Ole-Jan Iversen

Characterization of the a antigen of the c proteins of group B streptococci (GBS) using a murine monoclonal antibody

A murine monoclonal antibody raised against the a antigen of the group B streptococcal c proteins was analysed by immunofluorescence and whole‐cell ELISA against a collection of 22 c protein‐producing GBS. All the strains showing fluorescence and reactivity in ELISA turned out to be a antigen‐carrying strains as defined by polyclonal rabbit antisera, while none of the strains producing only the p antigen was positive. Western blot analyses of the a antigen released into the culture medium of growing bacteria suggest that the a antigen is present as distinct proteins of variable molecular weights. The upper limit of the molecular weights varies considerably from one strain to another, from approximately 200 kD to 70 kD. With all strains, the bands seen by the MAb occurred at regularly spaced intervals of about 10 kD throughout the gel. Some strains gave rise to 15–16 bands, while others gave rise to only one or two bands. The present investigation suggests that α antigens include several, probably identical, repeating subunits of approximately 10 kD. The epitope recognized by the MAb seems to be located on a 10‐kD fragment, and in addition, it appears to be surface located, making the MAb a suitable tool in serodiagnostic work.

The major internal protein p27, of a retrovirus-like particle participates in immune complex formation in psoriasis

SummaryThe major internal protein, p27, of a retrovirus-like particle isolated from the urine of a patient with psoriasis has been purified and used in an indirect ELISA to detect human antibodies against the virus antigen. Rabbit anti-p27 antiserum has been applied to detect p27 antigen present in clinical specimens.p27 and anti-p27 antibodies have been demonstrated in extracts from psoriatic scales. Insignificant amounts of free anti-p27 antibodies are present in serum, but both p27 and anti-p27 antibodies have been detected in circulating immune complexes obtained from serum or synovial fluid from patients with psoriatic arthritis.

The psoriasis-associated antigen, pso p27, is expressed by tryptase-positive cells in psoriatic lesions

Received: 3 May 1994 Key words Psoriasis 9 pso p 27 9 Antigen 9 Mast cells 9 Inflammation A main feature of psoriasis is an inflammatory reaction in the dermis with deposits of immune complexes and infil- tration of inflammatory cells. Recently, we presented evidence that a major antigen in the immune reactions in psoriasis is expressed by a sub- population of dermal cells [1], and that this antigen is probably identical to the psoriasis-associated antigen, pso p27 [2, 3]. Pso p27 is a protein isolated from psoriatic scale, and we have previously described the presence of complement-activating autoantibodies against it [3, 4]. The biological role of this protein has not been identified, but preliminary analyses show that pso p27 has an N-ter- minal amino acid sequence which differs from known se- quenced human proteins (Iversen et al., unpublished ob- servations). In the present study we attempted to characterize the dermal cells expressing the pso p27 antigen. Punch biopsies (6 mm) were obtained under local anaesthesia from lesions of eight patients suffering from static chronic plaque psoriasis. After fixation in ethanol, the biopsy specimens were embedded in paraffin as de- scribed elsewhere, and ultrathin sections (2-3 ~tm) were then prepared [51. Harvima et al. [6] have demonstrated that the presence of tryptase activity in psoriatic lesions is a specific marker for mast cells. To identify mast cells we used an enzyme- histochemical staining method using Z-Gly-Pro-Arg- MNA (Bachem, Bubendorf, Switzerland) as substrate [6, 7]. The thin sections prepared from the skin specimens were incubated with protease inhibitor LBTI (Sigma) for 30 rain [6], followed by incubation with (0.1 mM) Z-Gly- Pro-Arg-MNA and 0.4 mg/ml Fast Black K salt in 0.1 M Tris HC1 for 15 min at room temperature [7] and finally photographed with a Nikon FX-35WA. O.-J, Iversen (5~). H. Lysvand 9 T. Jacobsen - K. Bergh B. A. Lie Department of Microbiology, University Hospital, N-7006 Trondheim, Norway

Surprisingly high levels of anaphylatoxin C5a des Arg are extractable from psoriatic scales

Water-soluble extracts from psoriatic scales and normal human skin were prepared using either phosphate-buffered saline, pH 7.2, or 0.1M carbonate buffer, pH 10.8. Anaphylatoxin C5a des Arg was quantified using a novel sandwich enzyme-linked immunosorbent assay (ELISA) using neoepitope-specific monoclonal antibodies. Alkali was about five to eight times more efficient than PBS in extracting C5a des Arg from scales, probably via dissociation of bound C5a des Arg. C5a des Arg concentration in scales from three patients suffering from psoriasis vulgaris varied between 2.5 and 4.6 ng/mg scale. No C5a des Arg was detectable in normal skin extracts. The biological activity of alkali-extractable C5a des Arg, i.e. chemotaxis, was preserved. The concentration of C5a des Arg relative to the concentration of albumin was taken as a parameter of the degree of complement activation in the psoriatic lesions, and was found to be more than six times higher than values attained in serum after maximum complement activation by zymosan. We conclude that complement activation may play a quantitatively important role in the inflammatory process in psoriasis.

The major internal protein, p27, of a retrovirus-like particle is expressed in blood lymphocytes from psoriatic patients

SummaryRetrovirus-like particles were isolated from the urine of a patient with psoriasis. The major internal protein, p27, in these particles was isolated by immunosorbent chromatography and gel filtration on a Sephacryl S-300 column in 6m guanidine hydrochloride. The protein was purified to homogeneity as judged by SDS-PAGE. A hyperimmune serum with specificity for p27 was obtained by vaccination of a rabbit with purified p27 antigen. This antiserum was used to examine blood lymphocytes for the expression of p27 antigen by indirect immunofluorescence. Between 0.1 and 1 per cent of the lymphocytes obtained from patients with psoriasis showed a bright cytoplasmatic (and membrane) fluorescence while no p27 positive cells could be detected in the preparations from the healthy controls (frequency <0.01 per cent). Among the p27 positive psoriatic cells were lymphocytes with markers for T cells, B cells and NK cells.

Measurement of complement activation in rabbit plasma or serum using monoclonal antibodies against C5a.

Eight murine monoclonal antibodies (MoAb) raised against the major zymosan‐induced chemotactic factor in rabbit serum were found to neutralize the chemotactic activity induced by lipopolysaccharides (LPS) and antigen antibody complexes. A 15 kDa antigen was identified in plasma incubated with LPS by immunoblot analysis with MoAb. This is similar to the molecular weight of the major zymosan‐induced chemotactic factor. Both the generation of this 15 kDa antigen and chemotactic activity were abrogated in a heat‐inactivated plasma. A cross‐reaction to human C5a was demonstrated for three MoAb (5H8B9, 4B1C11, and 2A5E3) in an indirect enzyme‐linked immunosorbent assay (ELISA) of partially purified C5a and by the isolation of zymosan‐induced chemotactic activity by affinity chromatography. MoAb 5H8B9 and 4B1C11 were able to neutralize the chemotactic activity in human zymosan‐activated scrum. MoAb 2A5E3 was able to bind 125I‐labelled human C5a des Arg. We conclude that these MoAb are directed against rabbit C5a. MoAb 5B2C5 and 2B1A2, which are directed to different antigenic binding sites on C5a, may be applied in a sandwich ELISA for the detection and quantification of C5a des Arg in rabbit serum or plasma. The sandwich ELISA can be performed directly on serum or plasma samples without having to precipitate native C5. Complement activation is demonstrated by measuring the increased generation of C5a des Arg in rabbit plasma or serum activated with LPS, zymosan, antigen‐antibody complexes, or cobra venom factor.

Evaluation of monoclonal antibodies in serovar classification of group B streptococci (GBS)

We have produced murine MAbs against the c protein from GBS and selected one anti‐cα and one anti‐cβ MAb for serovar classification of c‐protein‐producing strains. A total of 61 GBS isolates, classified by serotype‐specific rabbit antisera, were tested in an immunofluorescent assay and whole‐cell ELISA. The MAbs permitted classification of the isolates into 13 serovars. A total of 34 strains contained the cα antigen, seven strains the cβ antigen, and 20 strains both the cα and the cβ antigen. The distribution of GBS with regard to c‐protein serovars was identical to that recorded when rabbit anti‐cα and ‐cβ sera were used. The results show that the MAbs are directed against common epitopes on cα and β, respectively. The MAbs will be useful in the serovar determination of clinical GBS isolates and in the study of the structure and immunobiology of c proteins.

Participation of antigens related to the psoriasis associated antigen, pso p27, in immune complex formation in patients with ankylosing spondylitis.

Analysis of five serum samples and three synovial fluids from patients with ankylosing spondylitis (AS) and five serum samples from healthy blood donors for the presence of antibodies cross reacting with the Fc part of rabbit IgG (rheumatoid factors (RFs] using an isotype specific, enzyme linked immunosorbent assay (ELISA) showed only insignificant amounts of free RFs, while IgG RFs were observed in alkaline dissociated circulating immune complexes (CICs). Only insignificant amounts of free antibodies reacting with the psoriasis associated antigen pso p27 could be detected in the samples, while extensive amounts of IgG antibodies and moderate amounts of IgM antibodies reacting with pso p27 were detected in alkaline dissociated CICs from the patients. Pso p27 has been reported to share a common determinant with the Fc part of human IgG. Removal of the RF activity from the CICs of patients with AS by absorption with IgG resulted in a decrease of the anti-pso p27 activity. Monoclonal anti-pso p27 antibodies in a sandwich ELISA were used to detect antigens cross reacting with pso p27. A positive reaction was observed in all serum CICs and in one of the synovial fluid CICs. The data indicate that antigens related to pso p27 participate in CIC formation in AS and may also be responsible for the elicitation of rheumatoid factors in patients with AS.

Rabbit antibodies against the major internal protein of a retrovirus-like particle bind to epidermal cells in psoriatic skin

SummaryA rabbit antiserum against the major internal protein, p27, of a psoriasis associated retrovirus-like particle has been used in indirect fluorescence microscopy of biopsies from psoriatic skin. The analysis indicate expression of p27 antigen in epithelial cells in psoriatic lesions and in clinically uninvolved psoriatic skin. A reaction of the antiserum with dermal vessel walls in the lesion was also observed.

Preparative Isolation of Immune Complexes from Serum by Sucrose Gradient Ultracentrifugation

Immune complexes were isolated by ultracentrifugation in sucrose gradients (20‐65% (w/w)). The centrifugation procedure was demonstrated 10 be isopycnic. The banding density of the complexes was influenced by the chemical nature and molecular size of the antigen and by the antigen to antibody ratio. The method was applied for preparative isolation of immune complexes from patients with systemic lupus erythematosus. rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and uncomplicated psoriasis.