Further studies on the ultrastructure of dimeric IgA of human origin.

Human colostral IgA and myeloma dimer IgA were purified and examined in the electron microscope using a modified technique of negative staining. Both types of preparation contained double Y-shaped structures of the dimensions: Fab region, 35 x 70 A, and the sum of the two Fc regions, 40 x 140-155 A. Colostral IgA as well as myeloma dimer IgA molecules showed a tendency of bending at the point where the Fc regions joined. Secretory component bound to dimer IgA produced no visible alteration of the molecule. Mild reduction and alkylation of colostral IgA yielded single Y-shaped 7S monomers with the dimensions, Fab, 35 x 70 A, and Fc, 40 x 65-70 A.

Ultrastructural studies of human and rabbit alpha-M-globulins.

Electron micrographs of isolated human alpha(2)M-molecules, obtained by the negative contrast technique, revealed morphologically homogenous structures resembling a graceful monogram of the two letters H and I. The modal values for the length and width of the alpha(2)M particles were 170 A and 100 A, respectively. Purified rabbit alphamacroglobulins contained about 80% alpha(1)M- and 20% alpha(2)M-globulins. The isolated rabbit alpha(1)M- and alpha(2)M-molecules were morphologically indistinguishable from one another and from human alpha(2)M-molecules. Preliminary immunoprecipitation studies demonstrated that the two rabbit alphaM-globulins were antigenically different. Sedimentation constant determinations gave s(20, w) values of 18.8 and 18.2 for rabbit alpha(1)M and alpha(2)M, respectively.

The ultrastructure of normal and pathological IgM immunoglobulins.

Free IgM immunoglobulins were examined in the electron microscope using the negative contrast technique. Normal human and rabbit IgM and Waldenström macroglobulins were indistinguishable from one another and revealed flexible spider-like particles with five appendages joining a central ring. The average total span of the molecules was 300 A. The appendages were about 125 x 30 A; the central ring had an outer diameter of approximately 100 A and an inner diameter of 40 A. Some purified 19S IgM preparations tended to form massive aggregates (>/=50S) which, when examined in the electron microscope, revealed enormous clumps of IgM molecules whose appendages were entangled with one another. Electron microscopy of reduced-alkylated IgM revealed total absence of intact spider-like molecules. The predominating structure observed was a round electron-dense knob about 50 A in diameter which in some cases had a fine fiber-like extension with approximate dimensions 100 x 15 A. Rabbit and human IgM molecules with antibody activity to poliovirus dried in sodium tungstosilicate on a carbon film as in preparation for electron microscopy were shown to retain nearly 100% of their poliovirus neutralizing activity after redissolving in a physiological buffer.

Ultrastructure of papain and pepsin digestion fragments of human IgM globulins.

The ultrastructure of papain and pepsin-digested products of human IgM globulins has been analyzed. Papain digestion was performed both in the presence and absence of cysteine. The Fcmicro fragment was found to represent the central ring structure in the intact IgM molecule, plus a minor part of the appendages extending from the ring. The Fcmicro ring structure was occasionally seen to be composed of dimers of short rods, probably identical with the endpieces of two micro-chains. Such dimeric structures, released from the intact Fcmicro rings, had a tendency to aggregate sidewise, producing complexes of varying size. The dimensions of the Fcmicro fragments were: outer diameter approximately 85 A, inner diameter about 40 A. The length of the protrusions varied from 20-30 A. The Fabmicro preparations contained long strands of sidewise aggregated, short rod-shaped fragments. No aggregates were seen in the F(ab'')(2)micro preparations. The two Fab''micro units in the dimeric F(ab'')(2)micro fragments were usually parallel to each other. The dimensions of the Fabmicro and F(ab'')(2)micro fragments were 50-80 A x 30 A and 75-80 A x 55 A, respectively. These findings provide morphological evidence that the C-terminal ends of the micro-chains (the Fcmicro fragment) make up the central ring structure in the IgM molecule. They further indicate that the F(ab'')(2)micro fragments constitute about (3/4) of the appendages extending from this ring structure.

Ultrastructure of secretory and high-polymer serum immunoglobulin A of human and rabbit origin.

Electron micrographs of immunoglobulins A from human and rabbit colostrum, which were purified on tall agarose columns, revealed Y-shaped molecules (125 by 140 angstroms). The linear dimensions of the arms were 55 to 75 by 25 to 30 angstroms. A molecular model is postulated in which two immunoglobulin A monomers are superimposed on each other in a close-packed state with the secretory piece inserted in the constant region of the alpha-chains. High-polymer (11 or 13S) immunoglobulin A molecules (total span 100 to 110 angstroms) from human serum were composed of four arms (50 to 55 by 20 angstroms) joined at a contrast-rich center.

Ultrastructure of gamma-M immunoglobulin and alpha macroglobulin: electron-microscopic study.

Electron microscopy of purified Waldenström macroglobulins and normal human and rabbit gammaM immunoglobulins revealed spider-like structures with five legs varying in length and often joining a central ring. Usually only the central more rigid part of this structure (about 150 by 170 angstroms) was clearly visible, but occasionally particles were seen with longer very flexible legs having a total span of about 350 angstroms. Molecules of gammaM antibody retained antibody activity during preparation for electron microscopy. Human and rabbit alpha macroglobulins revealed more rigid symmetric structures (100 by 200 angstroms) which resembled the Russian letter .

Calcium-dependent and -independent binding of the pentraxin serum amyloid P component to glycosaminoglycans and amyloid proteins: enhanced binding at slightly acid pH.

Serum amyloid P component (SAP), a member of the pentraxin family of proteins, binds calcium-dependently to several ligands including glycosaminoglycans (GAG's). We have investigated the influence of pH on the Ca2(+)-dependent binding of SAP to solid phase GAG's and amyloid fibril proteins (AA and beta2M) by ELISA. An increase in the dose-dependent binding of SAP to heparan sulfate, AA-protein and beta2M was observed as the pH decreased from 8.0 to 5.0. Furthermore, a lower, but significant Ca2(+)-independent binding of SAP to heparan sulfate, dermatan sulfate, AA protein and the amyloid precursor protein beta2M was observed. This binding was also enhanced at slightly acid pH, most pronounced at pH 5.0. The results of this study indicate that SAP can exhibit both Ca2(+)-dependent and -independent binding to ligands involved in amyloid fibril formation and that the binding is enhanced under conditions of slightly lowered pH.

ELISA quantitation of IgG subclass antibodies to dietary antigens.

The IgG subclasses of human antibodies against 2 dietary antigens, ovalbumin (OA) and beta-lactoglobulin (BLG), were studied by ELISA using monoclonal anti-human IgG subclass antibodies. Under the assay conditions used, the anti-IgG subclass antibodies were subclass specific. Quantitative estimates of the subclass antibodies were obtained by reference to a 'capture' assay using F(ab')2 anti-light chain antibody as ligand and IgG myelomas as standards. The validity of these estimates was supported by antibody quantitation using the Farr assay. In healthy adults with serum anti-OA or anti-BLG antibodies, anti-OA antibodies were found mainly in the IgG1 (9/11) and IgG4 (6/11) subclasses, whereas 5 sera showed high levels of IgG2 antibodies. In contrast, the IgG subclass distribution of anti-BLG antibodies was predominantly IgG4 (10/10).

A standardized method for quantitating the complement-mediated immune complex solubilizing capacity of human serum.

A standardized radioassay for measuring the complement-mediated immune complex solubilizing capacity (CMSC) and the initial kinetics of the solubilization (IKS) reaction is described. The total complement (C)-mediated solubilizing capacity was determined after incubation of diluted serum and 125I-BSA-anti-BSA. Percentage C-mediated solubilization (CMS) was measured after centrifugation by determining the distribution of radioactivity. The dependency of CMSC upon factors such as serum dilution and buffer system used, amount of IC added to serum, serum storage conditions and centrifugation conditions was investigated in order to optimize the assay. The CVt of the standardized assay was 0.10-0.17 depending upon the CMSC level measured. Treatment which inactivates C factors (heating), interferes with C activation (EDTA) or activates and consumes C components (zymosan) markedly reduces the CMSC. Preliminary investigation of pathological sera showed that both IKS and CMSC were clearly reduced in SLE sera. By contrast, rheumatoid arthritis sera exhibited normal IKS and only marginal reduction in CMSC.

An automatic multi-programmed affinity chromatographic system.

An automatic immunospecific affinity-chromatographic system for continuous operation is described. The system comprises time-controlled sample application, washing and elution steps and automatic dialysis of eluted fractions. The applicability of the system is illustrated by the purification of pregnancy zone protein on immunosorbent gel.

ELISA for evaluating the incorporation of plasma derived complement split-products C3b/iC3b into solid-phase immune complexes.

An ELISA that measures plasma derived complement (C) split-products C3b/iC3b deposited on solid-phase immune complexes during C activation is described. Plates are coated with BSA, anti-BSA and plasma is added. Deposited C3b/iC3b is then detected by biotinylated anti-C3c-antibodies, avidin-alkaline phosphatase and para-nitrophenylphosphate. A novel feature is that the assay measures residual C activation capacity rather than in vivo generated C activation products. The assay was applied to plasma from 250 healthy blood donors. No difference in activation capacity of either the alternative (AP) or classical pathway (CP) with regard to age or gender was demonstrated. The total coefficient of variation was <5.7%. The ELISA procedure was compared to a standard hemolytic complement CH(50) assay using plasma from 23 out-patients with systemic lupus erythematosus (SLE). There was a weak correlation between the two assays for both C pathways, but neither the ELISA nor the CH(50) assay showed any correlation with the diagnostic ACR-criteria for SLE. However, the capacity of the CP was significantly reduced in SLE out-patients compared to healthy blood donors (P<0.0001).

Affinity chromatographic purification of the pregnancy zone protein.

An immunospecific affinity chromatographic method for purification of human pregnancy zone protein (PZP) directly from serum is described. Highly purified goat-anti-human PZP-immunoglobulin was applied as a ligand. Recovery of PZP varied from 56--75%. The impurities constituted maximally 5--10% of the total protein in the eluate. The purification factor was approximately 100.

An immunoprecipitation-dissociation technique for large scale antibody purification and an antigen consumption electroimmunoassay for antibody quantitation. A model study with antibodies to pregnancy zone protein.

A simple immunoprecipitation--dissociation technique for large scale purification of antibodies is described, which comprises selective denaturation of the antigen and recovery of the antibody fraction by exclusion chromatography at low pH. Its use is illustrated by the purification of antibodies to pregnancy zone protein. A purification factor of about 60 was achieved. An antigen consumption electroimmunoassay was also developed which permits quantitative determination of the antigen binding activity of antibodies with a given specificity. The methods have general application.

Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll).

A standardized, reproducible two-step method for separation of human peripheral blood monocytes on continuous Percoll gradients has been developed. The first step involves separation of mononuclear cell on Percoll of density 1.075 g/ml and the second step separation of monocytes from lymphocytes on a continuous Percoll gradient with a starting density of 1.075 g/ml for the formation of the gradient. The average yield during a 10 month period of daily routine use has been 74 +/- 17% (mean +/- 1 S.D.), and the average purity 63 +/- 10%. Ninety to 95% of the monocytes are viable after separation as judged from trypan blue exclusion and by ingestion of latex particles and sensitized sheep erythrocytes. The separation takes about 3 h and the total number of monocytes obtained from 40 ml of blood is in the range of 10-15 x 106. The procedure has been reliable with 3-4% separation failures, mainly due to bacterial or fungal growth in Percoll suspension or media. The contaminating cells are exclusively lymphocytes, predominantly T-lymphocytes (90-95%), when citrate is used as anticoagulant. Heparin can not be used as anticoagulant, as there appears to be a dose-dependent formation of thrombocyte aggregates which contaminate the monocytes, and result in poor separation.

Double-decker rocket immunoelectrophoresis for direct quantitation of complement C3 split products with C3d specificities in plasma.

A double-decker rocket immunoelectrophoresis (DD-RIE) method for direct quantitation of complement split products with C3d determinants in human plasma is described. The usefulness of the DD-RIE method for monitoring C3 activation has been assessed and compared with conventional crossed immunoelectrophoresis (CIE) for C3c determination in a patient with iatrogenic septic shock and patients with rheumatoid arthritis. In contrast with CIE the DD-RIE method is quantitative by reference to a standard curve based on an internal reference C3d preparation and its sensitivity and assay capacity are superior to CIE. All reagents and antibody preparation are commercially available and the production of standards is easy. No overlapping was observed between C3d values in plasma from healthy persons and patients with active classical rheumatoid arthritis. The DD-RIE is highly suitable for routine use in laboratories of clinical immunology.

A method for preparation of immunosorbents by direct coupling of dissociated immune complexes.

A simple and rapid method of preparing immunosorbents by use of isolated immune complexes is described. Dissociated immune complexes may be directly coupled to activated Sepharose and an immunosorbent gel with complementary molecular populations of immunospecifically purified ligands is obtained. The antigen binding capacity of insolubilized antibodies was about 50% of the antigen bound at equivalence using liquid phase precipitation. The batch technique described may be scaled up and the only limitation is the availability of the ligands. The usefulness of the technique is illustrated in two model systems.

The third complement factor (C3) and its in vivo cleavage products: interaction with lectins and precipitation with polyethylene glycol.

Five molecular forms of C3 expressing D but not C epitopes were identified following in vivo activation of the complement system. Examination of concanavalin A (Con-A) reactivity in crossed immunoelectrophoresis revealed that native C3, C3c and the beta mobile form 4 of C3d were completely precipitated by 100 micrograms Con A/cm2. The alpha-1 mobile form 1 of C3d did not interact with Con A, whereas the alpha-2 mobile forms 2 and 3 were retarded in electrophoretic migration by Con A. Native C3, C3c, and forms 4 and 5 of C3d were precipitated by 12% (w/v) polyethylene glycol (PEG). Form 1 of C3d was soluble in these PEG concentrations, whereas forms 2 and 3 were partially precipitated.

Partial characterization of circulating macromolecular beta 2-microglobulin complexes by density gradient ultracentrifugation and page-blotting.

beta 2-microglobulin (beta 2m)-containing, rapidly sedimenting peaks were seen when sera of patients with 3% polyethylene glycol (PEG)-insoluble beta 2m were fractionated by sucrose density gradient ultracentrifugation. No such material was found in serum of a healthy volunteer without 3% PEG-insoluble beta 2m. MW of beta 2m-containing high molecular weight fractions ranged from 1.6 X 10(5) to 2.1 X 10(6). When some fractions were recentrifuged a majority of beta 2m was recovered at the original sedimentation position. Analysis of high molecular weight beta 2m-containing peaks by SDS-PAGE followed by electroblotting revealed only monomeric beta 2m indicating that beta 2m was not covalently bound in the high molecular weight material.

An in vitro cellular system for generation of AA-amyloid.

Different conditions for establishing a cell culture system for generation of AA-amyloid were investigated. The most effective system was based on peritoneal macrophages from CBA/J mice that had received repeated injections of Hammersten casein, with subsequent cultivation of the cells at high density, high levels of acute phase serum, and neutral pH. Staining with Congo red, thioflavin T, and anti-AA revealed amyloid-like structures associated with macrophage clusters. The structures increased in number and size from day 2 to 6 of cell cultivation. The concentration of apoSAA in the culture medium fell markedly in the amyloid-producing cell cultures, while the SAP concentration was not reduced. The described cell culture system can be useful in studies of the influence of chaperone molecules and other factors or the formation and degradation of amyloid fibrils.

Enhanced binding of immune complexes processed by erythrocyte CR1 (CD 35) receptors to purified CR2 (CD 21) receptors from tonsillar mononuclear cells.

The binding of immune complexes (IC) opsonized by serum complement (C) and IC processed by CR1 (CD 35) receptors on human erythrocytes (E) to purified CR2 (CD 21) receptors was compared. Soluble CR2 was prepared from tonsillar mononuclear cells and purified by antibody affinity chromatography. Solid phase CR2 as well as CR2 subjected to PAGE and blotted onto nitro-cellulose membranes bound 125I-labelled BSA anti-BSA IC which had been opsonized by C and processed by CR1 up to ten times more efficiently than IC reacted with serum only. Radiolabelled monomeric C3d also bound to solid phase CR2. The binding of IC to purified and solid phase bound CR2 could be inhibited by anti-CR2 antibodies or by preincubation of the IC with polyclonal antibodies reacting with C3d or C3b/iC3b. Thus, both C3dg and iC3b appeared to mediate binding of IC to CR2. Preincubation of solid phase CR2 with purified monomeric C3d did not inhibit the subsequent binding of E-CR1 processed IC. The data indicate that E-CR1 have an important role in generating IC which bind effectively to CR2 receptors on B lymphocytes.