Immunochemical characterization of a 150 000 dalton human fibroblast surface glycoprotein.

The characterization of a human fibroblast surface glycoprotein, visualized by crossed immunoelectrophoresis using rabbit antibodies against whole fibroblasts, is described. The antigen is synthesized by fibroblasts in culture and was localized both intracellularly and at the cell surface. It was highly antigenic and was detected only in human cells of mesenchymal origin. The glycoprotein occurred in two different forms with alpha 2 and beta electrophoretic mobility. The slow migrating amphiphilic beta form was localized at the cell surface and showed a single protein band with an apparent molecular weight of 150 000 in SDS-polyacrylamide gel electrophoresis. By external papain treatment of intact viable cells, a water-soluble molecule was released with a reduced molecular weight (140 000) and an increased electrophoretic mobility as compared to the native membrane component. This hydrophilic form was also present intracellularly in fibroblasts not treated with exogeneous proteases. The observation that the detergent-solubilized beta form was irreversibly converted to a more anodic form by incubation of whole cell extract at acidic pH, suggested that the intracellular protein represented a lysosomal degradation product of native internalized fibroblast surface glycoprotein.

Membrane asymmetry. A survey and critical appraisal of the methodology. I. Methods for assessing the asymmetric orientation and distribution of proteins.

This and the companion article are aimed at surveying the methods used for the study of membrane asymmetry. The techniques employed for the assessment of the asymmetric distribution and orientation of membrane proteins are reviewed in this article, whereas those pertaining to the unequal distribution of lipids are detailed in the companion paper. The use of immunological techniques and lectins, functions of proteins and their perturbations, chemical reagents, enzymatic isotopic labeling and enzymatic cleavage of membrane proteins and physical techniques are discussed and illustrated using recent examples of their application. Whenever appropriate, problems involving crypticity and non-availability or non-reactivity of functional sites, relevant chemical functions or protein fragments to appropriate ligands, reagents or modifying enzymes are envisaged and possible modification of the exposure of proteins during preparation of ghosts and other drawbacks are discussed, the use of different techniques and control experiments in conjunction is recommended for a more realistic assessment of the distribution and orientation of proteins.

The immunochemical approach to the characterization of membrane proteins. Human erythrocyte membrane proteins analysed as a model system.

1. Crossed immunoelectrophoresis was used for extensive characterization of individual proteins of human erythrocyte membranes solubilized in non-ionic detergent. 2. The precipitates were assigned to extrinsic or intrinsic proteins. 3. Four glycoproteins were identified by their lectin binding behaviour, whilst five proteins were affected by neuraminidase, indicating them to be sialoglycoproteins. 4. Enzymatic activity is retained in the solubilized system and the presence of acetylcholinesterase and an ATPase was demonstrated. The formation of phosphorylated membrane proteins on incubation with [32P]ATP was demonstrated by autoradiography on the immunoelectrophoresis plates. 5. Five proteins located on the outer cell surface were identified by antibody binding to intact cells. These same proteins were degraded by proteolytic enzymes in intact cells but only three of them were labelled by lactoperoxidase-catalysed 125I-iodination. 6. Analysis of erythrocyte membrane proteins using quantitive immunoelectrophoresis yields results concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis.

Detection of amphiphilic proteins and peptides in complex mixtures. Charge-shift crossed immunoelectrophoresis and two-dimensional charge-shift electrophoresis.

Charge-shift electrophoresis has been suggested as a simple and novel method for differentiating between emphiphilic and hydrophilic proteins (Helenius, A. and Simons, K. (1977) Proc. Natl. Acad. Sci. U.S. 74, 529-532.) This communication reports on the combination of charge-shift electrophoresis with second dimensional quantitative immunoelectrophoresis, and on a two-dimensional modification of the charge-shift electrophoresis technique. From results obtained with unfractionated human plasma proteins and human erythrocyte membrane proteins we conclude that these modifications reliably permit detection of amphiphilic proteins and peptides in complex mixtures.

Effect of transgenic expression of human alpha 1-acid glycoprotein (AGP) on the glycosylation of human and mouse AGP in various transgenic mouse sera.

The occurrence and the glycosylation of human alpha 1-acid glycoprotein (AGP) was studied in two classes of transgenic mice expressing either the A, B and B' genes (ABB'-mice) or only the A gene of human AGP (A-mice). The glycosylation of the human AGP molecules in the transgenic mouse sera was compared with the glycosylation of mouse AGP in the same animal and with human AGP in normal human serum by studying their heterogeneity in binding to concanavalin A (Con A), using crossed affino immunoelectrophoresis (CAIE) with Con A as the affinocomponent in the first dimension gel. Three to four different glycosylated fractions of human as well as mouse AGP were revealed by this method in all the transgenic mouse sera. A close relationship was apparent between the heterogeneities in Con A binding of human and mouse AGP in the same transgenic mouse. The magnitude of this so-called Con A reactivity was, however, strongly dependent on the transgenic mouse studied. Especially within the group of ABB'-mice dramatic changes in Con A reactivity were found when the human AGP genes were expressed. This indicates in the first place that the oligosaccharide chains of the human AGP molecules expressed also mouse-specific features. Secondly, and more importantly, these findings indicate that the expression of the human AGP genes affected the glycosylation process of the transgenic mouse liver. This organ is the source of the AGP forms occurring in serum. We do not know whether this effect has been caused by the introduction or the expression of the human gene(s) or by the presence of human AGP in the Golgi system or in serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Crossed-immunoelectrophoretic study on human renal brush border membrane vesicles.

The human kidney brush border membrane proteins were studied by crossed-immunoelectrophoresis. An antiserum against membrane vesicles was raised in rabbits and used in establishing a reference immunoelectrophoregram with the antigens released by Triton X-100. Among the precipitates observed, the following hydrolases were identified by zymogram staining: Microvillus aminopeptidase (EC 3..4.11.2), gamma-glutamyltransferase (EC 2.3.2.2), maltase (EC3.2.1.20) and trehalase (EC 3.2.1.28). Depletion of the antiserum with sealed, right-side-out vesicles was performed. No precipitates could be seen when the Triton X-100 extract was electrophoresed in a gel containing the depleted antibody. It is therefore suggested that the precipitation of membrane components by the complete antibody is mainly due to externally-located determinants and that the precipitates of the reference pattern correspond to membrane components pointing, at least in part, towards the tubular lumen. Evidence was also noted for a differential removal of antibodies directed against the different antigens. Such an observation could not be explained by the antigen accessibility nor by its amount in the membrane. Parallel crossed-immunoelectrophoresis of Triton X-100 and papain extracts gave rise to an "identity" pattern for only some antigens, particularly for microvillus aminopeptidase and maltase. It is thus strongly suggested that the papain-released form of these enzymes bears nearly all the antigenicity of the whole molecule.

Templates for antiserum application in immunoelectrophoresis and two-dimensional electroimmunodiffusion.

Two forms of template are described for use with flat-cast agarose gels to simplify and improve immunoelectrophoresis and two-dimensional electroimmunodiffusion tests.

A single step immunoelectrophoresis method for the quantitation of complement C3c in biological fluids.

An intermediate gel rocket immunoelectrophoresis (IRI) suitable for direct quantitation of complement split products with C3c specificity is described. The technique represents a simple, quick, and reproducible method for the assessment of complement activity in biological fluids in diseases. The advantages of the quantitative approach of the IRI method as compared to the conventional crossed immunoelectrophoresis (CIE) are the increased sensitivity, specificity, and precision of the method; also, the assay capacity is superior to CIE. The IRI-C3c assay is therefore highly suitable as a scientific tool, and may also be useful for routine laboratory investigations.

A technical improvement for crossed immunoelectrophoresis.

A simple antibody overlay technique for crossed immunoelectrophoresis is described. After the initial electrophoresis of antigens, the gel is overlaid with antibody-impregnated filter paper. This makes it necessary to pour only one gel, simplifying technique as compared with the conventional method.

Disc-crossed immunoelectrophoresis. A simple "laying-on" technique permitting the use of commercially available agarose.

A technique, disc-crossed immunoelectrophoresis, is described to provide a simple and convenient means of performing electrophoresis in polyacrylamide gel in a discontinuous buffer system (disc electrophoresis) followed by electrophoresis into antibody-containing agarose gel. A simple washing of the polyacrylamide gel in distilled water combined with a "laying-on" method, seems to be a satisfactory and rapid way to obtain immunoprecipitation patterns of high quality using commercially available agarose in the second electrophoresis.

Integration of monoclonal antibodies in quantitative immunoelectrophoresis by indirect immunoprecipitation.

Characterization of the specificity of monoclonal antibody in crossed immunoelectrophoresis has been achieved by mixing the monoclonal antibody with mycobacterial antigen in the circular antigen well. After electrophoresis in the first dimension, the separated antigens and antigen complexed with monoclonal antibody were run into an intermediate gel containing rabbit anti-mouse immunoglobulin and a top gel with polyvalent rabbit anti-BCG immunoglobulin. Monoclonal antibody with bound antigen precipitated in the intermediate gel while the other antigens precipitated in their reference positions. The method was simple, efficient and sensitive. Selected monoclonal antibodies were used to demonstrate the characteristic features of the method. In rocket immunoelectrophoresis the rocket height of a monoclonal antibody may be significantly altered by adding the relevant antigen. This principle can be exploited when the polyvalent antibodies do not precipitate the antigen, and it may be used for efficient screening of monoclonal hybridoma culture supernatants. This approach may permit the quantitation of an antigen when only monoclonal antibody is available.

Plasma C3d/C3 quotient as a parameter for in vivo complement activation.

Plasma levels of C3 and C3d in 30 healthy donors and 58 patients with systemic lupus erythematosus were determined by rocket immunoelectrophoresis and Con-A affinoimmunoelectrophoresis. Significantly decreased levels of C3 were found in approximately 50% of the SLE sera. Slight increases of free C3d were found in 55%, and marked increases in 9% of the SLE sera; the remaining SLE sera did not show increased C3d levels. When results were expressed as quotients of plasma C3d/C3, a significant pattern emerged and 70% of the SLE sera clearly fell outside the normal range. 10% of the SLE sera had normal C3d/C3 indices but had markedly depressed C3 levels. We conclude that in cases where total serum C3 is not markedly reduced (less than 60% of normal), the C3d/C3 quotient may be a more sensitive parameter for assessing in vivo complement activation than C3 or C3d determinations alone.

Crossed immunoelectrophoresis of mouse serum.

Because crossed immunoelectrophoresis (X-IEP) is quantitative and offers more information than immunoelectrophoresis (IEP), we selected X-IEP to study various physiologic and pathologic effects on the antigen composition of mouse serum. We therefore needed and X-IEP map of normal mouse serum. This paper presents one that we have developed, along with brief descriptions of how the mouse serum antigens were identified. Cross-reactivity between several mouse and human serum antigens was especially helpful. Some data from using ratios of the areas of precipitation of detected antigens to that of an internal standard antigen, alpha-macroglobulin, are presented. They show that X-IEP readily detects quantitative abnormalities among any of the several serum antigens detected by this technique, and that 'normal' values for these antigens in one population of mice can be established in the same way of using ratios with just a few analyses.

Measurement of the complement C3 breakdown product C3d by rocket immunoelectrophoresis.

A method is described for quantitative measurement of C3d in plasma and synovial fluid by the use of rocket immunoelectrophoresis after fractionation of the samples with 22% polyethylene glycol. This method has the advantage over radial immunodiffusion of being more sensitive (detecting C3d down to 3 mg/l) whilst proving equally reproducible. Investigations indicate that the collection of blood samples in EDTA prevents in vitro activation of C3 even after storage for up to 6 h at room temperature and up to 12 weeks at -70 degrees C. Elevated levels of C3d were found in a proportion of SLE and RA plasma samples and in synovial fluids from patients with inflammatory synovitis. It is suggested that C3 conversion in vivo may be assessed by measurement of C3d by the technique described, and when used in conjunction with measurements of complement components and immune complexes, offers a means of investigating complement catabolism by the classical and alternative pathways.

Antigen overlay technique for line immunoelectrophoresis and crossed-line immunoelectrophoresis of thermolabile antigens.

A simple antigen overlay technique for line immunoelectrophoresis and crossed-line immunoelectrophoresis is described. A blind gel is overlaid with antigen-impregnated filter paper instead to mix antigens with molten agarose and to pour them in a uniform sample gel. This makes it easier in line and crossed-line immunoelectrophoresis comparison of different antigen samples, simplifying technique as compared with the conventional method. Moreover, this technique is designated to obtain reproducible patterns from thermolabile proteins since they are unaffected by being heated.

Comparison of antigens and antisera in crossed immunoelectrophoresis by dual dilution.

The localization of individual precipitates in the crossed immunoelectrophoresis (CIE) pattern is an important feature for their identification, but in a given system this pattern may vary markedly when different antigen preparations or antisera are used. Two different antigen preparations or antisera may be compared by mixing them in various proportions in a set of four CIE plates. The amounts of the preparations to be compared are selected to obtain a gradual change in position of individual precipitates if differences are encountered. Similarities between antigen or antibody preparations are recorded when the positions of precipitates remain unchanged. The technique is particularly valuable for comparing complex patterns with a large number of precipitate lines.

A simple technique for increased sensitivity in immunoelectrophoresis by the use of antiserum against IgG.

Precipitates in two-dimensional immunoelectrophoresis (2D-IE) plates can be amplified by the application of an antiserum against IgG after the second dimension run. Thereby, the sensitivity of the IE method is increased; faint precipitates and antigen-antibody reactions close to but below the level of forming recognizable precipitates are made visible. The advantage of the method was demonstrated for a multilinear Mycobacterium phlei system.

C3 nephritic factor determination. A comparison between two methods.

C3 nephritic factor (NEF), an IgG autoantibody to the alternative pathway C3 convertase, is usually measured by crossed immunoelectrophoresis (CI) but recently a reliable haemolytic assay (HA) was described by Rother (1982). This method is more specific than CI because it is negative in sera with immune complexes, SLE and sera incubated with IgG aggregates. The haemolytic assay is sensitive enough to detect NEF antibody in serum from patients with only slightly low C3 levels and NEF negatives by CI. The haemolytic assay is easy to perform and reproducible, the interassay coefficient of variation being 10.7% compared to 64% in the CI. The intra-assay coefficient of variation in CI was 28% compared to 5.5% in the haemolytic assay. The haemolytic method enabled us to study the kinetic effects of NEF on C3b.Bb bound to sheep erythrocytes, and the lysis mediated by ShE.C3b.Bb.NEF complex. Also the C and NEF binding to sheep erythrocytes was studied.

Counterimmunoelectrophoresis (CIE) for the detection of anti-liver-kidney microsome (LKM) antibodies in the sera of patients with chronic liver disease.

A counterimmunoelectrophoresis (CIE) test for the detection of liver-kidney microsome specific antibodies in human sera is described. By testing different subcellular preparations the LKM antigen was found in the membranes of the smooth endoplasmic reticulum subfraction. The antigen was sensitive to trypsin digestion and behaved as an anionic protein in the experimental conditions used in the test. All sera positive for LKM in immunofluorescence gave a precipitin line of identity while none of the control sera gave a positive reaction. The CIE titers ranged between neat and 1/4096. A significant correlation was observed between the LKM titers obtained in immunofluorescence and those obtained in CIE. Moreover, by absorption experiments, it was concluded that the antigen preparation reactive in CIE was able to abolish the immunofluorescence pattern of LKM positive sera on rat liver and kidney sections. The LKM target antigen, although previously considered a structural protein of microsomal membranes, was shown to solubilize spontaneously during the isolation of microsomal membranes. Counterimmunoelectrophoresis appears to be an appropriate test for anti-LKM antibodies in human sera.