Purification and partial characterization of an IgM-like serum immunoglobulin from Atlantic salmon (Salmo salar).

An IgM-like immunoglobulin was isolated from pooled sera collected from healthy Atlantic salmon. The immunoglobulin was purified by means of gel filtration followed by ion-exchange chromatography. It eluted from the ion-exchange column as two distinct peaks, but the two IgMs seems to be identical in their molecular natures except for net charge. The molecular weight of the unreduced (native) IgM was determined to be approximately 800 kilo Daltons (kD) when estimated by dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). However, when the molecular weight of the native IgM was estimated by gel filtration, a molecular weight of 1000 kD was obtained. Furthermore, the molecular weights of the heavy and the light chains were estimated by SDS-PAGE analysis to be about 72 and 27 kD respectively. The amount of IgM was found to range from 80 to 130 mg/100 ml serum. Isoelectric focusing demonstrated that the major part of the IgM molecules focused between pH 5 and pH 6.

Intrathecal synthesis of antibodies to staphylococcal antigens in multiple sclerosis patients.

Human serum and cerebrospinal fluid (CSF) antibodies against three staphylococcal antigens were measured using the ELISA technique. Antibody levels in sera from patients with multiple sclerosis (MS) were lower than those found in sera from patients with other neurological diseases (OND) and from healthy individuals (N). The CSF antibody mean titres for patients with relapsing remittent MS were about twofold higher than those found for the other groups. Both serum and CSF antibodies were of the IgG class while IgM could only be detected in serum samples. Elevated IgG-indexes, indicating intrathecal immunoglobulin synthesis, were found only among individuals in the MS patient groups. Some of the patients also had elevated indexes of specific antibodies to the staphylococcal antigens used, i.e. lipoteichoic acid (LTA), peptidoglycan (PG) and beta-ribitol teichoic acid (beta-RTA). This indicates that some MS patients have synthesized staphylococcal antibodies intrathecally.

The mitogenic properties of lipoteichoic acid from Staphylococcus aureus.

Lipoteichoic acid (LTA) isolated from Staphylococcus aureus Cowan I induced an in vitro blastogenic response in isolated mononuclear cells (MNC). Seventeen of 20 normal donors responded strongly, with peak response after 3-5 days of incubation. Significant stimulation was also obtained with human cord blood MNC from 3 donors, further indicating that the stimulation was due to a general mitogenic effect. The mitogenicity of deacylated LTA (dLTA) was similar to that of LTA. Binding of LTA to isolated MNC was demonstrated by an indirect immunofluorescent technique staining with specific rabbit anti-LTA antibodies and by rosette inhibition experiments. Results obtained in cell fraction experiments indicated that LTA had a monocyte dependent mitogenic effect on T lymphocytes.

Antibodies to lipoteichoic acid from Staphylococcus aureus. Specificity of murine monoclonal and human antibodies.

Two monoclonal antibodies against staphylococcal lipoteichoic acid (LTA) were made by fusing P3X63Ag8 myeloma cells and splenocytes from mice immunized with purified LTA. Both were isotyped as being IgM kappa. Their specificities were determined by enzyme-linked immunosorbent assays indicating that both antibodies reacted with the glycerol-phosphate backbone, while one of them also had some affinity for the alanyl substituent. Antibodies in serum from 7 multiple sclerosis (MS) patients and serum and cerebrospinal fluid (CSF) from 7 non-MS patients apparently reacted with the sugar moiety of LTA. In contrast, CSF antibodies from 6 of the 7 MS patients and 1 of the 7 non-MS patients had affinity for the alanine residue. This non-MS patient also had serum antibodies against the alanine residue. None of the other sera tested appeared to contain such antibodies.

Antibodies to Staphylococcus aureus peptidoglycan and lipoteichoic acid in sera from blood donors and patients with staphylococcal infections.

An enzyme-linked immunoassay (ELISA) was used to detect antibodies in human sera to Staphylococcus aureus peptidoglycan (PG) and lipoteichoic acid (LTA). All the sera from the blood donors contained IgG antibodies to both substances. Among the sera from 34 patients with bacteriologically verified, serious S. aureus infections, 71 per cent contained significantly elevated levels of anti-PG antibodies and 76 per cent of anti-LTA antibodies. Among the sera from 38 patients with suspected but not bacteriologically verified staphylococcal infections, 58 per cent contained significantly elevated levels of anti-PG antibodies and 74 per cent of anti-LTA antibodies. The levels of antibodies to PG correlated well with the levels of antibodies to LTA, but the latter occurred over a broader range in the patient sera. Elevated antibody values were, however, also found in some patients with serious, non-staphylococcal infections. The diagnostic value of PG and LTA antibodies has to be further investigated.

Antibodies to staphylococcal lipoteichoic acid in serum and in cerebrospinal fluid from patients with multiple sclerosis.

Antibodies to staphylococcal lipoteichoic acid (LTA) were determined in serum and in cerebrospinal fluid (CSF) from a group of patients with multiple sclerosis (MS) and a group of healthy individuals. Sera from 11 out of 74 MS patients, i.e. 15 per cent, contained precipitating antibodies demonstrated by the double diffusion in agar test, compared to 48 out of 94 control sera, i.e. 51 per cent. By using an indirect haemagglutination test, antibodies to LTA were found in all sera from both patients and controls. On the other hand, LTA antibodies were detected in a higher proportion of the CSF samples from MS patients than from controls. A reduced serum to CSF ration of LTA antibodies compared with the ratio for a reference antibody was demonstrated in 7 out of 10 MS patients, suggesting a local synthesis within the central nervous system. Absorption of seven MS CSF samples with LTA removed one to three of the cathodic oligoclonal IgG bands demonstrated by agarose electrophoresis and reduced the IgG concentration.

Encephalitis induced in rabbits by staphylococcal lipoteichoic acid.

Rabbits were immunized with staphylococcal lipoteichoic acid (LTA) in Freund's adjuvant. After four injections (six weeks) the rabbits showed decreased activity and unsteadiness of the head. Two weeks after the sixth injection (ten weeks), two of five rabbits developed clinical signs of encephalitis with nystagmus, ataxia, general weakness, decreased activity, and dragging of the hind legs. The other three animals showed only mild symptoms. Neuropathological examination showed inflammatory infiltrates containing small lymphocytes and some plasma cells in the leptomeninges and within the perivascular spaces of the brain.

Immunoperoxidase and electron microscopy studies of staphylococcal lipoteichoic acid.

Immunoperoxidase technique together with electron microscopy shows that lipoteichoic acid (LTA) of Staphylococcus aureus Cowan I is attached to the membrane and penetrates the whole cell wall. A diffuse zone outside and no peroxidase reaction product inside the wall when whole cells were treated with antibody prior to embedding may indicate (i) that LTA is exposed to reaction with antibodies outside the wall, and (ii) that all or most of the anti-LTA antibodies used are of the IgM class and thus unable to penetrate the wall. Thin sections of strain Wood 46 showed the same picture as Cowan I, but treatment of whole cells before embedding gave no diffuse zone outside the wall. This may be due to a thicker wall as found by electron microscopy and/or shorter LTA-chains of strain Wood 46 than those present in the wall of Cowan I.

Antibodies in rabbits immunized with staphylococcal lipoteichoic acid.

Fractionated rabbit antiserum to staphylococcal lipoteichoic acid (LTA) was tested for reaction with homologous antigen by precipitation in agar gel, countercurrent immunoelectrophoresis, immunoperoxidase technique and electron microscopy. The antibodies to LTA present in the rabbit antisera examined were found to be of the IgM class.

Immunochemical studies on Staphylococcus aureus plasma membrane. 2. Antigenic properties.

Cytoplasma membrane and lipoteichoic acid (LTA) isolated from S. aureus Cowan I were examined serologically. LTA contains both alpha- and beta-glucosyl substituents at glycerol and most probably ester-linked alanine as well, all being antigenic determinants. In addition to LTA, the membrane contains a glycoprotein exhibiting antigenic determinant(s) in both the protein and sugar moieties.

Immunochemical studies on Staphylococcus aureus plasma membrane. I. Isolation and chemical characterization.

Cytoplasma membrane and lipoteichoic acid (LTA) were isolated from S. aureus Cowan I and analysed chemically. Pure membrane was obtained by using human IgG coupled to Sepharose, which then absorbed all cell wall fragments due to the interaction between IgG and protein A on the wall. LTA, shown to be a glucosylglycerol teichoic acid containing ester-linked alanine and pentadecanoic acid as the major fatty acid, was also present in the isolated membrane but only as a minor component. Other carbohydrate, protein and lipid components which were present as a chemical complex, dominated the membrane preparations.