125I Protein Research Articles

Anti-livin antibodies: novel markers of malignant gastrointestinal cancers

Livin represents apoptosis inhibitors and may be important in cancer. The aim of the study was to develop an anti-livin auto antibody assay and investigate its usefulness in the clinical practice in relation to gastrointestinal cancers (GIC). We studied sera obtained from 36 patients with GIC and 59 healthy controls. A solid-phase radioimmunoassay to detect anti-livin antibodies in serum was developed. Polipropylene tubes were coated with recombinant human livin, and 100-fold dilutions of sera were incubated in these tubes. On the next day, the tubes were decanted, washed and labeled 125-I protein A was added. After 2-hour incubation, the tubes were washed and radioactivity was measured using the gamma counter. We observed a statistically significant difference between the presence and levels of anti-livin antibodies in sera of patients with GIC and in control subjects. Anti-livin auto antibodies were detected in 9 patients with GIC. Of note, the level of anti-livin antibodies was significantly elevated in 25% of GIC patients. The presence of anti-livin antibodies was confirmed with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. A high prevalence of anti-livin antibodies in patients with GIC indicates that they may be useful in the diagnosis of these malignancies.

Protein adsorption on the poly(L-lactic acid) surface modified by chitosan and its derivatives

Surface modification of biomaterials has been adopted over the years to improve their biocompatibility. In this study, aiming to promote hydrophilicity and to introduce natural recognition sites onto poly(L-lactic acid) (PLLA) films, chitosan and its derivatives, carboxymethyl chitosan(CMC) and N-methylene phosphonic chitosan (NPC), were used to modify the surface of PLLA films by an entrapment method. Radiolabeled (125I) proteins were used to measure the amount of protein adsorbed to PLLA surfaces. Fibronectin (Fn) was used to study the protein adsorption on the modified PLLA surfaces, including isotherm adsorption and adsorption kinetics of single protein, competitive adsorption of binary proteins system and serum multi-proteins and the desorption behavior in serum solution. The results showed that in the isotherm adsorption, Fn had a larger adsorption capacity on the CS-modified surface at lower concentrations, but had a high adsorption capacity at CMC-modified surface at higher concentrations. In the study of absorption kinetics, Fn had a fastest adsorption equilibrium and a highest equilibrium adsorption capacity at the CS-modified surface, while it was opposite at the PCS-modified surface. When BSA and serum were added, it had the greatest effect on the adsorption of Fn on the PCS-modified surface. After 6 hours soaking in the desorption study, Fn had reached desorption equilibrium on all the modified surfaces, which had different effects on the desorption rate and the remaining percentage of Fn.

Immunoreactivity of Common Red Blood Cell Antigens in Cytoskeleton-Free Red Blood Cell Microvesicles

Background Cytoskeleton-free microvesicles can be generated from normal red blood cells (RBCs). These RBC vesicles maintain a representative sampling of the lipid bilayer and several membrane proteins. We investigated RBC antigen segregation and persistence of immunoreactivity in RBC microvesicles. Methods Cytoskeleton-free RBC microvesicles were generated from erythrocytes expressing known RBC antigens. Antigen segregation into vesicles was documented by immunospecific antigen-antibody binding using IgG eluates and 125I Protein A. 125I Protein A counts per minute (cpm) ratios between antigen-positive vesicles sensitized with 11 eluates compared with those of vesicles incubated with normal human serum are reported. Results Cytoskeleton-free RBC microvesicles preserved the antigenic expression of the original RBCs. Differences in cpm between eluate-sensitized vesicles and those incubated with normal human serum ranged from 3:1 for the Fy b antigen to 65:1 for the D antigen. Conclusions This study demonstrates that molecules bearing common RBC antigens segregate into microvesicles, fully preserving their epitope-bearing activity. The major proteins of the RBC cytoskeleton are not required for such antigen expression.

The Procoagulant State of the VX-2 Tumor in Rabbit Lung In Vivo

During their growth, many malignant solid tumors elicit a hemostatic response in the host. In this report, the fluxes of various rabbit plasma hemostatic proteins into pulmonary VX-2 tumors have been measured in vivo. VX-2 cells were contained within a small rabbit plasma clot which was injected intravenously into rabbits. At 28 d, each rabbit was injected intravenously with two radiolabeled (131I, 125I) proteins selected from fibrinogen, prothrombin, antithrombin-alpha, heparin cofactor II, or plasminogen-I or -II. After allowing the labeled proteins to circulate for 10-200 min, each rabbit was perfused with Krebs-Henseleit solution and the lungs excised. Solid tumors were isolated, weighed and measured for radioactivity content. A mean of 14 tumors/pair of lungs with a mean tumor weight of 0.3 g was obtained. Radioactivity per g of tumor was related to radioactivity/ml of blood at exsanguination for each protein at each time interval. Maximum flux rates were calculated (as pmol/g of tumor/min): Fibrinogen, 65.0; prothrombin, 53.0; antithrombin-alpha, 24.1; HCII, 17.2; plasminogen-II, 5.0; and plasminogen-I, 3.2. Using immunocytochemical staining, fibrin(ogen) was distributed heterogeneously within the VX-2 tumor, appearing largely in the perinodular region and in the necrotic core. From the net fluxes of these proteins, the profile of chronic hemostasis associated with the VX-2 tumor was shown to differ markedly from the hemostatic response that occurs after an acute vascular injury.

Identification of a protein linked to the genomic and subgenomic mRNAs of feline calicivirus and its role in translation.

125I protein labelling of oligo(dT)-selected RNA from feline calicivirus (FCV)-infected cells revealed that the genomic and 2.4 kb subgenomic RNAs of FCV are linked to a 15 kDa protein (VPg). Proteinase K treatment of FCV RNA, to remove VPg, led to a decrease in the translatability of the RNA, but there was no obvious change in the site of RNA initiation. Addition of the cap analogue 7-methylGTP to in vitro translations had no effect on the translation of FCV RNA, suggesting that FCV RNA is translated by a cap-independent mechanism. Further evidence that FCV RNA is translated by an unusual mechanism was obtained by translating FCV RNA in vitro at a range of K+ concentrations. FCV RNA was able to direct translation at K+ concentrations at which cellular RNA translation was inhibited.

Ethanol Enhances Desialylation of Transferrin by Liver Endothelial Cells in the Rat

Desialylated transferrin is emerging as a reliable index of alcoholism, and liver endothelial cells are known to partially desialylate transferrin. The effect of a single intraperitoneal injection of ethanol on the desialylation of transferrin in the rat was studied. In pulse-chase experiments, fully sialylated diferric transferrin labeled with 125I (protein moiety) or 3H (sialyl residues) was incubated with isolated, fractionated liver endothelial cells from rats that were given ethanol. The supernatants then were subjected to column chromatography using RCA120-agarose to separate and quantify the desialylated fraction of transferrin. Saline-treated animals served as controls. Incubation of the endothelial cells derived from the liver of ethanol-treated rats resulted in a 263% increase in desialylation level of transferrin. Our data indicate that administering ethanol in rats enhances the desialylation of transferrin by rat liver endothelial cells.

The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of >200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, κ), the other by clone 12C11 (IgM, λ). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2. MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.

Phagocytosis of phenylhydrazine oxidized and G-6-PD-deficient red blood cells: the role of cell-bound immunoglobulins

In this study, the role of Igs in the recognition and removal of oxidatively damaged human red blood cells (RBCs) was investigated. Phagocytosis of normal RBCs exposed to the oxidative hemolytic agent phenylhydrazine (Phz) and of glucose-6-phosphate dehydrogenase (G6PD)- deficient RBCs by murine macrophages was examined. A 40-fold increase in phagocytosis of RBCs treated with 3 mmol/L Phz was obtained both in the absence and presence of autologous serum, indicating that binding of autologous antibodies to the oxidized cells is not essential for phagocytosis. Yet, a basal number of IgG molecules was found to be present on the RBCs, as determined both by binding of 125I protein A and fluorescein isothiocyanate-antihuman Ig to the cells. Macrophage Fc receptors were found to be involved in the recognition of the RBCs, because phagocytosis was partially inhibited by incubating macrophages with bovine serum albumin (BSA) anti-BSA complexes, aIg (aggregated Igs), and anti-Fc receptor II monoclonal antibodies. Galactose/mannose inhibited phagocytosis of oxidized RBCs additively to aIg. Because phagocytosis was decreased when Phz-RBCs were incubated with F(ab')2 fragments of antihuman antibodies, it is suggested that the basal amount of Igs bound to the cells plays a role in the recognition of Phz- RBCs. G6PD-deficient RBCs were recognized and phagocytosed by murine macrophages without preexposure to oxidants in vitro (mean of 19 RBCs/100 macrophages). This phagocytosis was not affected by the addition of serum and was inhibited by incubating macrophages with galactose/mannose and the various Fc receptor blockers. A positive correlation between hemoglobin content and the number of cell-bound Igs to each patient erythrocytes was found. These results support the involvement of both an Fc and a lectin-like macrophage receptor in the recognition and phagocytosis of Phz-oxidized and G6PD-deficient RBCs and suggest opsonization as a possible physiologic process for the removal of severe damaged RBCs.

Phagocytosis of Phenylhydrazine Oxidized and G-6-PD-Deflcient Red Blood Cells: The Role of Cell-Bound Immunoglobulins

AbstractIn this study, the role of Igs in the recognition and removal of oxidatively damaged human red blood cells (RBCs) was investigated. Phagocytosis of normal RBCs exposed to the oxidative hemolytic agent phenylhydrazine (Phz) and of glucose-6-phosphate dehydrogenase (G6PD)-deficient RBCs by murine macrophages was examined. A 40-fold increase in phagocytosis of RBCs treated with 3 mmol/L Phz was obtained both in the absence and presence of autologous serum, indicating that binding of autologous antibodies to the oxidized cells is not essential for phagocytosis. Yet, a basal number of IgG molecules was found to be present on the RBCs, as determined both by binding of 125I protein A and fluorescein isothiocyanate-antihuman Ig to the cells. Macrophage Fc receptors were found to be involved in the recognition of the RBCs, because phagocytosis was partially inhibited by incubating macrophages with bovine serum albumin (BSA) anti-BSA complexes, alg (aggregated Igs), and anti-Fc receptor II monoclonal antibodies. Galactose/mannose inhibited phagocytosis of oxidized RBCs additively to alg. Because phagocytosis was decreased when Phz-RBCs were incubated with F(ab′)2 fragments of antihuman antibodies, it is suggested that the basal amount of Igs bound to the cells plays a role in the recognition of Phz-RBCs. G6PD-deficient RBCs were recognized and phagocytosed by murine macrophages without preexposure to oxidants in vitro (mean of 19 RBCs/100 macrophages). This phagocytosis was not affected by the addition of serum and was inhibited by incubating macrophages with galactose/mannose and the various Fc receptor blockers. A positive correlation between hemoglobin content and the number of cell-bound Igs to each patient erythrocytes was found. These results support the involvement of both an Fc and a lectin-like macrophage receptor in the recognition and phagocytosis of Phz-oxidized and G6PD-deficient RBCs and suggest opsonization as a possible physiologic process for the removal of severe damaged RBCs.

Characterization of an optimal protein A-gold complex: Improvement of the labeling efficiency

By using 125I protein A, we have prepared complexes of this immunoprobe with colloidal gold particles having diameters ranging from 5 to 15 nm. Various concentrations of protein A (from 35 to 1000 nM) of known specific activity were employed in order to obtain a spectrum of conjugates differing not only in sizes, but also in amounts of protein A they carry. After ultracentrifugation through a step gradient (15, 30 and 70% w/v) of sucrose, the protein A-gold complexes were quantitatively recovered from the most dense layer, and diluted in a 0.01 M phosphate buffer containing 0.01% polyethylene glycol. The conjugates did not contain free protein A, as proven by the lack of radioactivity in the intermediate sucrose layers. By correlating the radioactivity of the complexes with their particle densities (inferred from the optical absorbance at 520 nm), the number of protein A molecules bound per gold particle was calculated for each experimental condition.

Characterization of the murine T cell receptor for IgE (Fc epsilon RII). Demonstration of shared and unshared epitopes with the B cell Fc epsilon RII.

Antigenic relationships between the low affinity Fc epsilon R present on murine B and T lymphocytes were studied. A rat mAb (B3B4) and two polyclonal antisera produced by immunizing with the murine B lymphocyte Fc epsilon RII were examined for their ability to inhibit binding of IgE to murine B or T lymphocytes, using an IgE-specific rosette assay. One polyclonal antiserum (goat-anti-mouse Fc epsilon R) inhibited binding of IgE to both B and T lymphocytes, whereas another polyclonal antiserum (rabbit-anti-mouse Fc epsilon R) and the rat mAb inhibited the binding of IgE to B lymphocytes but did not influence the binding of IgE to T lymphocytes. When lymphocytes were surface labeled with 125I, 49-kDa and 38-kDa IgE-binding proteins were immunoprecipitated from B lymphocyte lysates by B3B4 and from B and T lymphocyte lysates by the goat antiserum. Taken together, these results suggest that the Fc epsilon R present on murine B and T lymphocytes are structurally related receptors that share some, but not all, epitopes.

An apocrine membrane antigen with polarized distribution and hormonally regulated expression in human endometrial and mammary carcinoma cell lines.

The topographical distribution and characteristics of an apocrine epithelial differentiation antigen (AEA) were studied in one endometrial adenocarcinoma cell line (HEC-1-B) and two mammary carcinoma cell lines (MCF-7 and T47-D), using an antiserum raised against glycoproteins which had been isolated from human milk fat globule membranes. Immunofluorescent staining of HEC-1-B and MCF-7 cells grown in monolayers, or of histological sections of cells grown in fibrin sponges, revealed a strictly polar distribution of the antigen. The antigen was present only in the dorsal-apical cell membrane. In contrast, T47-D cells grown under identical conditions displayed a nonpolar membrane distribution of the antigen. Detergent lysates of HEC-1-B, MCF-7 and T47-D cells, surface-labeled by the PI(sodiumetaperiodate)-NaB3H4 method, were immunoprecipitated and analysed by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). Two closely spaced bands having an apparent MW of 315-290 kD (kilodalton) (HEC-I-B), 330-295 kD (MCF-7) and 320-270 kD (T47-D). The surface expression of the antigen was found to be hormonally regulated. Cultivation of HEC-1-B and MCF-7 in the presence of prolactin increased the amount of antigen. The T47-D cells responded only weakly to prolactin, but displayed enhanced antigen expression after treatment by estrogen and/or progesterone, as quantified by 125I protein A radioimmunoassay. The behaviour of the membrane antigen reported here provides a new and interesting marker for the differentiation and maintenance of polarity in cultured malignant cells of secretory epithelial origin.

Application of a nitrocellulose immunoassay for quantitation of proteins secreted in culture media

A macro-dot immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglass. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and 125I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cpm) and protein concentration was found. Intra- and intertest reproducibility was excellent (C.V.'s < 7%). By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogenous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally defined medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer.

Concentration of 125 Globulins in Intestinal Tissues of Immunized and Non-Immunized Chickens Infected with Eimeria acervulina

Humoral antibody response to Eimeria infections has been well established (Rose, 1982. In The biology of the Coccidia, P. L. Long (ed.). University Park Press, Baltimore, Maryland, pp. 329-371); however, the role of these antibodies in the intestine has only been speculative. Leakage of serum components into the intestine occurs during coccidial infection, but the extent of leakage is not known because only semi-quantitative methods such as dye leakage have been used to measure it. During a primary infection, leakage occurred 3.5-7 hr after Eimeria acervulina or Eimeria praecox oocysts were given orally (Long, 1968, Parasitology 58: 691-700). Leakage was not observed at 24-48 hr post-inoculation, but does occur at 72-120 hr post-inoculation, coinciding with the eruption and multiplication of merozoites in epithelial cells. Previous results to determine permeability changes in immunized birds have been equivocal because of the lack of sensitivity of the dye leakage assay (Rose et al., 1975, Parasitology 71: 357-368). These studies did, however, reveal that the permeability change to sporozoite invasion was somewhat species-specific and that the changes were not induced by non-viable coccidial preparations. We have used a sensitive, direct method to evaluate the leakage of serum proteins into the intestine of Eimeria-immunized and non-immunized chickens, using radioiodinated (125I) chicken globulins. Serum globulins were precipitated with Na2SO4, dialyzed, and lyophilized from clarified, filtered chicken serum (Garvey et al., 1977, Methods in immunology. W. A. Benjamin, Inc., Reading, Massachusetts, pp. 218220). The lyophilized proteins were radioiodinated (125I, Amersham) (Greenwood et al., 1963, Biochemistry Journal 89: 114-123). lodinated protein was separated from free iodine by passage over a Sephadex PD 10 column. Fractions on the ascending limb of the protein peak were consolidated and diluted to 1 x 106 cpm/0.5 ml with phosphate-buffered saline (pH 7.6, 0.1 M). Three groups of 3-wk-old Leghorn chicks were used in this experiment. Group 1 (n = 9) was immunized by inoculating each bird with 5,000 sporulated oocysts of E. acervulina at 3 wk and 4 wk. Group 2 (n = 4) and group 3 (n = 4) were not immunized. Groups 1 and 2 were challenged with 1 x 107 oocysts of E. acervulina at 6 wk. Group 3 was not challenged. Three-and-one-half hr after challenge, each bird from all groups was injected intravenously with radioiodinated (1251) chicken globulins. Thirty min after injection of the 125I proteins, all birds were killed by intra-cardiac injection of sodium pentobarbital. The duodenal loop and upper jejunum were excised, cut into 6 equal sections, weighed, and placed into tubes filled with 0.85% NaCl solution. After determining the amount of radioactivity, the gut pieces were removed and radioactivity in the saline was determined. The latter measurement was taken as an indication of extravascular globulin. Four hr after challenge, there were striking differences in the amount of iodinated protein associated with the intestinal tissues (Fig. 1). Nonimmunized chickens had significantly higher

Demonstration of antibodies binding to autologous and allogeneic leukemic cells in childhood ALL. Evidence for a common ALL antigen(s).

The humoral immune response to autologous leukemic cells was investigated in childhood ALL using a 125I protein A binding assay. In 5/7 patients antibodies were demonstrated at diagnosis and in 3/7 cases also after chemotherapy. Sera from 2/3 patients, which bound significantly to autologous leukemic cells, did not bind significantly to autologous remission cells. In allogeneic experiments sera bound significantly to ALL leukemic cells (6/7 positive combinations), but not to AML leukemic cells (8/8 negative combinations). We propose that ALL sera contain antibodies binding to autologous leukemic cells and that they are directed against a common ALL antigen(s).

Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity.

Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.

The retention of 125I‐labeled plasma membrane proteins in bovine spermatozoa cryopreserved in egg‐yolk citrate extender

AbstractCryopreservation of bovine sperm in egg‐yolk citrate extender (EYC) usually maintains fertility. Since plasma membrane proteins are important for the fertilizing potential of sperm, the possible loss of membrane proteins from sperm subjected to cryopreservation in EYC was evaluated. Sperm were washed and labeled with 125I without significantly reducing motility. Radiolabeled sperm were a) held for 2 hr at 22°C in N‐2‐hydroxyethylpiperazine‐N'‐ethanesulfonic acid (HEPES)‐buffered saline containing 1% polyvinyl alcohol, b) cooled to 5 °C in glycerol‐free EYC and held for 3 hr, or c) frozen‐thawed in EYC containing 7% glycerol. Sperm were solubilized and proteins were separated by electrophoresis under denaturing conditions. Freeze‐thawing dislodged most egg‐yolk proteins from the spermatozoal plasma membrane that were bound to and retained by sperm that only were cooled to 5 °C. Autoradiography resolved 11‐18 bands of 125I polypeptides. There was no difference (P &gt; 0.05) in the amount of 125I protein retained by frozenthawed and cooled sperm. However, the radioactivity in two polypeptide bands (MW = 105 K and 24.2 K) was less (P &lt; 0.05) for sperm held at 22 °C in HEPES‐buffered saline. Thus, holding sperm in buffered saline at 22 °C resulted in a greater loss of 125I proteins from the plasma membrane than did cryopreservation of sperm in EYC. Cryopreservation did not induce greater loss of 125I proteins from the plasma membrane than simply cooling sperm to 5 °C in EYC.

Visualization of the Insulin Receptor by Immunoblotting

Insulin receptors from rat hepatoma cells (Fao) and human placenta were partially purified by detergent solubilization and lectin purification. The insulin receptor preparations were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under reducing or nonreducing conditions. The proteins were transferred to nitrocellulose paper and the electrophoretic blots were treated with human anti-receptor autoantibodies, rabbit antibody to purified insulin receptor, or a monoclonal antibody to human insulin receptor. The nitrocellulose paper was then treated with 125I protein A or 125I second antibody followed by autoradiography. The rabbit polyclonal antiserum and one of the human autoantibodies recognized both the alpha (Mr = 135,000) and beta (Mr = 95,000) subunits after transfer from a SDS gel to nitrocellulose paper. On transfers from nonreduced gels, several high-molecular species were labeled ranging from Mr = 200,000 to Mr = 330,000. Similar high-molecular bands of the receptor were seen if highly purified human placental receptor, as well as partially purified receptor from rat or human origin, were used. As little as 0.1-0.5 microgram of pure receptor could be detected by this technique. Treatment of the receptor with neuraminidase (50 mU/ml) before gel electrophoresis resulted in a 50% increase in intensity of intact receptor and about a 70% increase in the labeling of the alpha-subunit of the receptor, but no change in labeling of the beta-subunit. The monoclonal antibody used, as well as two other human autoantibodies, did not recognize the receptor after transfer to nitrocellulose paper.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of sequential immune complexes in infective endocarditis by Western blot analysis.

A patient with cutaneous vasculitis during infective endocarditis due to Lactobacillus casei was studied. Immune complexes (IC) were isolated from serum at the time of diagnosis and after 4 wk of therapy. Purification of IC used differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. In situ radioiodination of IC was performed, followed by SDS-polyacrylamide gel electrophoresis (PAGE). Anti-IC antisera were raised in rabbits by immunization with purified IC. IC were characterized by SDS-PAGE followed by electrophoretic transfer to nitrocellulose, incubation with antiserum and then with 125I protein A, and autoradiography. Although early and late IC differed quantitatively, there were no differentiating immunochemical features. Both IC contained a 60,000 dalton component that did not react with preimmune serum nor with anti-normal human serum. This component reacted with antiserum rendered specific for L. casei by affinity chromatography. The restricted antigen-antibody representation in IC contrasted with a wider panel of antibody activity in patient serum. The Western blot analysis proves to be an ideal method for the characterization of IC because of its sensitivity, dissociative capability, and preservation of immunoreactivity. IC isolated at a time removed from the original antigenic challenge may provide insight into the nature of the inciting antigen.

Differential binding of IgG anti-D and IgG autoantibodies to reticulocytes and red blood cells.

125I IgG anti-D binding to reticulocytes obtained by density fractionation is reduced relative to that bound to all other red cell (RBC) fractions. Maximum D antigen reactivity occurs following reticulocyte maturation with no detectable change in D reactivity of mature RBC throughout their life span. Reticulocytes have in the range of about 60% of the content of mature RBC. Previously reported increased anti-D agglutinability and binding to old RBC it is not due to an intrinsic increase in D antigen with age, but results from an 'apparent' decrease in anti-D binding to young RBC fractions due to reticulocyte enrichment. IgG RBC autoantibodies obtained by elution from the RBC of eight Coombs-positive blood donors, probably associated with alpha-methyldopa (alpha-MD) administration, showed decreased binding to reticulocytes as determined by 125I protein A (PA). Reticulocytes bound about 70% of the IgG bound to mature RBC, indicating that the membrane antigenic determinant defined by these autoantibodies was incompletely expressed in the reticulocyte. This difference in IgG autoantibody binding between reticulocytes and mature RBC is similar to the decreased D antigen content of reticulocytes and consistent with an autoantibody determinant associated with the Rh complex. Direct testing of density fractionated Coombs-positive RBC in four out of five patients with autoimmune haemolytic anaemia (AIHA) showed reduced quantities of IgG on reticulocytes. The distribution of IgG between reticulocytes and mature RBC may be useful in serologically characterizing patients with AIHA and in identifying subpopulations of patients with this disorder.