Staphylococcal Protein A-Sepharose Research Articles

Anti-β2-Microglobulin Lymphocytotoxic Autoantibodies in Systemic Lupus Erythematosus

Abstract The occurrence of anti-beta-2-microglobulin (β2m) lymphocytotoxic antibodies was investigated in 81 sera obtained from 63 patients with systemic lupus erythematosus (SLE). β2m-binding activity, measured by precipitation of radiolabeled β2m in polyethylene glycol, was found significantly higher in SLE than in control sera. Chromatography of SLE sera on polyacrylamideagarose gel AcA 3/4 showed that most of the β2m-binding activity was recovered in the IgG peak. This activity could be adsorbed on staphylococcal protein A-Sepharose immunosorbent. Complement-dependent lymphocytotoxicity was detected at 15°C in 32 SLE sera, of which 19 could be inhibited by addition of 1 ng β2m. A strong association was found between lymphocytotoxicity inhibition by β2m and the presence of β2m-binding material, suggesting that both assays detected β2m autoantibodies. The presence of β2m antibodies was not dependent on the age or sex of the patients, was not correlated with titers of antinuclear antibodies or rheumatoid factors, and was weakly associated with Clq-binding activity. Inhibition experiments indicated that the amount of β2m required to inhibit lymphocytotoxicity or 125I-β2m binding by SLE sera was much higher than that sufficient to inhibit binding by heterologous sera, suggesting that SLE autoantibodies were of low avidity and might be directed against the β2m determinant(s) accessible in the HLA-β2m complex and at the cell surface.

Immunoprecipitation of the Insulin Receptor: A Sensitive Assay for Receptor Antibodies and a Specific Technique for Receptor Purification*

Autoantibodies that inhibit the binding of insulin to its receptors and elicit insulin-like biological responses are found in the sera of certain patients with severe insulin resistance. In the present study, five of these antireceptor sera inhibited binding of insulin to the Triton-solubilized insulin receptor at titers comparable to those for inhibition of binding to whole cells. Studies with the highest titer serum indicated that inhibition of insulin binding to the solubilized receptor was due to a reduction in the number of available binding sites, whereas with receptors in the intact membranes, the predominant effect of this antireceptor serum was to decrease receptor affinity. In addition to their ability to inhibit insulin binding, all of the sera were capable of completely precipitating the [125I]insulin-receptor complex in an indirect immunoprecipitation assay; immunoprecipitation required the addition of either antihuman immunoglobulin G (IgG) or Staphylococcal protein A-Sepharose. Insulin anti...

Characterization of Immune Complexes in Serum by Adsorption on Staphylococcal Protein A: Model Studies and Application to Sera of Rats Bearing a Gross Virus-Induced Lymphoma

Abstract A procedure to isolate soluble IgG-containing immune complexes from serum was developed, which combined Sephadex G-150 filtration with affinity chromatography on Staphylococcal protein A-Sepharose. The effectiveness of the method was shown by isolating model immune complexes of 125I bovine plasma albumin (BPA) and antibody to BPA from serum. Functionally intact antibody and antigen were recovered from these complexes by chromatography on Sephadex G-150 at low pH and the degree of purification was demonstrated by NaDod.SO4 polyacrylamide slab gel electrophoresis (PAGE) and autoradiography of the immune complexes and their components. Application of these methods to a Gross virus-induced rat lymphoma model tested their potential for characterizing naturally occurring circulating immune complexes (CIC). Sera from these animals were shown by Raji cell immunofluorescence to contain high concentrations of CIC 15 to 21 days after tumor inoculation. CIC could be isolated from these sera by the procedures developed for model complexes. Analysis by PAGE indicated that they contained primarily a protein of apparent m.w. 79,000 complexed with IgG. This putative antigen component of CIC (79,000 daltons) reacted with a rabbit antiserum directed against the murine leukemia virus (MuLV) envelope glycoprotein, gp70. Techniques using protein A affinity chromatography may also be applicable to characterization of both antigen and antibody components of CIC in human diseases including cancer.

Staphylococcal protein A-sepharose columns and the characterization of measles virus-specific polypeptides in persistently infected cells

Viral polypeptides in extracts prepared from [ 35S]methionine-labeled human epithelial carcinoma cells persistently infected with measles virus were reacted with virus-specific antisera. Microcolumns of staphylococcal protein A linked to Sepharose were used to isolate antigen-antibody complexes that contained few contaminating host polypeptides. Measles virus polypeptides in these complexes could be identified readily on sodium dodecyl sulfate-polyacrylamide gels even when antiserum with low reactivity toward the viral antigens was used.

IgG subclass studies of C3 nephritic factor

Antisera to the IgG subclasses, IgG1, IgG3, and IgG4, were insolubilized on Sepharose 6B and used to immunoabsorb five nephritic factor (NeF)-containing sera. Specific MgCl 2 eluates from the immunoabsorbents were evaluated for NeF activity. In one instance, NeF activity was of the subclasses IgG3 and IgG4; a second was only IgG3; a third was IgG1, IgG3, and IgG4; and two were only IgG1. The serum with only IgG3 NeF was also absorbed with staphylococcal protein A-Sepharose. NeF activity was present in both the absorbed serum and in the IgG eluted from the absorbent. This suggests that NeF activity may also reside in IgG2. These data demonstrate that IgG subclasses of NeFs vary from patient to patient, as do light-chain types.

Methods for separation of long-acting thyroid stimulator (LATS) from sera containing LATS and TSH

Methods were devised for separation of long-acting thyroid stimulator (LATS) from TSH in serum containing both thyroid stimulators by using Rivanol, concanavalin A (con A), or staphylococcal protein A. When 3–5 volumes of 0.5% Rivanol solution were mixed to serum containing TSH or LATS activity, LATS activity remained mainly with IgG in the supernatant fraction. On the contrary, TSH activity was precipitated. When 10 mg con A was added to 1 ml test serum, almost all TSH activity was precipitated, but LATS activity remained in the supernatant fraction, which consisted mainly of IgG and albumin. Almost all LATS activity and part of the TSH activity were precipitated by addition of more than 7.5% polyethylene glycol (PEG), which was therefore not useful for separation of the stimulators in serum. Affinity chromatography on staphylococcal protein A-Sepharose was also found to separate the two thyroid stimulators in serum. By this method LATS-immunoglobulin bound to the protein A column, but no binding of the biologic and immunologic activity of TSH was observed. The protein A method seems the most useful of these four methods for complete separation of both stimulators.

STUDIES ON IMMUNE RESPONSES TO LARVAL CESTODES IN MICE. INCREASED SUSCEPTIBILITY OF CERTAIN MOUSE STRAINS AND HYPOTHYMIC MICE TO TAENlA TAENIAEFORMIS AND ANALYSIS OF PASSIVE TRANSFER OF RESISTANCE WITH SERUM

Various inbred strains of mice vary markedly in their susceptibility to the larvae of the cestode, Taenia taeniaeformis. Males are generally more susceptible than females and the most susceptible common inbred mouse strains are those which are deficient in C5 and/or C4 components of complement. However, no genetic evidence is yet available to implicate loci controlling complement levels in susceptibility/resistance, and multiple genetic factors appear to be operative. Hypothymic, nu/nu ("nude") mice of the relatively resistant mouse strain, BALB/c, are highly susceptible in that cystic larvae in the liver develop in large numbers and more rapidly than in intact BALB/c.nu/+litter-mates. Cyclophosphamide pretreatment also increases the susceptibility of relatively resistant strains of mice in terms of both the number and size of liver cysts. Hypothymic and intact mice can be protected, absolutely, by an injection of serum from infected intact mice, provided the serum is given to recipient mice close to the time of oral egg administration. The protective activity of immune serum is absorbed totally by staphylococcal protein A-Sepharose columns and can be abolished by treatment of recipients with cobra venom factor. Cyst fluid from established larvae facilitates the activity of subhaemolytic amounts of guinea pig complement in a standard direct PFC assay. The data suggest that complement-fixing antibodies are responsible for inhibition of establishing larvae in mice and that one method of protection for established cystic larvae involves the alteration of host complement activity within the cyst.

Conjugation of antibodies with fluorochromes: Modifications to the standard methods

A number of modifications to the standard procedures for coupling of fluorochromes to antibodies are described. The suggested procedures result in economies of time, labour and materials, and allow the reliable production of high quality conjugates. The modifications include the use of staphylococcal protein A-Sepharose for (a) a simple one-step procedure for preparation of IgG from whole serum, (b) removal of undigested IgG after pepsin treatment, and (c) concentration of dilute solutions of IgG. Many other details of coupling procedures are discussed, and modifications suggested. Optimally coupled antibodies were separated by linear salt gradient elution from DEAE-Sephadex, and the effects of fluorescein and tetramethyl rhodamine on the antibody isoelectric point studied.