scholarly journals Antibody produced against isolated Rh(D) polypeptide reacts with other Rh-related antigens

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1622-1626
Author(s):  
K Suyama ◽  
J Goldstein
Keyword(s):  
Gel Electrophoresis ◽  
High Titer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rabbit Antibody ◽  
Immune Precipitation ◽  
D Cells ◽  
D Antigen ◽  
Related Proteins

Rh(D) antigen-containing polypeptide was prepared by immune precipitation of intact cDE/cDE erythrocytes by using a high-titer preparation of polyclonal anti-D. when isolated Rh(D) polypeptide was administered to rabbits, antibody was produced that was unresponsive toward Rh-positive and -negative cells but reacted strongly with the immunogen in enzyme-linked immunosorbent assay-type immunobinding and Western blot immunostaining assays. Rabbit antibody also immunostains isolated Rh(c) polypeptide as well as the Rh antigen-containing components of sodium dodecyl sulfate-polyacrylamide gel electrophoresis- separated membrane proteins from Rh(D)-positive (cDE/cDE,CDe/CDe), Rh(D)-negative (cde/cde,Cde/Cde), and -D-/-D- cells. It does not react with any membrane protein from Rh-null regulator type cells, thus indicating a specificity for Rh-related proteins. We have also been able to demonstrate that polyclonal and monoclonal anti-D preparations that do not immunostain isolated Rh(D) polypeptide will react with it in our immunobinding assay.

scholarly journals Antibody produced against isolated Rh(D) polypeptide reacts with other Rh-related antigens

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1622-1626 ◽  
Author(s):  
K Suyama ◽  
J Goldstein
Keyword(s):  
Gel Electrophoresis ◽  
High Titer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rabbit Antibody ◽  
Immune Precipitation ◽  
D Cells ◽  
D Antigen ◽  
Related Proteins

Abstract Rh(D) antigen-containing polypeptide was prepared by immune precipitation of intact cDE/cDE erythrocytes by using a high-titer preparation of polyclonal anti-D. when isolated Rh(D) polypeptide was administered to rabbits, antibody was produced that was unresponsive toward Rh-positive and -negative cells but reacted strongly with the immunogen in enzyme-linked immunosorbent assay-type immunobinding and Western blot immunostaining assays. Rabbit antibody also immunostains isolated Rh(c) polypeptide as well as the Rh antigen-containing components of sodium dodecyl sulfate-polyacrylamide gel electrophoresis- separated membrane proteins from Rh(D)-positive (cDE/cDE,CDe/CDe), Rh(D)-negative (cde/cde,Cde/Cde), and -D-/-D- cells. It does not react with any membrane protein from Rh-null regulator type cells, thus indicating a specificity for Rh-related proteins. We have also been able to demonstrate that polyclonal and monoclonal anti-D preparations that do not immunostain isolated Rh(D) polypeptide will react with it in our immunobinding assay.

scholarly journals Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1491-1497 ◽  
Author(s):  
C Bloy ◽  
D Blanchard ◽  
P Lambin ◽  
D Goossens ◽  
P Rouger ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Gel Electrophoresis ◽  
Human Monoclonal Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Binding Studies ◽  
Sds Page ◽  
Immune Precipitation ◽  
D Antigen

Abstract A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.

scholarly journals Human monoclonal antibody against Rh(D) antigen: partial characterization of the Rh(D) polypeptide from human erythrocytes

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1491-1497 ◽  
Author(s):  
C Bloy ◽  
D Blanchard ◽  
P Lambin ◽  
D Goossens ◽  
P Rouger ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Gel Electrophoresis ◽  
Human Monoclonal Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Binding Studies ◽  
Sds Page ◽  
Immune Precipitation ◽  
D Antigen

A human monoclonal anti-Rh(D) antibody produced by an Epstein-Barr virus (EBV)-transformed B-cell line (IgG1(lambda), clone H2D5D2) has been purified on protein A-Sepharose column and used for binding studies and immune precipitation of the blood group rhesus (Rh) antigens. Scatchard plot analyses show that the 125I-labeled antibody (iodo-gen procedure), binds to 1.09 X 10(5), 0.43 X 10(5), and 0.32 X 10(5) antigen sites on each D--/D--, R2R2 and R1R1 RBC, respectively, with an association constant of approximately 0.6 X 10(8) mol/L-1. Immune precipitation studies indicate also that the Rh(D) antigen of the Rh(D)-positive RBCs is carried by a 29 kd polypeptide as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). No material could be precipitated from Rh(D)-negative or Rhnull RBCs. These results indicate that the monoclonal and the polyclonal human anti-Rh(D) behave similarly. A sample (Blo., presumed genotype R2r or R0R2) showing an increased number of antigen sites (0.76 X 10(5)/cell) and a high binding constant (5.7 X 10(8) mol/L-1) was used, as well as D--/D-- RBCs, for further purification of the 29-kd component. Extraction by Triton X-100 (0.1% to 5%) of the immune complexes formed between the membrane-bound Rh(D) antigens and the monoclonal antibody as well as a direct quantitative estimate of the 29- kd component, suggest that the Rh(D) polypeptide is loosely bound to the skeleton, since less than or equal to 80% can be solubilized from the membrane. In similar conditions, glycophorin A showed a slight association with the Triton-insoluble residue, whereas glycophorin B was easily and completely extracted. In contrast, both the minor RBC sialoglycoproteins, glycophorin C and glycoprotein gamma, remained predominantly bound to the membrane skeleton. The purified Rh(D) polypeptide obtained from Blo. and D--/D-- RBCs by immunoprecipitation and preparative gel electrophoresis was homogenous as judged by SDS- PAGE. Amino acid composition indicated that the Rh(D) protein contained sulfhydryl groups which are essential for biological activity.

Using Cell-Based Activity Assays In Support Of Carryover Calculations In Multi-Product Facilities

2021 ◽  
Vol 27 (4) ◽  
Author(s):  
Gabriella del Hierro ◽  
Emily Holz ◽  
Ed Contreras ◽  
Pauline Che ◽  
Shelley Elvington ◽  
...  
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Immunosorbent Assay ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Analytical Techniques ◽  
Polyacrylamide Gel ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Cell ◽  
Therapeutic Antibodies

The calculation of cleaning carryover limits in multi-product facilities can be based on the inactivity of molecules after exposure to cleaning conditions if the inactivation of active molecules can be demonstrated. The demonstration of inactivation has been addressed in several publications that have shown degradation and/or denaturation using different analytical techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay, which directly or indirectly demonstrate that the product residue is no longer active. In this paper, authors expand the assay options by demonstrating the use of molecule-specific cell-based activity assays, which provide a “catch all” measurement of sample bioactivity, to assess the inactivation of therapeutic antibodies after exposure to cleaning conditions.

PAP, a pancreatic secretory protein induced during acute pancreatitis, is expressed in rat intestine

1993 ◽  
Vol 265 (4) ◽  
pp. G611-G618 ◽  
Author(s):  
J. L. Iovanna ◽  
V. Keim ◽  
A. Bosshard ◽  
B. Orelle ◽  
J. M. Frigerio ◽  
...  
Keyword(s):  
Acute Pancreatitis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Secretory Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rat Intestine ◽  
Mrna Concentration ◽  
Initial Content ◽  
Pancreatitis Associated Protein

The pancreatitis-associated protein (PAP) is a lectin-related secretory protein present in small amounts in the rat pancreas and rapidly overexpressed during the acute phase of pancreatitis. We demonstrate in this report that PAP is also expressed in rat intestine. A cDNA library from rat jejunum was probed with pancreatic PAP cDNA. The inserts of the selected recombinant clones corresponded to a transcript whose nucleotide sequence was identical to that of pancreatic PAP mRNA. The transcript was detected in duodenum, jejunum, ileum, and colon. A protein with same molecular mass (16 kDa) and pI (8.2) as pancreatic PAP was actually immunodetected in ileum homogenate after separation by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Intestinal PAP was immunolocalized to the epithelial cells of the lower part of the villi. The protein accounted respectively for 0.02, 0.05, and 0.1% of soluble proteins in duodenum, jejunum, and ileum homogenates, as measured by enzyme-linked immunosorbent assay, and could not be detected in stomach and colon. Influence of fasting and feeding on PAP mRNA concentration was analyzed in ileum. Concentration decreased by 81 and 94% after animals were fasted for 24 and 48 h, respectively. Feeding restored the initial content within 6 h. On the other hand, intestinal PAP mRNA concentration was not altered during acute pancreatitis

scholarly journals Identification of the Wheat Curl Mite as the Vector of the High Plains Virus of Corn and Wheat

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1161-1166 ◽  
Author(s):  
Dallas L. Seifers ◽  
Tom L. Harvey ◽  
T. J. Martin ◽  
Stanley G. Jensen
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Triticum Aestivum L ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Plains ◽  
Serological Relationship ◽  
Hordeum Vulgare L ◽  
Wheat Curl Mite ◽  
Aceria Tosichella

Wheat with virus-like symptoms (extracts containing a 33-kDa protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, negative in enzyme-linked immunosorbent assay to wheat streak mosaic virus, and not infectious in a backassay to other wheat) reacted positively to antiserum made against a protein purified from symptomatic corn infected with the High Plains virus (HPV), indicating a serological relationship between the corn and wheat pathogens. The wheat curl mite (WCM, Aceria tosichella Keifer) was identified as the vector of the virus and caused persistent infection of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) in greenhouse experiments. The HPV was recovered in the field from naturally infected wheat where the number of HPV-infected plants decreased with increasing distance from the WCM source in volunteer wheat.

scholarly journals Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

2003 ◽  
Vol 10 (3) ◽  
pp. 383-387 ◽  
Author(s):  
Masahiro Yoneda ◽  
Takao Hirofuji ◽  
Noriko Motooka ◽  
Koji Nozoe ◽  
Kayoko Shigenaga ◽  
...  
Keyword(s):  
Immune Responses ◽  
Gel Electrophoresis ◽  
Early Onset ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Healthy Control ◽  
Specific Antigens

ABSTRACT Bacteroides forsythus is one of the important periodontopathic bacteria, and this microorganism is known to have an S-layer outside the outer membrane. The S-layer-like antigens were recently isolated from B. forsythus, and they were found to be 270- and 230-kDa proteins in the envelope fraction. In this study, these proteins were confirmed to be specific to B. forsythus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they were clearly recognized by sera from patients with adult and early-onset periodontitis in Western immmunoblot analysis. We compared the immunoglobulin G (IgG) responses against the purified S-layer-like antigen by enzyme-linked immunosorbent assay. IgG responses against this antigen were low in healthy control subjects, but they were significantly higher in subjects with adult and early-onset periodontitis. Together with the fact that the IgG responses against the crude extract of B. forsythus did not rise significantly in patients with periodontitis, S-layer-like proteins are considered to be specific antigens of B. forsythus and may play an important role in the progression of periodontitis.

scholarly journals Transit of alpha-mannosidase during its maturation in Dictyostelium discoideum.

1985 ◽  
Vol 101 (6) ◽  
pp. 2063-2069 ◽  
Author(s):  
L Wood ◽  
A Kaplan
Keyword(s):  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Percoll Gradient ◽  
Density Fractions ◽  
Physical Evidence ◽  
Immune Precipitation

We proposed that Dictyostelium discoideum contains two linked pools of mature alpha-mannosidase (Wood, L., R. N. Pannell, and A. Kaplan, 1983, J. Biol. Chem., 258:9426-9430). To obtain physical evidence for these pools, cells were pulse-labeled with [35S]methionine, homogenized, and subjected to Percoll gradient centrifugation. After immune precipitation of alpha-mannosidase, its polypeptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and detected by fluorography. After a 30-min pulse with [35S]methionine, the precursor and small amounts of cleaved enzyme were detected in a low density fraction (1.04 g/ml). Subsequently, cleaved enzyme was transferred to higher density fractions (1.05 and 1.07 g/ml) that were enriched in lysosomal enzymes. The half time for formation of the 1.07 g/ml pool was approximately 45 min, whereas formation of the 1.05 g/ml pool was not detected until 1.5 h after the pulse. The transfer of mature forms out of the 1.04 g/ml pool was inhibited by monensin (3.5 microM). Thus, alpha-mannosidase precursor appears to be cleaved in a prelysosomal organelle. The data also indicate that starving cells secrete precursor directly from this organelle to the extracellular space, whereas cleaved forms are first transferred into lysosomes before they are secreted. Furthermore, 2 h after starvation, the secretion of mature forms ceases even though both transit of mature forms between the two pools and secretion of precursor continues. From this we inferred that the cessation of secretion of mature forms is due to a halt in fusion of lysosomes with the plasma membrane and that precursor follows a different route to the plasma membrane.

scholarly journals Plasma Proteolytic Cascade Activation during Neonatal Cardiopulmonary Bypass Surgery

2018 ◽  
Vol 118 (09) ◽  
pp. 1545-1555 ◽  
Author(s):  
Susan Maroney ◽  
Julie Peterson ◽  
Wes Zwifelhofer ◽  
Nicholas Martinez ◽  
Ke Yan ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Protease Inhibitors ◽  
Gel Electrophoresis ◽  
Surgical Procedure ◽  
Heart Defects ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heparin Administration ◽  
Operative Recovery

Background Neonates undergoing cardiopulmonary bypass (CPB) surgery to correct congenital heart defects often experience excessive bleeding. Exposure of blood to artificial materials during CPB may activate coagulation, complement and inflammatory pathways. In addition, the surgical stress placed on the haemostatic system may result in cross-activation of other plasma proteolytic cascades, which could further complicate physiological responses to the surgical procedure and post-operative recovery. Plasma protease inhibitors undergo distinct conformational changes upon interaction with proteases, and, thereby, can serve as endogenous biosensors to identify activation of the different proteolytic cascades. We tested the hypothesis that changes in the concentration and conformation of protease inhibitors regulating plasma proteolytic cascades during neonatal CPB are associated with post-operative bleeding. Patients and Methods Plasma samples from 44 neonates were obtained at four time points across the surgical procedure. Anti-thrombin, antitrypsin, anti-chymotrypsin, anti-plasmin, C1-inhibitor and tissue factor pathway inhibitor (TFPI) concentrations and conformations were evaluated by enzyme-linked immunosorbent assay, transverse urea gradient gel electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Results/Conclusion The most striking changes were observed following heparin administration and were associated with the appearance of inactive forms of anti-thrombin and an increase in the plasma concentration of TFPI. Changes in anti-thrombin and TFPI remained evident throughout surgery and into the post-operative period but were not different between patients with or without post-operative bleeding. The concentration of antitrypsin decreased across surgery, but there was no significant accumulation of inactive conformations of any inhibitor besides anti-thrombin, indicating that widespread cross-activation of other plasma proteolytic cascades by coagulation proteases did not occur.

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