scholarly journals Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242376
Author(s):  
Naw May Pearl Cartee ◽  
Soo Jung Lee ◽  
Simon G. Keep ◽  
Michael M. Wang
Keyword(s):  
Monoclonal Antibodies ◽  
Peptide Sequence ◽  
Western Blots ◽  
Multiple Protein ◽  
Reversible Redox ◽  
Protein Sulfhydryl ◽  
Specific Proteins ◽  
Context Dependent ◽  
Protein Sulfhydryl Groups

Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.

Antibodies against Specific Proteins of and Immobilizing Activity against Three Strains of Borrelia burgdorferi Sensu Lato Can Be Found in Symptomatic but Not in Infected Asymptomatic Dogs

2000 ◽  
Vol 38 (7) ◽  
pp. 2611-2621 ◽  
Author(s):  
Joppe W. R. Hovius ◽  
K. Emil Hovius ◽  
Anneke Oei ◽  
Dirk J. Houwers ◽  
Alje P. van Dam
Keyword(s):  
Borrelia Burgdorferi ◽  
Acute Phase ◽  
Bactericidal Activity ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blots ◽  
Cell Enzyme ◽  
Specific Proteins ◽  
Different Strains

In an area where Lyme disease is endemic in The Netherlands all dogs had positive titers by whole-cell enzyme-linked immunosorbent assay and appeared to be naturally infected by Borrelia burgdorferi sensu lato. To compare the antibody responses of symptomatic dogs and asymptomatic controls, we performed Western blots and in vitro immobilization assays to study antibody-dependent bactericidal activity. Strains from three different genospecies were employed as the antigen source: B. burgdorferi strain B31,Borrelia garinii strain A87S, and Borrelia afzelii strain pKo. Antibodies against flagellin (p41) and p39 for three strains were found in sera from both symptomatic and asymptomatic dogs and were therefore considered to be markers of exposure. Antibodies against p56 and p30 of strain B31, against p75, p58, p50, OspC, and p<19 of strain A87S, and against p56, p54, p45, OspB, p31, p26, and p<19 of strain pKo were found significantly more frequently in sera from symptomatic dogs younger than 8 years when the first symptoms were observed than in those from age-matched controls (P < 0.01). These antibodies were not found in preclinical sera and appeared during development of disease. Antibodies against OspA of strains B31 and A87S were only seen in acute-phase and convalescent sera from three dogs that recovered from disease. Incubation with 25% normal canine serum did not result in the immobilization of strains B31 and pKo, but partial immobilization of strain A87S (61% ± 24% [standard deviation] at 5 h) occurred. Seven of 15 sera from symptomatic dogs but none of the sera from 11 asymptomatic dogs had antibody-dependent immobilizing activity against one of the strains. Consecutive sera from one of these dogs immobilized two different strains. Antibody-mediated bactericidal serum was not seen before onset of disease, was strongest in the acute phase of disease, and fluctuated during chronic disease. From seven out of eight symptomatic dogs Borrelia DNA was amplified by PCR; in three of them the bactericidal activity was directed against one of the genospecies amplified from that dog; however, four PCR-positive dogs lacked bactericidal activity. In conclusion, dogs with symptomatic canine borreliosis have more-extensive antibody reactivity against Borrelia, as shown by both Western blotting and immobilization assays.

scholarly journals Variable major proteins of Borrellia hermsii.

1982 ◽  
Vol 156 (5) ◽  
pp. 1312-1324 ◽  
Author(s):  
A G Barbour ◽  
S L Tessier ◽  
H G Stoenner
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Antigenic Variation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Relapsing Fever ◽  
Western Blots

Borrelia hermsii, a relapsing fever agent, manifests antigenic variation in vivo and in vitro. We studied three mouse-passaged serotypes of strain HS1 (7, 14, and 21) and a HS1 derivative obtained after multiple in vitro passages (C serotype). All four serotypes had two major proteins in whole cell lysates fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One major protein species (pII) had the same apparent subunit molecular weight (or approximately 3.9 X 10(4) in all the serotypes. In contrast, the other abundant protein in lysates, pI, had a different apparent molecular weight in each serotype. In one gel the molecular weights of pIc, pI7, pI14, and pI21 were 1.9, 4.2, 4.1, and 4.0 X 10(4), respectively. Serotype-specific mouse antisera bound to both hemologous and heterologous pIIs, to homologous pI, but not to heterologous pI in Western blots. Hybridomas were raised from spleens of mice infected with B. hermsii. Monoclonal antibodies were identified by immunofluorescence assays using whole organisms. Monoclonal antibodies specific for serotype 7 (H1826) or for serotype 21 (H3326) bound only to pI7 or pI21, respectively, in Western blots. The surface location of the pI was suggested not only by the immunofluorescence studies but also by the labeling of pI7 and pI21 when whole cells of serotypes 7 and 21 were incubated with 125I in the presence of Iodogen. Under the same circumstances, pII was relatively poorly labeled. These studies have identified the variable pI proteins of B. hermsii as serotype-specific antigens. A change from one pI to another may be the basis of antigenic variation of Borrelia species during relapsing fever.

Changes induced by cadmium administration on thyroxine deiodination and sulfhydryl groups in rat liver

1993 ◽  
Vol 138 (2) ◽  
pp. 219-224 ◽  
Author(s):  
B. Paier ◽  
K. Hagmüller ◽  
M. I. Noli ◽  
M. Gonzalez Pondal ◽  
C. Stiegler ◽  
...  
Keyword(s):  
Body Weight ◽  
Rat Liver ◽  
Colorimetric Method ◽  
Amyl Alcohol ◽  
Sulfhydryl Groups ◽  
Serum Hormone ◽  
Protein Sulfhydryl ◽  
Normal Rat ◽  
Protein Sulfhydryl Groups

ABSTRACT The effects of cadmium on 5′-deiodination of thyroxine (T4) by rat liver and on the hepatic concentration of non-protein sulfhydryl groups (NPSH) were studied in Wistar rats of 200–250 g body weight. A group of ten rats was injected with cadmium chloride (300 μg/100 g body weight i.p.) daily for 4 days. Another group of six rats received, in addition, dithiothreitol (DTT; 1 mg/100 g body weight i.p.) daily for the same period. A group of eight normal untreated rats served as control. T4 deiodination was also determined in aliquots of liver from untreated rats, with cadmium (2 or 5 mmol/l) and with or without DTT (0, 2·5, 5 or 10 mmol/l) plus 1 μCi 125I-labelled T4. Hepatic NPSH were measured by a colorimetric method employing dithioldinitrobenzoic acid. Homogenates were incubated for 90 min at 37 °C and chromatographed in a tertiary amyl alcohol: hexane: ammonia (2 mol/l) (10: 1: 12) system. Cadmium-injected rats showed a significant (P <0·01) decrease in T4 deiodination and in the generation of 125I (P <0·01) and tri-iodothyronine (T3) (P <0·02). NPSH were also decreased (P <0·02). Administration of DTT restored T4 deiodination and NPSH to normal. In-vitro addition of cadmium or DTT to normal rat liver homogenates induced similar effects on the degradation of T4. Serum concentrations of T4 (P <0·01) and T3 (P <0·01) declined significantly in cadmium-injected rats, whereas DTT administration failed to normalize serum hormone levels. The data suggest that cadmium may have decreased 5′-deiodinating activity through binding to sulfhydryl groups of 5′-deiodinase as it does in other enzymes. The effects on serum T4 concentrations may be unrelated to those on 5′-deiodinase. Journal of Endocrinology (1993) 138, 219–224

scholarly journals RELATION BETWEEN WATER PERMEABILITY AND INTEGRITY OF SULFHYDRYL GROUPS IN MALIGNANT AND NORMAL CELLS

1961 ◽  
Vol 9 (4) ◽  
pp. 733-745 ◽  
Author(s):  
Morris Belkin ◽  
Walter G. Hardy
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Intraperitoneal Administration ◽  
Malignant Cell ◽  
Sulfhydryl Groups ◽  
Ascites Tumor ◽  
Protein Sulfhydryl ◽  
Protein Sulfhydryl Groups

When malignant cells, animal and human, were exposed in vitro to solutions of heavy metals or other selected compounds, three types of cell blebs were produced: (1) acentric blebs, arising from one side of the cell, e. g., by chlormerodrin, meralluride sodium, mercuric chloride; (2) symmetrical blebs; which completely enveloped the cell, e. g., by strong silver protein, auric chloride, p-chloromercuribenzoate; (3) scallop blebs, numerous small spherical elevations which completely covered the cell, e.g., by N-ethyl-maleimide, trivalent arsenicals, iodoacetamide. As indicated by vital stains and morphologic appearance, the blebs arose in healthy cells. They also can be made to appear in vivo in ascites tumor cells by intraperitoneal administration of a blebbing agent. All the bleb-producing chemicals have the property of reacting with protein-sulfhydryl groups by alkylation, oxidation or mercaptide formation. The three bleb types have been induced in 8 mouse and 2 rat ascites tumor cells; in 4 human and 1 mouse malignant cell lines; and in 3 normal cell lines grown in tissue culture. In contrast, cells from normal solid tissues of liver, lung, spleen, kidney, testis and brain from mouse, rat and rabbit failed to produce blebs. A possible interpretation for these observations is presented.

scholarly journals New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250175
Author(s):  
Daniel F. Lusche ◽  
Deborah J. Wessels ◽  
Ryan J. Reis ◽  
Cristopher C. Forrest ◽  
Alexis R. Thumann ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cancer Cell ◽  
Cell Types ◽  
Western Blots ◽  
Cd44 Isoforms ◽  
Cellular Processes ◽  
Transmembrane Glycoprotein ◽  
Extracellular Region

CD44 is a transmembrane glycoprotein that binds to hyaluronic acid, plays roles in a number of cellular processes and is expressed in a variety of cell types. It is up-regulated in stem cells and cancer. Anti-CD44 monoclonal antibodies affect cell motility and aggregation, and repress tumorigenesis and metastasis. Here we describe four new anti-CD44 monoclonal antibodies originating from B cells of a mouse injected with a plasmid expressing CD44 isoform 12. The four monoclonal antibodies bind to the terminal, extracellular, conserved domain of CD44 isoforms. Based on differences in western blot patterns of cancer cell lysates, the four anti-CD44 mAbs separated into three distinct categories that include P4G9, P3D2, and P3A7, and P3G4. Spot assay analysis with peptides generated inEscherichia colisupport the conclusion that the monoclonal antibodies recognize unglycosylated sequences in the N-terminal conserved region between amino acid 21–220, and analyses with a peptide generated in human embryonic kidney 293 cells, demonstrate that these monoclonal antibodies bind to these peptides only after deglycosylation. Western blots with lysates from three cancer cell lines demonstrate that several CD44 isoforms are unglycosylated in the anti-CD44 target regions. The potential utility of the monoclonal antibodies in blocking tumorigenesis was tested by co-injection of cells of the breast cancer-derived tumorigenic cell line MDA-MB-231 with the anti-CD44 monoclonal antibody P3D2 into the mammary fat pads of mice. All five control mice injected with MDA-MB-231 cells plus anti-IgG formed palpable tumors, while only one of the six test mice injected with MDA-MB-231 cells plus P3D2 formed a tiny tumor, while the remaining five were tumor-free, indicating that the four anti-CD44 mAbs may be useful therapeutically.

scholarly journals Selection and Characterization of Murine Monoclonal Antibodies to Staphylococcus aureus Iron-Regulated Surface Determinant B with Functional Activity In Vitro and In Vivo

2009 ◽  
Vol 16 (8) ◽  
pp. 1095-1104 ◽  
Author(s):  
Martha Brown ◽  
Rose Kowalski ◽  
Julie Zorman ◽  
Xin-min Wang ◽  
Victoria Towne ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibodies ◽  
Functional Activity ◽  
Indwelling Catheter ◽  
Lethal Challenge ◽  
Western Blots ◽  
Vitro Assay ◽  
Epitope Recognition

ABSTRACT In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (≥50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.

Non-specific binding of mouse IgG1 to Heligmosomoides polygyrus: parasite homogenate can affinity purify mouse monoclonal antibodies

Parasitology ◽  
1997 ◽  
Vol 114 (1) ◽  
pp. 79-84 ◽  
Author(s):  
M. ROBINSON ◽  
T. R. GUSTAD ◽  
S. MEINHARDT
Keyword(s):  
Monoclonal Antibodies ◽  
Adult Worm ◽  
Primary Infection ◽  
Cyanogen Bromide ◽  
Specific Binding ◽  
Affinity Column ◽  
Heligmosomoides Polygyrus ◽  
Specific Proteins ◽  
Mouse Lymphocytes

A characteristic feature of infections with the nematode parasite of mice Heligmosomoides polygyrus, is a marked IgG1 hypergammaglobulinaemia. A possible source for this immunoglobulin has recently been demonstrated, through evidence that H. polygyrus adult worm homogenate (AWH) can induce the in vitro production of non-specific IgG1 from mouse lymphocytes. To determine the interactions between this immunoglobulin and the parasite, the ability of IgG1 to bind to AWH of H. polygyrus was investigated. Protein (Western) blotting indicated that mouse monoclonal antibodies are able to bind non-specifically to selected parasite antigens. Furthermore, by binding H. polygyrus adult worm homogenate to cyanogen bromide (CNBr)-activated Sepharose CL-4B, an affinity column was prepared which could be used to efficiently purify mouse IgG1 monoclonal antibodies. These antibodies were eluted from the affinity column and still retained their original specificity. These results indicate that H. polygyrus not only induces the production of non-specific IgG1 by the host, it can also bind this immunoglobulin to its own specific proteins. Thus, it is possible that IgG1 produced during a primary infection with H. polygyrus may not entirely benefit the host.

Minimal contribution of cell-bound antibodies to the immunoscintigraphy of inflamed joints with 99mTc-anti-CD4 monoclonal antibodies

2002 ◽  
Vol 41 (03) ◽  
pp. 129-134 ◽  
Author(s):  
A. Wolski ◽  
E. Palombo-Kinne ◽  
F. Wolf ◽  
F. Emmrich ◽  
W. Becker ◽  
...  
Keyword(s):  
T Cells ◽  
Monoclonal Antibodies ◽  
Synovial Membrane ◽  
In Vitro Release ◽  
Peritoneal Macrophages ◽  
Lymphoid Organs ◽  
Whole Body ◽  
Free Form ◽  
Ankle Joints

Summary Aim: The cellular joint infiltrate in rheumatoid arthritis patients is rich in CD4-positive T-helper lymphocytes and macrophages, rendering anti-CD4 monoclonal antibodies (mAbs) suitable for specific immunoscintigraphy of human/ experimental arthritis. Following intravenous injection, however, mAbs are present both in the free form and bound to CD4-positive, circulating monocytes and T-cells. Thus, the present study aimed at analyzing the relative contribution of the free and the cell-bound component to the imaging of inflamed joints in experimental adjuvant arthritis (AA). Methods: AA rat peritoneal macrophages or lymph node T-cells were incubated in vitro with saturating amounts of 99mTc-anti-CD4 mAb (W3/25) and injected i.v. into rats with AA. Results: In vitro release of 99mTc-anti-CD4 mAb from the cells was limited (on average 1.57%/h for macrophages and 0.84%/h for T-cells). Following i.v. injection, whole body/joint scans and tissue measurements showed only negligible accumulation of radioactivity in inflamed ankle joints (tissue: 0.22 and 0.34% of the injected activity, respectively), whereas the radioactivity was concentrated in liver (tissue: 79% and 71%, respectively), kidney, and urinary bladder. Unlike macrophages, however, anti-CD4 mAb-coated T-cells significantly accumulated in lymphoid organs, the inflamed synovial membrane of the ankle joints, as well as in elbow and knee joints. Conclusion: While the overall contribution of cell-bound mAbs to the imaging of arthritic joints with anti-CD4 mAbs is minimal, differential accumulation of macrophages and T-cells in lymphoid organs and the inflamed synovial membrane indicates preferential migration patterns of these 2 cell populations in arthritic rats. Although only validated for 99mTc-anti-CD4 mAbs, extrapolation of the results to other anticellular mAbs with similar affinity for their antigen may be possible.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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