is described. The assay, performed in flexible 96-well microplates coated with Brucella abortus antigens, utilizes 125I-labeled staphylococcal protein A to detect antibody to Brucella abortus. The parameters of the assay have been analyzed using well recognized statistical methods. Least squares analysis of antigen concentration, antiserum dilution, antigen by antiserum and replicates within antigen by antiserum, estimated an r2 of 0.98, a coefficient of variation of 3.58 and a standard deviation of 0.10. Results of regression analysis of serum dilution versus antigen concentration (ranging from 635 micrograms/ml to 6.35 ng/ml) indicated that an antigen concentration of 6.35 micrograms/ml was the most efficient for describing antibody variability (r2 = 0.98, with a coefficient of variation = 3.28). Regression analysis also revealed a closer correlation between the radioimmunoassay with complement fixation test (r2 = 0.98) than with the standard tube test (r2 = 0.84). Detection of specific antibody assessed by radioimmunoassay was 4 to 64 fold more sensitive than the standard tube test titer and 16 to 32 fold more sensitive than the complement fixation test titer. The results described here indicate that this radioimmunoassay is very sensitive and may be capable of discriminating between false positives and false negatives, thereby, improving the diagnostic efficiency of Brucella serologic tests.

Cytotoxic tumor necrosis factor activity produced by equine alveolar macrophages: preliminary characterization.

Blood monocytes and alveolar macrophages (AM) were harvested from foals (aged 46 days to 6 months) and cultured in either medium alone or medium containing 10 micrograms/ml bacterial lipopolysaccharide (LPS). After 24 h, culture supernates were collected and analyzed for cytotoxic activity on sensitized L929 cells. Both monocytes and AM that had been treated with LPS produced significantly more cytotoxic activity than the same cell type exposed to medium lacking LPS. LPS-treated macrophages secreted significantly more cytotoxic activity (120 +/- 17.8 U/ml) than did LPS-treated monocytes (47.3 +/- 17.0 U/ml); however, constitutive production of cytotoxin by monocytes was higher (16.7 +/- 4.1 versus 1.2 +/- 1.2 U/ml). The identification of the cytotoxin as tumor necrosis factor (TNF) was strongly suggested by its reactivity with a rabbit antiserum directed against the N-terminal 15 amino acids of human TNF. TNF secretion by AM increased in a dose-dependent manner between LPS concentrations of 0.0001 and 1 microgram/ml, then leveled off. Most of the cytotoxic TNF activity produced by AM was secreted within the first 8 h after initial contact with LPS. Macrophage supernatant TNF was stable over a pH range of 6-11, but lost activity when kept at a pH less than 6. Equine TNF also was destroyed by exposure for 1 h to temperatures more than 60 degrees C. TNF bioactivity was recovered as a single peak after crude macrophage supernate was subjected to analysis by either anion exchange or gel filtration chromatography (molecular weight approximately 56,000).

Streptococcal Fc receptors. I. Isolation and partial characterization of the receptor from a group C streptococcus.

A receptor for the Fc region of immunoglobulin G was extracted from a group C streptococcus, purified and physicochemically characterized. The Fc receptor was extracted in high yield by lysis of the bacteria after infection with bacteriophage. The soluble receptor was purified to functional homogeneity by sequential chromatography on cellulose phosphate, DEAE, and selective elution from a column of immobilized human IgG. Four hundred micrograms of the functionally pure protein was obtained per gram (wet weight) of bacteria extracted. The affinity-purified receptor was functionally homogeneous in binding to the Fc region of human IgG; however, the product was heterogeneous on both non-denaturing and SDS polyacrylamide gels. Four major protein bands were observed, with the predominant form of the Fc receptor having an m.w. of 64,000 daltons. Antibody prepared against the major Fc receptor protein ( FcRc -II) was capable of reacting with all the fractions and completely inhibiting functional activity. The results of the competitive binding studies suggest that the purified Fc receptor behaves as a single receptor, and that the differences in charge and size were probably due to covalently bound cell wall constituents.

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.

Identification of distinct Fc-receptor molecules on streptococci and staphylococci.

The structural similarity between staphylococcal protein A and streptococcal Fc receptors was examined. Antibody to staphylococcal protein A, proven not to bind to protein A through Fc, was used to determine if the Fc receptors on 4 Fc-receptor positive streptococcal strains were antigenically related to staphylococcal protein A. Anti-protein A antibody bound to Staphylococcus aureus Cowan I, but failed to bind to any of the streptococci. Additionally, when the Cowan strain and 9 other Staphylococcus aureus isolates demonstrating various levels of IgG adsorption capacity were preincubated with antiprotein A antibody, the ability of these bacteria to adsorb IgG was completely inhibited. These results suggest that all the Fc receptors on Staphylococcus aureus are antigenically similar or identical to protein A. Fc receptors on streptococci, while sharing with staphylococcal protein A the capacity to bind to Fc of human IgG, were not antigenically crossreactive with protein A.

Effect of mouse passage on Fc receptor expression by group A streptococci.

The expression and stability of receptors for the Fc region of human IgG on the surface of group A streptococci was studied. Two strains were sequentially passed in mice 22 times. The Fc-receptor expression on one group A strain, 529, was unaltered while the expression of Fc receptor on a second, 64, was enhanced and approached the level of Fc-receptor expression of the protein A-rich Staphylococcus aureus Cowan strain. The level of Fc-receptor expression on this organism remained stable for over 18 months of laboratory subculture. Mouse passage did not result in the production of a soluble Fc receptor from either of the streptococcal strains. Heat extraction of the Fc-receptor-positive group A strain resulted in solubilization of an Fc-receptor activity which was functionally distinct from either staphylococcal protein A or the Fc receptor isolated from a group C streptococcus.

Column agglutination technology: the antiglobulin test.

A new system for typing and screening blood, based on the sieving effect of glass bead microparticles, has been developed. The test is performed in a microcolumn in which the red cell agglutinates are trapped in the glass bead matrix during centrifugation, and unagglutinated cells form a pellet at the bottom of the column. Anti-human globulin reagents were incorporated in the diluent and the new test system, column agglutination technology, was compared to conventional tube tests and low-ionic-strength method. Sera and plasmas (228 samples) were screened for red cell antibodies with two anti-human globulin reagents: one containing only anti-IgG and the other containing both anti-IgG and anti-C3b, -C3d. After initial testing, there was 94-percent agreement between column agglutination technology and tube tests, and after repeat testing, there was 97-percent agreement. The column agglutination technology anti-human globulin test eliminates the need to wash red cells, which decreases the overall test time. The test is easy to perform, and the results are more objective than those with tube and microplate methods.

Use of IgM monoclonal reagents licensed for tube tests in column agglutination technology.

BACKGROUND: Red cell (RBC) phenotyping using column agglutination technology (CAT) is currently limited by the reagents formulated in the system. To overcome this limitation, it was investigated whether monoclonal IgM reagents licensed for use with tube tests produced valid results with CAT. STUDY DESIGN AND METHODS: Commercial CAT, does not contain antisera, was used to evaluate Procedures A (40 microL of reagent and 10 microL of 4% RBCs) and B (50 microL of reagent and 50 microL of 0.8% RBCs) with or without incubation at room temperature. In Study 1, reagents were tested to determine whether potentiators inhibit the passage of antigen-negative RBCs through the column. In Study 2, CAT sensitivity was measured by the use of potency titrations to define a procedure for each reagent that matched or exceeded that of the tube method. In Study 3, the specificity of each reagent was determined in parallel with the CAT and tube tests. Typing of 1644 samples was performed. RESULTS: Study 1: Free passage was obtained with all reagents. Study 2: Immediate-spin methods using CAT produced the same results as the tube method. Study 3: With 8048 comparisons made, discrepant results were found in 32 transfused patients and in 6 cord blood samples, mainly with Lewis reagents. With comparison of CAT and the standard tube method, complete agreement was obtained with Kell reagents, 99.9-percent agreement with Kidd reagents, and 98.9-percent and 99.4-percent agreement with Lewis reagents. CONCLUSION: Most examined reagents seem suitable for use with CAT.