Detection of Monoclonal Antibody-Defined Colorectal Carcinoma Antigen by Solid-Phase Binding Inhibition Radioimmunoassay

Hybridoma ◽  
1981 ◽  
Vol 1 (1) ◽  
pp. 37-45 ◽  
Author(s):  
TONG H. CHANG ◽  
ZENON STEPLEWSKI ◽  
HENRY F. SEARS ◽  
HILARY KOPROWSKI
Keyword(s):  
Monoclonal Antibody ◽  
Colorectal Carcinoma ◽  
Solid Phase ◽  
Binding Inhibition ◽  
Solid Phase Binding

Turbidimetric assay of IgG with use of single monoclonal antibodies.

1986 ◽  
Vol 32 (5) ◽  
pp. 752-757 ◽  
Author(s):  
Y Maynard ◽  
M G Scott ◽  
M H Nahm ◽  
J H Ladenson
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Binding Assay ◽  
Solid Phase ◽  
Polyclonal Antiserum ◽  
Turbidimetric Assay ◽  
Group 2 ◽  
Solid Phase Binding ◽  
Low Ionic Strength ◽  
Group 1

Abstract We describe a turbidimetric assay for quantifying total immunoglobulin G (IgG) in serum with use of a single monoclonal antibody. The reaction, monitored by a centrifugal analyzer, is technically simple, rapid, and precise. Buffer of low ionic strength and polyethylene glycol are required for formation of detectable antibody-antigen complexes. We measured IgG concentrations in 49 polyclonal sera (Group 1) and 84 sera containing monoclonal IgG (Group 2) in assays in which we used either of two anti-IgG monoclonal antibodies (HG6 or HG8). Results compared well with those obtained with a nephelometric assay involving polyclonal antiserum, except for sera from four persons of Group 2 whose immunoglobulins were not detected by antibody HG6. HG6 bound IgG from these four sera in a solid-phase binding assay. HG6 and HG8 recognize epitopes on the Fab and Fc regions of IgG, respectively, and they do not compete for binding to the whole molecule. However, use of the two monoclonal antibodies combined failed to improve the sensitivity or range of the assay. We conclude that light-scattering assays of IgG can be validly performed with a single monoclonal antibody.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae

1989 ◽  
Vol 35 (3) ◽  
pp. 409-415 ◽  
Author(s):  
Anthony B. Schryvers ◽  
B. Craig Lee
Keyword(s):  
Binding Proteins ◽  
Solid Phase ◽  
Bacterial Species ◽  
Binding Activity ◽  
Intact Cells ◽  
Human Transferrin ◽  
Affinity Isolation ◽  
Lactoferrin Receptor ◽  
Solid Phase Binding ◽  
Branhamella Catarrhalis

Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin–agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.Key words: iron, Neisseria, transferrin, lactoferrin, receptor.

scholarly journals Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa

1992 ◽  
Vol 284 (3) ◽  
pp. 711-715 ◽  
Author(s):  
G Piétu ◽  
A S Ribba ◽  
G Chérel ◽  
D Meyer
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Fusion Proteins ◽  
Solid Phase ◽  
Platelet Glycoprotein ◽  
Rgd Sequence ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

Interaction of the A1 Subunit of Factor VIIIa and the Serine Protease Domain of Factor X Identified by Zero-length Cross-linking

1998 ◽  
Vol 80 (09) ◽  
pp. 418-422 ◽  
Author(s):  
Kirsty Lapan ◽  
Philip Fay
Keyword(s):  
Serine Protease ◽  
Heavy Chain ◽  
Solid Phase ◽  
Activated Protein C ◽  
Cross Linking ◽  
Factor X ◽  
Serine Protease Domain ◽  
C Terminus ◽  
Factor Viiia ◽  
Solid Phase Binding

SummaryWe have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

scholarly journals Generation of anti-type III pneumococcal polysaccharide hybridomas from mice with an X-linked B-lymphocyte defect.

1979 ◽  
Vol 150 (3) ◽  
pp. 698-702 ◽  
Author(s):  
K R Schroer ◽  
K J Kim ◽  
B Prescott ◽  
P J Baker
Keyword(s):  
B Cell ◽  
Somatic Cell ◽  
B Lymphocyte ◽  
Solid Phase ◽  
Cell Subset ◽  
Somatic Cell Hybrids ◽  
Spleen Cells ◽  
Type Iii ◽  
Solid Phase Binding ◽  
Free Antigen

(CBA/N X BALB/c male)F1 mice bear on X-linked defect making them totally unresponsive to T-independent (TI), TI-2 antigens such as type III pneumococcal polysaccharide (SSS-III). We found that somatic cell hybrids between CB nonresponder spleen cells and NS1 plasmacytoma cells secreted antibody specific for SSS-III. The solid-phase binding of such antibody was completely inhibited by the addition of free antigen (SSS-III) and the amount of antibody detected in culture fluids ranged from 10 ng/ml to 10 micrograms/ml. Eight hybridoma clones were identified; all make antibody of the IgM class. These results indicate that the X-linked defect does not result in a deletion of a B-cell subset which responds to TI-2 antigens.

PURIFICATION AND CHARACTERIZATION OF REACTIVE AND NON-REACTIVE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) FROM HUMAN PLACENTA

1987 ◽  
Author(s):  
M Philips ◽  
A G Juul ◽  
S Thorsen ◽  
J Selmer ◽  
L Thim
Keyword(s):  
Monoclonal Antibody ◽  
Plasminogen Activator ◽  
Human Placenta ◽  
Solid Phase ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Pai 1

Reactive and non-reactive forms of PAI-1 have been identified in various biological materials. The structural differences between these forms remain to be determined.A monoclonal antibody specific for a non-reactive PAI-1 and a monoclonal antibody reacting with both the reactive and nonreactive form of the inhibitor were obtained by immunization with a tissue-type plasminogen activator (t-PA)-PAI-1 complex (Philips et al., Thromb Haemostas 1986; 55:213-7). These antibodies were used for the isolation of reactive and non-reactive PAI-1 by solid-phase immunoadsorption from extracts of human placenta. The inhibitor preparations were further purified by HPLC. Reactive and non-reactive PAI-1 both migrated with a Mr ∼ 52,000 when analyzed by SDS-PAGE. Furthermore, the two inhibitor forms were indistinguishable by N-terminal sequence analysis. Two N-terminal sequences were found in about equal ammounts for both the reactive and non-reactive PAI-1. They were Ser-Ala-Val-His-His-Pro-Pro- and a two residues shorter sequence (Val-His-His-Pro-Pro-). These sequences are in agreement with the published cDNA sequence of PAI-1 and shows that the inhibitor is N-terminally heterogeneously processed. The second order rate constant (ki) for the reaction between reactive PAI-1 and single-chain t-PA was about 6 106 M-1s-1. Treatment with 4 M guanidinium-HCl partially converted the non-reactive PAI-1 to a reactive form exhibiting a similar k1 for inhibition of single-chain t-PA. SDS-PAGE showed that the t-PA-PAI-1 complex could be dissociated by 1,5 M NH4OH/ 39 mM SDS resulting in the release of a PAI-1 with approximately the same Mr as native PAI-1. This indicates either that t-PA does not cleave the inhibitor or that it cleaves a peptide bond close to the C-terminus.In conclusion a non-reactive and a reactive form of PAI-1 can be purified from placenta. The two forms are distinguishable by monoclonal antibodies but they show similar Mr′ls and the same N-terminal sequences.

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

1989 ◽  
Vol 35 (10) ◽  
pp. 2087-2092 ◽  
Author(s):  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Horseradish Peroxidase ◽  
Human Serum ◽  
Solid Phase ◽  
Sample Volume ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Peroxidase Conjugate

Abstract In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

Sign in / Sign up

Export Citation Format

Share Document