Anti-membrane Antibodies Research Articles

Goodpasture Syndrome with Neurological Condition Associated with anti-MBG Ab, Negative ANCA: Case Report

Goodpasture syndrome is a rare immunologically based clinical entity that is characterized by rapidly progressive glomerulonephritis, diffuse pulmonary hemorrhage, and the presence of circulating autoantibodies against the glomerular basement membrane (GBM). Concomitant central nervous system (CNS) manifestations are an unusual presentation of GSP in the absence of ANCA. We present a case of atypical course of the disease in a 48-year-old female patient who started with the presence of hemoptoic sputums and manifestations of uremic syndrome in the presence of high titres of glomerular basement anti membrane antibodies (Ac-anti-MBG) and negative ANCA, with a report of diffuse active extracapillary proliferative glomerulonephritis biopsy, of the pauciimmune type. After immunosuppressive treatment and plasmapheresis, the patient presented decreased anti-MBG Ab titres, as well as improvement of symptoms, however without recovery of renal function on hemodialysis, after 2 months he presents clonic-tonic seizures crisis, we conclude that PGS with neurological involvement is extremely rare, especially with negative ANCAs.

Anti-membrane Antibodies Persist at Least One Year and Discriminate Between Past Coronavirus Disease 2019 Infection and Vaccination.

The consequences of past COVID-19 infection for personal and population health are emerging, but accurately identifying distant infection is a challenge. Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline. We evaluated anti-membrane antibodies in COVID-19 naïve, vaccinated, and convalescent subjects to determine if they persist and accurately detect distant infection. We found that anti-membrane antibodies persist for at least a year and are a sensitive and specific marker of past COVID-19 infection. Thus, anti-membrane and anti-spike antibodies together can differentiate between COVID-19 convalescent, vaccinated, and naïve states to advance public health and research.

Anticancer activities of anti-membrane antibodies against colon carcinoma cells undergoing chemotherapy.

Chemotherapy combined with target therapy using antibodies against tumor cell membrane antigens may greatly increase the survival of cancer patients. Similar to autoantigens in autoimmunity diseases, certain membrane components may be more heterogeneous and create new determinants of antigens or haptens after chemotherapy. The aim of the current study was to prepare anti-membrane antibodies against colon carcinoma cells undergoing chemotherapy and examine their anticancer activities in vitro. After the colon carcinoma cells were treated by mimic chemotherapy, the synthesized poly-lysine was used as a carrier to link the membrane antigen or hapten with the covalent bond of carbodiimide bridging. It was affirmed by fluorescence-activated cell sorting under laser confocal microscopy that the vaccine with poly-lysine membrane-linked cells with a covalent bond was successfully engineered. Then, the cognate mice were vaccinated, and the anti-membrane polyclonal antibodies were prepared and validated for their activities. The anti-membrane polyclonal antibodies were effectively induced and prepared. Folliculus lymphaticus were found significantly increased in vaccinated mice, and B lymphocyte proliferation was also intensively stimulated by vaccine and generating antibodies. The polyclonal antibodies, exhibiting minimal endotoxicity, displayed intensive sensitivity, high affinity and strong specificity. They also elicited apoptosis and necrosis for wild type colon carcinoma cells and offered synergistic effect to repress the chemotherapy-resistant tumor cells. The poly-lysine-linked membrane for colon carcinoma cells undergoing chemotherapy could produce the anti-membrane polyclonal antibodies (promising as novel antibody molecules for target therapy) and generate an effective immune attack on the surviving cancer cells.

Anticancer Activities of Anti-Membrane Antibodies against Colon Carcinoma Cells Undergoing Chemotherapy

Chemotherapy combined with target therapy using antibodies against tumor cell membrane antigens may greatly increase the survival of cancer patients. Similar to autoantigens in autoimmunity diseases, certain membrane components may be more heterogeneous and create new determinants of antigens or haptens after chemotherapy. The aim of the current study was to prepare anti-membrane antibodies against colon carcinoma cells undergoing chemotherapy and examine their anticancer activities in vitro. After the colon carcinoma cells were treated by mimic chemotherapy, the synthesized poly-lysine was used as a carrier to link the membrane antigen or hapten with the covalent bond of carbodiimide bridging. It was affirmed by fluorescence-activated cell sorting under laser confocal microscopy that the vaccine with poly-lysine membrane-linked cells with a covalent bond was successfully engineered. Then, the cognate mice were vaccinated, and the anti-membrane polyclonal antibodies were prepared and validated for their activities. The anti-membrane polyclonal antibodies were effectively induced and prepared. Folliculus lymphaticus were found significantly increased in vaccinated mice, and B lymphocyte proliferation was also intensively stimulated by vaccine and generating antibodies. The polyclonal antibodies, exhibiting minimal endotoxicity, displayed intensive sensitivity, high affinity and strong specificity. They also elicited apoptosis and necrosis for wild type colon carcinoma cells and offered synergistic effect to repress the chemotherapy-resistant tumor cells. The poly-lysine-linked membrane for colon carcinoma cells undergoing chemotherapy could produce the anti-membrane polyclonal antibodies (promising as novel antibody molecules for target therapy) and generate an effective immune attack on the surviving cancer cells.

Identification of plasma membrane autoantigens in autoimmune hepatitis type 1 using a proteomics tool

Autoimmune hepatitis (AIH) is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components. Because AIH is a liver-specific disease, autoantibodies against plasma membrane antigens may be involved in its pathogenesis and have been reported; however, no definite identification has been described. We thus investigated the fine specificity of anti-hepatocyte plasma membrane autoantibodies in type 1 AIH (AIH-1) using a proteomic tool. A plasma membrane-enriched fraction was validated using enzymatic activity and western blot analysis experiments. Sera from AIH-1 patients (n = 65) and from 90 controls, that is, healthy blood donors (n = 40) and patients with systemic diseases (n = 20) or other liver diseases (n = 30), were studied by immunoblot performed with plasma membrane proteins resolved by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or 2-dimensional (2D) electrophoresis. Proteins contained in the immunoreactive spots were identified by sequences provided by ion-trap mass spectrometry. Hepatocytes probed with sera were also studied using confocal immunofluorescence and immunoelectron microscopy. The more prominent bands stained by patient sera were located at 38 kDa, 48, 50, 52 kDa, 62 kDa, 70 kDa, and a 95-kDa double band. Six proteins with known potential plasma membrane expression were identified: liver arginase (38 kDa), cytokeratins (CK) 8 and 18 (48-52 kDa), heat shock proteins (HSP) of 60, 70, 90 kDa, and valosin-containing protein (VCP) of 92 kDa. The presence of anti-membrane antibodies was confirmed by immunofluorescence and immunoelectron microscopy. Overall, our data demonstrate that liver arginase, CK 8/18, HSP 60, HSP 70, HSP 90, and VCP represent potential candidate targets on liver membrane for autoantibodies in AIH-1.

Autoimmunity to polymorphonuclears: functional consequences of the binding of antibodies to membrane and cytoplasmic target antigens of polymorphonuclear leukocytes.

Antineutrophil autoantibodies reacting with cytoplasmic antigens are associated with various types of vasculitides, whereas antibodies reacting with neutrophil membrane antigens are mostly related to autoimmune neutropenias. The aim of this study was the investigation of the effect of monoclonal antibodies (MoAbs) reacting with surface and cytoplasmic antigens of polymorphonuclear leukocytes (PMN) known to be targets for autoantibodies in human diseases. Blood of healthy volunteers was tested for several phagocytic functions in the presence of MoAbs against surface (CD16, CD11b, CD18, NB1) and cytoplasmic (proteinase 3; PR3) molecules. Candidacidal activity was significantly inhibited in the presence of all MoAbs but isotypic control. Phagocytic activity was inhibited by anti-CD11b and/or anti-CD18 MoAbs. Zymosan-induced chemiluminescence was reduced by MoAbs anti-CD16, CD18, and NB1, enhanced by anti-PR3 MoAb, and less enhanced by anti-CD11b. In conclusion, antimembrane antibodies diminished phagocytic functions at multiple steps; in contrast, anticytoplasmic MoAb promoted activation of oxidative burst in addition to impairment of microbicidal activity. This fact may be related to different pathogenic aspects of diseases associated with antimembrane and anticytoplasmic antibodies.

Liver cell-membrane specific antibodies in autoimmune hepatitis patients detected by flow cytometry

Abstract In this study, we identified autoantibody in patients with autoimmune hepatitis against liver cell membrane using flow cytometry. After incubation of one of the hepatoblastoma cell lines, Hep G2, with serum from a patient, and the addition of FITC-labeled anti-human Ig antibody, anti-membrane antibodies were analyzed by flow cytometry. By our method, the antibody in serum can react only with autoantigens on the cell surface. Furthermore, propidium iodide staining enabled us to exclude the possibility of crossreaction of antibodies against dead cells. The relative fluorescence intensity in 12 patients with autoimmune chronic active hepatitis was significantly higher than that in 20 normal subjects, six primary biliary cirrhosis (PBC), 18 chronic viral hepatitis, and 11 systemic lupus erythematosus (SLE). The normal level of fluorescence intensity provided by sera from the SLE patients indicated that antibody binding to the liver cell membrane was not derived from Fc-mediated immune complex capture. These findings demonstrated that this flow cytometric technique provides a simple and accurate method for the detection of autoantibodies against liver cell membrane.

Detection of anti-liver cell membrane antibody using a human hepatocellular carcinoma cell line

A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of 125I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

Antimyolemmal antibodies in myocarditis in children

Both in histologically proven (N = 3) and clinically diagnosed (N = 3) definite (peri)myocarditis in children, as well as in probable (N = 6) and possible inflammatory perimyocardial disease (N = 31), antibodies directed against the sarcolemma and myolemma (AMLAs) were demonstrated. In the acute phase AMLAs of the IgM class and complement fixation prevailed in the serum and the biopsy specimens. Complement dependent cytolysis by the patients' sera in the acute phase may simulate in vitro what could also be operative in vivo: a secondary immunopathogenesis due to cytolytic antimembrane antibodies.

Envelope proteins of avian infectious bronchitis virus: Purification and biological properties

Immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (IBV). The purified proteins were inoculated into rabbits to produce antisera. The rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. IBV-infected chickens produced antibodies to both the spike and membrane proteins. Both these antibodies were at their highest concentration about 9–11 days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. Neutralizing antibodies were at their highest concentration 21 days after inoculation. A second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. The neutralizing, anti-spike and anti-membrane antibodies all reached highest concentrations 7–11 days after this inoculation. The advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed.

Differential effects of insulin, antibodies against rat adipocyte plasma membranes, and other agents that mimic insulin action in rat adipocytes

The effects of insulin and several insulin-mimetic agents on rat adipocyte D-glucose metabolism were studied in an effort to determine if any of the insulin-mimetic agents could be used to define the mechanism of insulin action. Antibodies against rat adipocyte plasma membranes have been characterized as having insulin-mimetic effects on glucose transport and these effects may be caused by divalent clustering of cell surface antigens. In contrast to insulin, antimembrane antibodies had little stimulatory effect on D-glucose conversion to lipids in isolated rat adipocytes, under conditions where both reagents stimulated D-glucose oxidation. Among other insulin-mimetic agents tested, the reagents hydrogen peroxide and concanavalin A most closely resembled insulin in their ability to increase both [ 14C]-CO 2 and [ 14C]-lipid formation from [ 14C]D-glucose in rat adipocytes. Vitamin K5 and diamide had the unusual effect of inhibiting [1- 14C]D-glucose conversion to [ 14C]-lipids at a concentration that gave maximal stimulation of glucose oxidation by rat adipocytes. Analysis of the lipid components into which glucose derivatives were incorporated revealed that insulin increased D-glucose incorporation into both nonesterified fatty acids and triglycerides and H 2O 2 and concanavalin A had similar effects. These findings argue against the possibility that insulin and the antimembrane antibodies or the insulin-mimetic agents other than H 2O 2 and concanavalin A share the same mechanism of action.

Neutralization of erythroid burst-promoting activity in vitro with antimembrane antibodies

To investigate the relatedness of soluble and pelletable vesicular erythroid burst-promoting activity (BPA) present in lymphocyte- conditioned medium (LCM), we immunized rabbits with partially purified lymphocyte plasma membranes and tested the antisera for biological and immunologic crossreactivity with LCM and its component fractions. When preincubated with IgG purified from post-immune but not from preimmune serum, BPA expression by unseparated LCM, LCM-derived pellets, and supernatants was abolished in a dose-related fashion. As little as 0.001 mg/mL post-immune IgG reduced burst formation by 50%. Antimembrane IgG crossreacted on immunoblots with multiple components of both supernatants and pellets of LCM. Crossreactivity was also seen in LCM-derived supernatants that were subjected to ultracentrifugation. Soluble BPA was adsorbed from LCM supernatants incubated with antimembrane IgG-coated Staphylococcus aureus. Conversely, incubation of purified antimembrane IgG with intact circulating lymphocytes removed BPA-neutralizing effects from the antibody preparation. Antimembrane IgG incompletely suppressed erythroid colony-forming unit (CFU-E)-derived colony formation, an effect that could not be explained by alteration in erythropoietin sensitivity or action. There was no effect of the antibody preparation on erythroid differentiation of K562 cells or on CFU granulocyte/macrophage-derived colony growth, (CFU-G/M) by human or murine bone marrow. Taken together, our findings suggest that antibodies directed against lymphocyte plasma membranes react with both soluble and vesicular BPA, and that these physically separable erythroid growth factors may share antigenic determinants.

Neutralization of Erythroid Burst-Promoting Activity In Vitro With Antimembrane Antibodies

To investigate the relatedness of soluble and pelletable vesicular erythroid burst-promoting activity (BPA) present in lymphocyte-conditioned medium (LCM), we immunized rabbits with partially purified lymphocyte plasma membranes and tested the antisera for biological and immunologic crossreactivity with LCM and its component fractions. When preincubated with IgG purified from post-immune but not from preimmune serum, BPA expression by unseparated LCM, LCM-derived pellets, and supernatants was abolished in a dose-related fashion. As little as 0.001 mg/mL post-immune IgG reduced burst formation by 50%. Antimembrane IgG crossreacted on immunoblots with multiple components of both supernatants and pellets of LCM. Crossreactivity was also seen in LCM-derived supernatants that were subjected to ultracentrifugation. Soluble BPA was adsorbed from LCM supernatants incubated with antimembrane IgG-coated Staphylococcus aureus. Conversely, incubation of purified antimembrane IgG with intact circulating lymphocytes removed BPA-neutralizing effects from the antibody preparation. Antimembrane IgG incompletely suppressed erythroid colony-forming unit (CFU-E)-derived colony formation, an effect that could not be explained by alteration in erythropoietin sensitivity or action. There was no effect of the antibody preparation on erythroid differentiation of K562 cells or on CFU granulocyte/macrophage-derived colony growth, (CFU-G/M) by human or murine bone marrow. Taken together, our findings suggest that antibodies directed against lymphocyte plasma membranes react with both soluble and vesicular BPA, and that these physically separable erythroid growth factors may share antigenic determinants.

Immunofluorescent studies of the rat adipocyte cell surface

Antibodies against rat adipocyte plasma membranes have been shown to mimic insulin action in isolated adipocytes (Pillion & Czech, J. biol. Chem. Vol. 253, pp. 3761 – 3764, 1978). Immunofluorescent studies with antimembrane antibodies reveal capping on the adipocyte cell surface. Dose-response studies showed that the number of fat cells with obvious caps did not increase as the concentration of antimembrane antibodies was increased, whereas stimulation of glucose oxidation was proportional to the antibody concentration. At fairly high dilutions of antiserum there was no effect on rat adipocyte glucose oxidation, but caps were still visible, suggesting that there is not a direct correlation between capping and stimulation of adipocyte metabolism. At 15°C, it was found that capping was not significantly impaired, while the basal rate of glucose oxidation was reduced considerably. Both insulin and antimembrane antibodies were still able to stimulate glucose oxidation at 15°C, although the maximal rate of glucose oxidation which could be achieved at this temperature was considerably lower than that observed at 37°C. Fat cells that were fixed with paraformaldehyde before being exposed to antimembrane antibodies showed a similar number of caps to unfixed cells, suggesting that some fat cells had a polarized distribution of membrane antigens even before exposure to antimembrane antibodies. These results demonstrate for the first time that antibodies against the rat adipocyte plasma membrane, which are known to mimic insulin action on rat fat cells, can associate with antigens arranged non-randomly on the cell surface, but it appears unlikely that capping plays a critical role in the expression of this biological activity.

Humoral immunity in experimental immune carditis

An experimental model of carditis has been produced in the rhesus monkey by giving 12 weekly injections of a streptococcal membrane antigen. There was histological evidence of focal myocarditis, subendocardial infiltration with lymphomononuclear cells and in one animal myocardial granuloma formation. No valvular lesions were seen. Humoral immune responses monitored throughout the experiment showed that anti-heart antibodies appeared in circulation after the 2nd injection, attained maximal titres by the 6th injection, and thereafter the titres declined. The anti-membrane antibodies appeared slightly later and these titres kept rising till sacrifice. By the 6th injection there was evidence of complement consumption and appearance of circulating immune complexes. It is concluded that these immunological responses might have a role to play in the pathogenesis of carditis.

Production of insulinomimetic antibodies against rat adipocyte membranes by hybridoma cells.

SJL mice were injected intraperitoneally with adipocyte plasma membranes or with intrinsic membrane proteins obtained by extraction of plasma membranes with dimethylmaleic anhydride. Three days after the boost injection, the spleens were removed and fused with NS-a, a thioguanine-resistant myeloma cell line derived from P3X63 Ag8 (Balb/c). Following selection for hybrids with hypoxanthine, aminopterin, and thymidine, medium of the hybrid cells was tested for its ability to bind to the plasma membrane of the adipocyte and to stimulate the oxidation of D-(1-14C) glucose to 14CO2. Approximately 40% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with plasma membranes produced immunoglobulin that bound to adipocyte plasma membranes. About 30% of these mimicked the ability of insulin to stimulate the oxidation of D-(1-14C) glucose to 14CO2 in adipocytes. Media from 51% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with intrinsic membrane proteins produced immunoglobulin that bound to the plasma membrane and 48% of those stimulated glucose oxidation. The bioactivity of the hybrid cell media could be blocked by adsorption with intrinsic membrane proteins or by the removal of immunoglobulins using formalin-fixed Staphylococcus aureus. The hybrids generated in this study can be divided into three categories: 1) hybrids that secrete antibodies that can bind to plasma membranes and mimic insulin action of glucose transport; 2) hybrids that secrete antibodies that bind to plasma membranes but do not stimulate the oxidation of D-(1-14C) glucose to 14CO2; and 3) hybrids that produce no antimembrane antibodies. The data suggest that interaction of immunoglobulins with specific membrane proteins is essential in mimicking the action of insulin on glucose transport and oxidation in the rat adipocyte.

Immune derangement in patients with malignant melanoma.

The interaction between immune system and growing tumor can be expressed differently at different stages of the disease. This presentation covers three facets of these reactions in melanoma patients. A. The Primary Tumor. Time-lapse cinematography, with an analysis of lymphocyte movement demonstrated positive and negative chemotaxis against tumor tissues which correlated with their histological presence or absence within the primary tumor. B. The Regional Lymph Nodes. Histological examination of regional lymph nodes showed an increase in germinal center activity and B cell number, with a decrease in sinus histiocytosis and monocyte count as the tumor progressively invaded the node. This correlated with the elution studies, wherein the antimembrane antibody decreased and the anti-cytoplasmic antibody increased during the same period of progression. C. Humoral Immunity and Metastasis. Clinical metastasis heralded the decrease of anti-membrane antibodies with a rise in anti-immunoglobulins, especially anti-idiotypic antibodies and immune complexes containing tumor-directed antibody and either antigen or anti-immunoglobulin. This triad of anti-immunoglobulin, immune complexes and anergy as seen in other diseases with persistent antigenic stimulation, results in abnormal regulation and derangement.

Technical improvements in the mixed antiglobulin rosetting reaction with consequent demonstration of high numbers of immunoglobulin-bearing lymphocytes in viable preparations of human peripheral blood

A modification of the mixed antiglobulin rosetting reaction (MARR) to improve sensitivity as a test for immunoglobulin (Ig)-bearing human blood lymphocytes is described. A mean 5.7%, lymphocytes Ig-positive by the MARR when rosettes were formed in medium containing 0.2% bovine serum albumin (BSA), increased to 20% when rosettes were formed in 5% BSA or by incubating the lymphocytes or indicator erythrocytes with Vibrio cholerae neuraminidase before rosetting. Under these various rosetting conditions the MARR is as sensitive as the direct antiglobulin rosetting reaction (DARR). Further, with the MARR, false positive rosette formation due to unusual antimembrane antibodies can be excluded during the mixed antiglobulin rosetting procedure by use of blocking controls. Substitution of F(ab′) 2 antiglobulin for IgG anti-γ, anti-α and anti-μ did not reduce the number of lymphocytes demonstrable with the MARR, indicating that the MARR does not react with adsorbed Ig molecules on lymphocytes. Summation of the number of sheep erythrocyte (E) rosetting lymphocytes and mixed antiglobulin rosetting lymphocytes approximated 100%, yet in T-enriched preparations a maximum of 4% of lymphocytes were Ig-positive by the MARR, suggesting that null lymphocytes which have been reported to be E-negative and immunofluorescence-negative are B lymphocytes with surface Ig determinants.

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes: Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

1. 1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043 or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. 3. Dodecyl sulfate-treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane.

The Alternate Complement Pathway in Cell Damage: Antibody-Mediated Cytolysis of Erythrocytes and Nucleated Cells

Abstract Components of the alternate complement pathway present in C4 deficient guinea pig serum have been studied for their ability to lyse cells sensitized with cytotoxic antibody. Five different cell types, including erythrocytes, nucleated cells and tumor cells, were examined. Cytotoxic antibodies utilized were obtained from guinea pig, mouse, burro and rabbit sources. The specific assay systems included sheep erythrocytes-rabbit anti-Forssman antibody; TNP-coated sheep erythrocytes-rabbit anti-DNP antibody; guinea pig thymocytes-burro anti-thymus cell antibody; mouse thymocytes-mouse anti-ϑ antibody; guinea pig leukemia cells (L2C)-guinea pig anti-histocompatibility antibody. In all cases the alternate complement pathway mediated lysis inefficiently or not at all. The data indicate that in most situations the sequential participation of C1, C4 and C2 is requisite for the efficient lysis of cells sensitized with anti-membrane antibodies.