Binding of monoclonal antibodies to glomerular endothelium, slit membranes, and epithelium after in vivo injection. Localization of antigens and bound IgGs by immunoelectron microscopy.

The antigens recognized by seven monoclonal antibodies (MAbs) raised against rat glomerular proteins were localized, and the sites of binding of the MAbs after in vivo injection were determined by immunoelectron microscopy. The antigens were localized in situ by immunoperoxidase and immunogold labeling to different domains and microdomains of the glomerular endothelium and epithelium. 23A recognized an antigen expressed exclusively on the luminal (apical) domain of the endothelium. 5A (anti-podocalyxin) and 26C (anti-DPPIV) recognized antigens expressed on the apical domains of both the endothelium and podocytes. 13A, 14A, 20B (anti-gp330), and 27A recognized antigens restricted to podocytes in the glomerulus. The 13A antigen was present on their basal surface and the 27A and 14A antigens were expressed on both their apical and basal domains. The 14A antigen also was associated with the filtration slit membranes. All these MAbS bound to their antigens after injection in vivo. Those that recognize endothelial antigens were rapidly cleared from the circulation and rapidly disappeared from glomeruli, whereas those that recognize epithelial antigens persisted in the circulation and were detectable in glomeruli for hours or days. The sites of binding of the MAbs differed: 23A and 5A IgG (antipodocalyxin) bound exclusively to the luminal domain of the endothelium, whereas 26C IgG (anti-DPPIV) bound to both the luminal endothelial membrane and the apical and basal domains of podocytes. The MAbs that recognize podocyte antigens bound to different domains of the podocyte plasmalemma: 13A and 27A IgGs to the basal domain, 14A to the slit membranes, and 20B to coated pits on the entire plasma membrane. 27A IgG led to the formation of small subepithelial immune deposits that remained up to 10 days. It is concluded that 1) glomerular membrane proteins vary considerably in their distribution among plasmalemmal domains and microdomains of endothelial and epithelial cells; 2) virtually all structures in the glomerulus and all domains and micro-domains of the endothelium and podocyte are accessible to circulating antibodies; and 3) the fate of immune complexes formed by binding to glomerular components varies with the location of the antigen within the glomerulus, with those that bind to the basal domain and slit membranes of the podocyte persisting longer than the others.     

Preparative polyacrylamide gel electrophoresis in the isolation of the nephritogenic proteins of passive Heymann nephritis

To study kidney antigens involved in the formation of glomerular subepithelial immune deposits in passive Heymann nephritis polypeptides of 500, 130 and 105 kDa were isolated from rat kidney brush border (BB) membrane fraction using preparative polyacrylamide gel electrophoresis. Polyclonal antibodies raised against these proteins were specific for their respective antigens in immunoblotting. All three antisera bound to proximal tubular BB of kidney and to apical surfaces of several other epithelia as shown by indirect immunofluorescence on frozen sections of normal rat tissues. The anti-500 kDa and anti-105 kDa, but not the anti-130 kDa, antibodies also stained glomeruli and the anti-105 kDa antibodies also endothelial cells. After injection into rats the anti-500 kDa IgG bound to kidney glomeruli forming diffuse, granular deposits of rabbit IgG along the glomerular capillary walls, as shown by direct immunofluorescence. In electron microscopy the immune deposits were subepithelial and electron dense. The deposits remained in glomeruli for at least 60 days and increased with time. Deposits of C3 were not detected and proteinuria did not develop. The anti-130 kDa and the anti-105 kDa IgGs did not form glomerular deposits after in vivo injections. The results suggest that the 500 kDa and the 105 kDa proteins or related antigens are present in glomeruli and the 500 kDa protein is located on the epithelial side of the glomerular basement membrane. Circulating antibodies can bind to the 500 kDa protein forming immune complexes which rearrange and form electron dense deposits. The results further demonstrate that preparative gel electrophoresis is a useful technique for the isolation of kidney proteins of immunopathologic interest.     

Production of two novel monoclonal antibodies against human renal glomeruli and theri application to the immunohistochemical investigation of crescentic glomerulonephritis

Two monoclonal antiboides (MAbs) against human renal glomeruli, HEP1 and HBM1, were produced using isolated human glomeruli as an antigen. Immunohistochemical and immunoelectron microscopic analyses revealed that the recognition site of HEP1 is the cell surface of the visceral glomerularepithelial cells. HEP1 showed no reaction in the renal interstitium or in other tissues. HBM1 recognized an antigen present in the basal lamina of the renal glomeruli, Bowman's capsule, and partly in the basement membrane of renal tubules, but it was not reactive with the mesangial matrix or renal interstitium. The isotype of both MAbs was IgG1, k. The molecular weights of their corresponding antigens were 127–177 and 220kD, respectively. Besides human tissues, HEP1 also showed cross-reactions with antigens in the visceral glomerular epithelial cells of various animals other than mice. HBM1 recognized human antigen only. Using both MAbs, eight cases of crescentic glomerulonephritis were examined immunohistochemically. As a result, it was demonstrated that the crescents may not be formed by visceral glomerular epithelial cells. From this study, these two MAbs appear to be userful markers for the evaluation of renal glomerular disorders.     

Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli. I. Renal distribution.

The authors have previously reported the production of monoclonal anti-brush border antibodies defining two glycoprotein (gp) antigens of 90 and 330 kd which are also expressed on glomerular cells and may thus be of significance for in situ formation of immune complexes. The 330-kd antigen is involved in Heymann's nephritis, whereas the 90-kd antigen induces transient glomerular immune deposits. In this report, the authors describe the renal ultrastructural localization of the two antigens, which are both detectable on brush border and glomerular epithelial cells but assume contrasting patterns: gp 90 is diffusely present on the cell membranes, whereas gp 330 is concentrated in the intermicrovillar region and within the coated pits. Their localization within proximal tubular cells, on the membrane of intracytoplasmic vesicles, suggests that they are involved in endocytosis. In addition, gp 90 is expressed on endothelial cells lining glomerular capillaries. These observations may help us to understand the formation and different kinetics of immune glomerular deposits.     

Experimental membranous glomerulonephritis in rats induced with gp 700, a glomerular protein.

To investigate the antigenicity of glomerular cell products for the induction of Heymann nephritis (HN), eight Lewis rats were immunized with an isolated glomerular protein (GLP) with only limited tubular cell contamination. Four out of four rats immunized with 240 micrograms GLP showed proteinuria at week 12. Their kidney specimens taken at week 16 showed contiguous granular deposits of IgG along the glomerular basement membrane by direct immunofluorescence and corresponding electron-dense deposits; the findings were identical to those in classical HN induced by the brush border protein. By immunoprecipitation, IgG eluted from isolated glomeruli of rats that received 240 micrograms GLP precipitated only a 700 kd glycoprotein (gp 700) in GLP, whereas 330, 440, and 700 kd glycoproteins were precipitated in the crude preparation (Fx1A), which is derived mainly from renal tubules. Two different monoclonal antibodies (anti-gp 330 and 14C1) against gp 330 precipitated the antigens in the same fashion as the glomerular eluate. These data reveal that gp 700, gp 440, and gp 330 share epitopes and that the gp 700 is demonstrable both in the glomerulus and in tubular brush border but that gp 440 and gp 330 are present only in the brush border. In addition, glomerular gp 700 was shown to induce Heymann-type glomerulonephritis just as gp 330 did.     

Production of monoclonal antibodies specific to major allergens of Cryptomeria japonica pollen.

Monoclonal antibodies (MAbs) directed against Cryptomeria japonica pollen antigen (CPAg) were prepared. CPAg was precipitated with these MAbs and both MAbs CPA7 (IgG2a) and CPA9 (IgG1) recognized two major glycoproteins (m = 41 kDa and 46 kDa) of CPAg. MAb CPA7 or CPA9 was coupled to CNBr activated sepharose and affinity purification of the major allergen of CPAg from the crude extract was performed. The affinity purified CPAg bound IgE antibody present in patients with Cedar pollinosis.     

Rat glomerular cells do not express podocytic markers when cultured in vitro.

A panel of cell-type specific monoclonal and polyclonal antibodies and lectins was used to examine the early, morphologically epithelial outgrowth of rat renal glomerular cells in culture. The cell type-specific reactivity of the monoclonal antibodies has been previously verified on tissue sections of rat kidneys at light and electron microscopic levels. Morphologically distinct epithelial cells grew out from the isolated glomeruli within 3 days in culture, followed by the growth of morphologically typical stellate mesangial-like cells. Endothelial and mesangial cells were positively identified from the early cultures (up to 10 days) with antibodies to a 350 kD protein, dipeptidyl peptidase IV, podocalyxin, factor VIII, OX-43 and with Bandeiraea simplicifolia (BS-I B4) lectin, and with antibodies to smooth muscle actin, desmin, Thy1.1 antigens and with Ricinus communis (RCA-1) lectin, respectively. The antibodies recognizing podocytes in vivo (antipodocalyxin, anti-O-acetyl GD3 ganglioside, anti-gp330, anti-C3b complement receptor, anti-vimentin and anti-CALLA) consistently failed to bind to the predominant epithelial cells in early cultures, although these antibodies readily bound to the cells of the intact glomeruli remaining in culture. The attempts to augment the expression of cell-type specific epitopes by culturing glomeruli on various matrices or by enriching the medium with various growth factors, failed to induce podocytic epitopes on the growing epithelial cells. Glomeruli from newborn rats cultured in vitro, but were also constantly negative for the markers of podocytes. In addition, we cultured glomerular-like bodies from in vitro were induced metanephric mesenchymes but failed to obtain evidence of growing podocytes. However, the epithelial cells reacted with antibodies to thrombospondin and cytokeratin that react with the parietal epithelium of glomeruli on tissue sections. The results show that early glomerular cultures consist of mesangial, endothelial and presumably parietal epithelial cells readily identifiable by immunocytochemical methods. No podocytes could be grown under the various growth conditions tested. This suggests that glomerular podocytes are effectively growth arrested and call for new approaches to obtain these cells in culture.     

Autoantibodies to the laminin P1 fragment in HgCl2-induced membranous glomerulopathy.

Exposure to mercuric chloride induces the development of a membranous glomerulopathy with high proteinuria in DZB rats, in which immunoglobulin (Ig)G1 and IgG2a bound in the glomeruli were previously found to react with laminin of the EHS tumor and several unidentified glomerular basement membrane components. Monoclonal antibodies were prepared by fusing cervical and mandibular lymph node cells from a HgCl2-treated DZB rat with a nonsecreting mouse myeloma. Monoclonal antibodies were screened for reactivity with collagenase-digested glomerular basement membrane and kidney sections; upon subcloning, eight stable hybridomas were obtained, named MEC1 to MEC8. MEC2 (IgG1, kappa), MEC3 (IgM, kappa), and MEC5 (IgG1, kappa), as well as the polyclonal glomerular eluate, reacted preferentially with the P1 fragment of the laminin-1 (alpha 1 beta 1 gamma 1) isoform. MEC8 (IgM, kappa) reacted with the P1 and the E4 fragment of laminin. Both MEC6 (IgM, kappa) and MEC8 bound to actin and to various other, unidentified cellular antigens, indicating that MEC6 and MEC8 are polyreactive antibodies. MEC7 (IgM, kappa) bound to a cytoskeleton-linked cell membrane antigen, present on various epithelial cells and between heart muscle fibers and associated with small peripheral, intramuscular nerves. Several of the MEC monoclonal antibodies bound in vivo along the glomerular capillary wall. Although discrete electron-dense subepithelial immune aggregates were not detected and proteinuria was not induced, MEC3 localization changed from a continuous pattern into a fine granular pattern along the glomerular basement membrane, and focally along the TBM, upon passive transfer into naive DZB rats. These findings suggest a pathogenetic role for the P1 fragment of laminin either in the induction phase of HgCl2-induced membranous glomerulopathy as an immunogen or in the effector phase as a target antigen.     

Monoclonal antibodies against heavy and light chains of domestic mink IgG.

A panel of 26 monoclonal antibodies (MAbs) specific to mink IgG was produced and analyzed by ELISA, immunodiffusion assay (IDA) and immunoblotting assay. All the raised MAbs were directed against the isotypic IgG epitopes. Immunoblotting assay demonstrated that 11 MAbs reacted only with the Fc-fragments of IgG and 7 only with the light chains. Four antibodies bound to the Fab-containing fragments and failed to react with the Fc-fragments or isolated L-chains. Three MAbs did not react with IgG in IDA. Based on the results of IDA and cross-blocking assays, the MAbs were divided into 10 groups, with the MAbs of each group recognizing the same epitope. In IDA some MAbs were able to react with the epitopes which are common to the IgGs of some other representatives of Mustelidae family and also to some mammalian species remote from mink (dog, horse, pig, fox and rabbit).     

Expression of IgG Fc receptor antigens in placenta and on endothelial cells in humans. An immunohistochemical study.

Expression of leukocyte IgG Fc receptor (Fc gamma R) antigens by placenta, endothelial cells (EC) of normal tissues, and ECs of kidney and skin from subjects with immune complex diseases was studied immunohistochemically using anti-Fc gamma R monoclonal antibodies (MAb). Monoclonal antibodies against all three leukocyte Fc gamma R classes stained placental villous macrophages. Placental villous trophoblasts were stained intensely by anti-Fc gamma RIII MAb 3G8, while both anti-Fc gamma RI (MAb 32) and anti-Fc gamma RII MAbs IV3, KU79, CIKM5, 2E1, KB61, and 41H16) antibodies did not react with these cells. Anti-Fc gamma RII MAbs IV3, KU79, CIKM5, 2E1, KB61, and 41H16 immunostained placental villous capillary EC, in contrast to anti-Fc gamma RI MAb 32 and anti-Fc gamma RIII MAb 3G8, CLB-Granl, and B73.1, which did not bind. Anti-Fc gamma RI MAb 32, anti-Fc gamma RII MAb IV3 and CIKM5, and anti-Fc gamma RIII MAb 3G8 did not react with the ECs of tonsil, liver, kidney, spleen, intestine, lung, or uterus. Similarly no EC staining was seen with these four MAbs in 14 skin and 14 kidney biopsies from subjects with immune-complex diseases. Fc gamma R antigens are expressed constitutively only by placental villous ECs and are not induced on nonplacental ECs by immune-complex-mediated diseases.     

Identification of a 400-kd protein in the brush borders of human kidney tubules that is similar to gp330, the nephritogenic antigen of rat Heymann nephritis.

The nephritogenic antigen of Heymann nephritis (HN)--a well-studied experimental rat model disease of human membranous glomerulonephritis (MGN)--was recently shown to be a 330-kd glycoprotein (gp330) which is present in the membranes of both the rat tubular brush borders and of podocytes. Because the pathogenic antigen(s) of MGN are unknown, the authors have searched for a gp330-like molecule in human kidney and for its role in MGN. The authors here report that a membrane protein (apparent molecular weight 400 kd) is present in human kidney which is immunologically cross-reactive with rat gp330. By immunoelectron microscopy (using rabbit anti-rat gp330 IgG or a monoclonal anti-400-kd IgG) this molecule is similarly localized in human proximal tubules, but it is absent from the podocytes of human glomeruli. The 400-kd molecule is not detected in the glomerular immune deposits of 30 biopsies of MGN. It is proposed that this is due to the lack of the 400-kd protein in human glomeruli which prevents the formation of initial 400-kd anti-400-kd IgG immune complexes in situ.     

Antibodies against mesangial cells and their secretory products in chronic renal allograft rejection in the rat.

Antibodies or cell-mediated immunity can cause chronic rejection of vascularized organ grafts, but the nature and specificity of the antigen(s) involved has remained elusive. We have previously demonstrated the presence of antibodies against cryptic glomerular basement membrane antigens and undefined antigens in the mesangial area in rats with chronic renal allograft rejection. Current experiments were designed to study the post-transplant antibody response against cultured mesangial and endothelial cells in rats with chronic rejection using flow cytometry, indirect immunofluorescent staining, immunoelectron microscopy, confocal microscopy, and Western blots. The results were compared with those obtained with alloantisera raised by immunization with cultured mesangial cells. Post-transplant and post-immunization sera contained IgG antibodies against trypsinized mesangial cells detected by flow cytometry. Indirect immunofluorescent studies using mesangial cells grown on coverslips showed autoantibody binding to cytoplasmic granules in cultures early after plating whereas staining of later cultures showed antibody binding in an interrupted, web-like pattern on the outside of the cells. Immunoelectron microscopy showed autoantibody binding to intracellular secretory granules and to cell surface focal adhesion plaques. The latter finding was confirmed in double-labeling experiments with an antiserum against vinculin. Western blots with mesangial cell culture supernatants demonstrated autoantibody reactivity with antigens in the 40-kd and 60- to 70-kd range, and immunoprecipitation identified these molecules as biglycan and decorin. Absorption of the sera with mesangial cell culture supernatant removed most of the antibodies except those that gave a punctate staining with the mesangial cell surface. However, not all immunostaining of mesangial cells could be explained by antibodies against biglycan and decorin. Post-transplant sera, furthermore, contained low-titered antibodies against endothelial cells. We conclude that rats with chronic renal transplant rejection produce a strong autoantibody response against mesangial cell focal adhesion plaques and proteins secreted by these cells in culture. Such antibodies may cause local damage and interfere in the tissue repair process after injury.     

Monoclonal antibodies reactive with K1-encapsulated Escherichia coli lipopolysaccharide are opsonic and protect mice against lethal challenge

Seven murine monoclonal antibodies (MAbs) directed against O-side-chain determinants of the K1-encapsulated Bortolussi strain of Escherichia coli (O18:K1:H7) were evaluated for their in vitro and in vivo activities. All the MAbs reacted well in Western blots against E. coli O18 lipopolysaccharide antigens. Two MAbs of the immunoglobulin G (IgG) class promoted in vitro opsonophagocytosis and protected mice lethally challenged with bacteria. Two IgM MAbs showed partial protection, although they had no in vitro opsonic activity, and the remaining three IgM MAbs showed no apparent functional activities. Monoclonal IgG antibodies against bacterial lipopolysaccharide can be opsonic and protective in spite of the presence of the K1 capsule on the bacterium.     

Identification of spirochetes related to Treponema pallidum in necrotizing ulcerative gingivitis and chronic periodontitis.

BACKGROUND. Spirochetes are commonly associated with periodontal disease, but it is not known whether these treponemes are pathogenic or merely opportunistic. We sought to determine whether spirochetes present in periodontal disease share antigens thought to be unique to spirochetes that are known pathogens. METHODS. We examined dental plaque from 24 healthy subjects, from ulcerative sites in 17 patients with ulcerative gingivitis, and from areas of involvement in 19 patients with chronic periodontitis, using an immunocyto-chemical technique with monoclonal antibodies against pathogen-specific determinants on 47-kd and 37-kd molecules from Treponema pallidum subspecies pallidum. Serum was tested against T. pallidum by immunoblotting and by serologic assays for syphilis. RESULTS. Spirochetes with a pathogen-specific epitope on a 47-kd molecule were not found in plaque samples from any of the 24 healthy subjects, but they were identified in plaque samples from 11 of 17 patients with ulcerative gingivitis (P less than 0.001) and from 10 of 19 patients with periodontitis (P less than 0.01). Monoclonal antibodies directed against a 37-kd molecule reacted with spirochetes in plaque samples from 1 of 14 controls, from all 11 patients with gingivitis from whom samples could be obtained (P less than 0.001), and from 14 of 19 patients with periodontitis (P less than 0.001). Five of 18 normal subjects had IgG against 47-kd and 37-kd molecules, but none had IgG against 14-kd or 12-kd molecules from T. pallidum subspecies pallidum. Among 19 patients with ulcerative gingivitis, IgG was identified against 47-kd molecules in 15, against 37-kd molecules in 12, against 14-kd molecules in 4, and against 12-kd molecules in 15. CONCLUSIONS. The spirochetes found in dental plaque from patients with ulcerative gingivitis or chronic periodontitis have antigens that are thought to be unique to pathogenic treponemes. This close antigenic relation suggests that T. pallidum or a closely related organism may be involved in the pathogenesis of periodontal disease.     

A plasma membrane antigen of rat glomerular epithelial cells. Antigenic determinants involving N-linked sugar residues in a 140-kilodalton sialoglycoprotein of the podocytes.

The aim of the present study was to investigate the antigenic make up of the plasma membrane of rat glomerular visceral epithelial cells (GEP). A crude plasma membrane fraction (PM) was extracted by 1% sodium dioxycholate from isolated rat glomeruli. PM was digested with neuraminidase (NRD) and purified by the Helix pomatia agglutinin (HPA)-affinity column. When studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the HPA-affinity purified plasma membrane fraction (HPA-PM) formed two bands, a main band of 160 kilodaltons (kd) and a smaller band of 40 kd. By Western blot analysis, the antibodies raised in a rabbit against HPA-PM (RbAHPA-PM) reacted only with the 160-kd protein of HPA-PM, or with the relevant 140-kd protein of PM when PM was digested by NRD. The 140-kd protein of PM was reactive with wheat germ agglutinin and, when treated with NRD, was reactive with HPA and peanut lectin. The 160-kd protein of HPA-PM was degraded by endoglycosidase-F and lost its reactivity with RbAHPA-PM. These results suggest that RbAHPA-PM react with antigenic sites involving N-linked sugar residues of a 140-kd sialoglycoprotein, presumably podocalyxin. Immunohistochemical studies using normal rat kidney tissues treated with NRD as substratum showed that RbAHPA-PM bound to the free surface of GEP but not with soles of the foot processes or with other structures of the kidney. In rats intravenously injected with NRD and subsequently with RbAHPA-PM, antibodies were rapidly fixed (within 1 hour) to the free surface of GEP. Immunofluorescence study showed that RbAHPA-PM also reacted with human glomeruli after treatment with NRD. These results suggest that GEP express surface sugar residues that are potential targets for direct immunologic attack.     

Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats

Summary Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular-fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double-staining techniques of rat kidney sections obtained from normal and rabbit anti-FX1A-injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti-FX1A antibody. Western blot analysis have shown that the rabbit anti-rat FX1A IgG and the rat anti-rat KF3 IgM antibodies are directed against the same renal tubular-derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.     

Comparison of antigenic targets involved in antibody-mediated membranous glomerulonephritis in the mouse and rat

Membranous glomerulonephritis in the mouse can be induced by a single injection of an antiserum against homologous, pronase-digested, renal tubular antigens (TAPron). In indirect immunofluorescence studies on normal mouse and rat kidneys it has now been found that the antiserum reacts strongly with the visceral epithelia of the mouse in a homogeneous pattern, while a faint granular staining is seen in the rat glomerulus against a homogeneous background. After injection in rats, a classic passive Heymann nephritis could be induced. By immunoprecipitation of radiolabeled rat renal brush borders (BB) it could be shown that anti-TAPron antisera contain antibodies to 330-kd and 90-kd BB proteins expressed by rat glomeruli. With the use of two monoclonal antibodies specific for the 330- and 90-kd proteins the homogeneous binding observed in rat and mouse glomeruli could be related to the 90-kd antigen, whereas the coarse irregular staining observed in rat glomeruli was only related to the 330-kd antigen. Immunoglobulins eluted from glomeruli of rats bound to rat glomeruli and reacted only with the 330-kd protein. They did not bind to mouse glomeruli. Discrete localization in coated pits, multivesicular bodies, and endoplasmic reticulum of the visceral epithelia was seen in immunoelectron-microscopy. The results presented thus demonstrate that immune deposits induced in the rat by anti-TAPron antibodies are related to antibodies specific for the 330-kd antigen, ie, the classic Heymann antigen. By contrast, immune deposits observed in the mouse are related to antibodies specific for a 90-kd protein.     

Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids

Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.     

IgA-associated renal diseases: Antibodies to environmental antigens in sera and deposition of immunoglobulins and antigens in glomeruli

Levels of IgA1, IgA2, IgM, and IgG antibodies specific for 10 ubiquitous food and bacterial antigens were examined by radioimmunoassay in the sera of 29 patients with IgA-associated renal diseases and 22 normal individuals. No significant differences were observed between patient and normal groups in the levels of IgA1 antibodies, and IgA2 antibodies were detected in only a few individuals in either group. Minor differences in IgM or IgG antibodies were seen against some antigens. Significant positive correlations between IgA1 and IgG and between IgA1 and IgM antibodies to casein were found in the patient group. Analysis of the molecular form of serum IgA1 antibodies revealed that although the pattern of polymeric and monomeric forms varied between individuals and between antibody specificities, there was no preponderance of one form in either patient or normal groups. Examination of kidney biopsies from 50 patients with IgA-associated renal diseases revealed that IgA1 represented the predominant subclass deposited in the glomerular mesangium; glomeruli from three patients contained both IgA1 and IgA2. Seventy-eight percent of the patients also had deposits of IgM, although IgA and IgM deposits did not always coincide. When IgG was present in glomeruli (45% of patients), the IgG1 subclass predominated. J chain was detectable in glomeruli of only four patients. C3 was detected in glomeruli of 95% of the patients, although the distribution of C3 did not always coincide with that of IgA. Indirect immunofluorescence staining with rabbit antisera to various environmental antigens showed that milk protein antigens could be deposited in association with IgA in the glomerular mesangium.