scholarly journals Affinity purification and refined structural characterization of terminal deoxynucleotidyltransferase

1985 ◽  
Vol 231 (1) ◽  
pp. 105-113 ◽  
Author(s):  
S A Fuller ◽  
A Philips ◽  
M S Coleman
Keyword(s):  
Monoclonal Antibody ◽  
Specific Binding ◽  
Affinity Purification ◽  
Single Step ◽  
Immunoaffinity Column ◽  
Tissue Slices ◽  
Terminal Transferase ◽  
Antigenic Sites ◽  
High Binding Affinity

A total of 56 stable murine hybridoma monoclones that produce homogeneous antibodies against human or calf terminal deoxynucleotidyltransferase have been established. All of the antibodies exhibited specific binding to various Mr forms of terminal transferase and eight possessed neutralizing activity. Results are presented that permitted characterization of ten of these antibodies with respect to their immunoglobulin class, their recognition of calf or human terminal-transferase Mr species by immunoblotting techniques and their recognition of distinct antigenic sites. Terminal transferase was purified in a single step by using an immunoaffinity column constructed with a monoclonal antibody exhibiting a high binding affinity for the enzyme. Single monoclonal antibodies were also used to bind selectively to terminal-transferase antigen in tissue slices and individual cells.

scholarly journals Preparation and characterization of a d-myo-inositol 1,4,5-trisphosphate-specific antibody

1995 ◽  
Vol 311 (3) ◽  
pp. 1009-1014 ◽  
Author(s):  
W R Shieh ◽  
C S Chen
Keyword(s):  
Binding Sites ◽  
Inositol Phosphates ◽  
Specific Binding ◽  
Affinity Purification ◽  
Competitive Elisa ◽  
Inositol 1,4,5 Trisphosphate ◽  
Ic50 Values ◽  
High Concentrations ◽  
Differential Affinity

Antibodies against Ins(1,4,5)P3 were raised by immunizing rabbits with two types of InsP3-BSA conjugates which were synthesized by covalently coupling Ins(1,4,5)P3 to the carrier protein via alkyl linkages. The anti-Ins(1,4,5)P3 antibody was detected by a novel ELISA using Ins(1,4,5)P3-immobilized microtitre plates. Both antiserum preparations showed specific binding with Ins(1,4,5)P3, with titres of 1:4000. Most inositol phosphates, including Ins1P, Ins(4,5)P2, Ins(1,3,4)P3, Ins(1,5,6)P3, Ins(1,2,5,6)P1, Ins(3,4,5,6)P4, Ins(1,3,4,5,6)P5, InsP6, and PtdIns(4,5)P2, did not exhibit significant molecular interactions with the antibodies. Ins(1,3,4,5)P4, however, cross-reacted with these antibodies with one-third of the affinity as that of Ins(1,4,5)P3, in part due to the largely shared structural motifs. The differential affinity was significantly improved by affinity purification on Ins(1,4,5)P3-agarose. The affinity-purified antibody displayed IC50 values of 12 nM and 730 nM for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 respectively, according to a competitive ELISA; these values are in line with those reported for the Ins(1,4,5)P3 receptor. The modes of ligand recognition at the binding sites of these two types of biomolecules are, however, different. Moreover, although the ligand binding was interfered with by multivalent anions such as ATP4-, HPO4(3-) and SO4(2-) at high concentrations, no inhibition was noted with heparin, an antagonist of the Ins(1,4,5)P3 receptor.

scholarly journals Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase

1994 ◽  
Vol 301 (2) ◽  
pp. 551-556 ◽  
Author(s):  
P Glynn ◽  
D J Read ◽  
R Guo ◽  
S Wylie ◽  
M K Johnson
Keyword(s):  
Affinity Purification ◽  
Order Rate Constant ◽  
Single Step ◽  
Neuropathy Target Esterase ◽  
Serine Esterase ◽  
Western Blots ◽  
Sds Page ◽  
Covalent Inhibitors ◽  
Serine Esterases

We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases.

Preparation and Characterization of scFv-Coupled Immunoaffinity Column for Purification of Fibrinolytic Enzyme from Bacillus subtilis Natto-89

2020 ◽  
Vol 9 (2) ◽  
pp. 104-110
Author(s):  
SunIl Choe ◽  
CholJin Kim ◽  
UnHui Yun ◽  
HyonGwang Li ◽  
CholHo Kim
Keyword(s):  
Binding Capacity ◽  
Affinity Purification ◽  
Coupling Efficiency ◽  
Immunoaffinity Column ◽  
Single Chain ◽  
Dynamic Binding ◽  
Dynamic Binding Capacity ◽  
Sepharose 4B ◽  
Dissociation Constant Kd

Background: The focus of this study was to prepare and characterize the single-chain variable fragment antibody (scFv)-coupled immunoaffinity column for purification of subtilisin BRC. Methods: The scFv against subtilisin BRC was immobilized onto CNBr-activated Sepharose 4B. Adsorption isotherm for subtilisin BRC on scFv-BRC-coupled Sepharose 4B was obtained and calculated the maximum binding capacity. The extraction conditions, including eluting solution, the concentration of eluting solution and flow rate, were optimized. Under the optimized eluting conditions, the dynamic binding capacity of the immunoaffinity column was determined. Results: The scFv-BRC-coupled Sepharose 4B for immunoaffinity purification of subtilisin BRC was prepared. The coupling efficiency was about 78.4%, e.g. about 8 mg of scFv-BRC was covalently coupled to 1 g CNBr-activated Sepharose 4B. The maximum equilibrium binding capacity (qm) and dissociation constant (Kd) of the immunoaffinity column for subtilisin BRC were 3.01 mg/mL and 0.465 mg/mL, respectively. The immunoaffinity chromatography conditions were optimized and the subtilisin BRC was purified 3.29-fold with 55.6%. Conclusion: The subtilisin BRC was effectively purified with high purity using scFv-BRC-coupled Sepharose 4B and the dynamic binding capacity of the column was determined. These results suggested that scFv-BRC can be used as a ligand for affinity purification of subtilisin BRC.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Purification of Urokinase from Complex Mixtures Using Immobilized Monoclonal Antibody Against Urokinase Light Chain

1983 ◽  
Vol 49 (01) ◽  
pp. 024-027 ◽  
Author(s):  
David Vetterlein ◽  
Gary J Calton
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Culture Media ◽  
Biological Fluids ◽  
Single Step ◽  
High Yield ◽  
Step Method ◽  
Commercial Preparation ◽  
E Coli ◽  
Tissue Culture Media

SummaryThe preparation of a monoclonal antibody (MAB) against high molecular weight (HMW) urokinase light chain (20,000 Mr) is described. This MAB was immobilized and the resulting immunosorbent was used to isolate urokinase starting with an impure commercial preparation, fresh urine, spent tissue culture media, or E. coli broth without preliminary dialysis or concentration steps. Monospecific antibodies appear to provide a rapid single step method of purifying urokinase, in high yield, from a variety of biological fluids.

Immunochemical characterization of anti-islet cell surface monoclonal antibody from nonobese diabetic mice

Diabetes ◽  
1986 ◽  
Vol 35 (5) ◽  
pp. 517-522 ◽  
Author(s):  
J. Hari ◽  
K. Yokono ◽  
K. Yonezawa ◽  
K. Amano ◽  
S. Yaso ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Islet Cell ◽  
Diabetic Mice ◽  
Immunochemical Characterization ◽  
Nonobese Diabetic ◽  
Nonobese Diabetic Mice
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