A simple method for the determination of affinity and binding site concentration in receptor binding studies

Akera, Tai; Cheng, Ven-Jim K.

doi:10.1016/0005-2736(77)90132-8

Cited by 136 publications

(39 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For the calculation, a molecular mass of 27,000 g/mol for scFv6H4 was assumed. METH and amphetamine concentrations in serum and urine were determined by liquid chromatography-tandem mass spectrometry as described previously (Hendrickson et al, 2004 (Akera and Cheng, 1977) using the computer software program Origin (OriginLab Corp., Northampton, MA). Pharmacokinetic analysis was performed with the software package WinNonlin (Pharsight, Mountain View, CA).…”

Section: Single-chain Antibody Against Meth 127mentioning

confidence: 99%

Development and Preclinical Testing of a High-Affinity Single-Chain Antibody against (+)-Methamphetamine

Peterson¹,

Laurenzana²,

Atchley³

et al. 2008

J Pharmacol Exp Ther

View full text Add to dashboard Cite

Chronic or excessive (ϩ)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single-chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, light chain, K d ϭ 11 nM) and found to have similar ligand affinity (K d ϭ 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified, and formulated as a naturally occurring mixture of monomer (ϳ75%) and dimer (ϳ25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n ϭ 5) were implanted with 3-day s.c. osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [ 3 H]scFv6H4 tracer. Serum pharmacokinetic analysis of METH and [3 H]scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0 to 480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t 1/2z of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t 1/2z (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together, these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum.There are currently nearly 20 monoclonal antibody (mAb) medications approved by the United States Food and Drug Administration and over 20 more in early clinical or preclinical trials (Holliger and Hudson, 2005). These medications include full-length IgG mAbs, along with five mAb fragments as Fab, F(abЈ) 2 (antigen binding fragments of IgG), or singlechain variable fragment (scFv) proteins.IgG mAbs are typically chimeric, humanized, or fully human proteins and are administered to patients requiring a long-acting antagonist with minimal extravascular penetration (Bazin-Redureau et al., 1997). IgG mAb is best for this purpose because it has a terminal elimination half-life (t 1/2z ) ranging from approximately 3 to 26 days. The longest t 1/2z values are usually achieved when the antibody does not bind to tissue sites and is not prematurely cleared due to antigenicity. When a short duration of action and greater extravascular penetration are needed, a significantly smaller fragment like Fab (t 1/2z ranging from 0.5-21 h; Trang, 1992) or scFv (t 1/2z ranging from minutes to hours; Goel et al., 2000) is used. In particular, rat pharmacokinetic studies of an antianthrax toxin scFv report a t 1/2␣ of ϳ5 min (Maynard et al., 2002). In addition, PCKN studies of a scFv in mice report t 1/2␣ values of 2.7 and 1 min Willuda et al., 2001). It is possible that a short-acting scFv could be used to

show abstract

Section: Single-chain Antibody Against Meth 127mentioning

confidence: 99%

Development and Preclinical Testing of a High-Affinity Single-Chain Antibody against (+)-Methamphetamine

Peterson¹,

Laurenzana²,

Atchley³

et al. 2008

J Pharmacol Exp Ther

View full text Add to dashboard Cite

show abstract

“…All data were corrected for nonspecific binding determined in the presence of an excess (10 Ϫ6 M) of unlabeled motilin. The dissociation constant (K d ) was calculated from the displacement curves fitted to the equation of Akera-Cheng by computer (Akera and Cheng, 1977).…”

Section: Motilin Receptor Binding Studiesmentioning

confidence: 99%

Interaction of the Growth Hormone-Releasing Peptides Ghrelin and Growth Hormone-Releasing Peptide-6 with the Motilin Receptor in the Rabbit Gastric Antrum

Depoortere

Thijs²,

Thielemans³

et al. 2003

J Pharmacol Exp Ther

View full text Add to dashboard Cite

The structural relationship between the motilin and the growth hormone secretagogue receptor (GHS-R), and between their respective ligands, motilin and ghrelin, prompted us to investigate whether ghrelin and the GHS-R agonist growth hormonereleasing peptide-6 (GHRP-6), could interact with the motilin receptor. The interaction was evaluated in the rabbit gastric antrum with binding studies on membrane preparations and with contraction studies on muscle strips in the presence of selective antagonists under conditions of electrical field stimulation (EFS) or not. Binding studies indicated that the affinity (pK d ) for the motilin receptor was in the order of ghrelin (4.23 Ϯ 0.07) Ͻ GHRP-6 (5.54 Ϯ 0.08) Ͻ motilin (9.13 Ϯ 0.03). The interaction of ghrelin with the motilin receptor requires the octanoyl group. Motilin induced smooth muscle contractile responses but ghrelin and GHRP-6 were ineffective. EFS elicited on-and off-responses that were increased by motilin already at 10 Ϫ9 M, but not by 10 Ϫ5 M ghrelin. In contrast, GHRP-6 also enhanced the on-and off-responses. The motilin antagonist Phe-cyclo[Lys-Tyr(3-tBu)-␤Ala-] trifluoroacetate (GM-109) blocked the effect of GHRP-6 on the off-responses but not on the on-responses. Under nonadrenergic noncholinergic conditions, the effects of motilin and GHRP-6 on the on-responses were abolished; those on the off-responses were preserved. All responses were blocked by neurokinin (NK) 1 and NK 2 antagonists. In conclusion, ghrelin is unable to induce contractions via the motilin receptor. However, GHRP-6 enhances neural contractile responses, partially via interaction with the motilin receptor on noncholinergic nerves with tachykinins as mediator, and partially via another receptor that may be a GHS-R subtype on cholinergic nerves that corelease tachykinins.

show abstract

“…24 The binding of a fixed concentration of [ -0,5 a = B.C 05 /a where C o 5 is the concentration of unlabeled ouabain that caused a 50% reduction of the previously bound labeled ouabain, a is the labeled ouabain concentration, and B a is the equilibrium binding of the labeled ouabain in the absence of unlabeled ouabain.…”

Section: Methodsmentioning

confidence: 99%

“…Binding of [ 3 H]ouabain (19.5 Ci/mmol; New England Nuclear Corporation, Boston, Massachusetts) was carried out by the filtration method as previously described 23 except that a displacement assay 24 was used to determine the apparent K D as a measure of the affinity of Na + ,K + -ATPase for ouabain. Ouabain binding was carried out in the presence of 100 mM NaCl, 10 mM KC1, 5 mM MgCl 2 , 5 mM Na 2 ATP, 25 mM histidine, 0.4 mM NADH, and 1 mM phosphoenolpyruvate.…”

Section: Methodsmentioning

confidence: 99%

Decrease in Na+,K+-ATPase activity and [3H]ouabain binding sites in sarcolemma prepared from hearts of spontaneously hypertensive rats.

Lee¹,

Schwartz²,

Adams³

et al. 1983

Hypertension

View full text Add to dashboard Cite

SUMMARY Na+ ,K+-ATPase activity, phosphorylation, and [ 3 H]ouabain binding in sarcolemma isolated from spontaneously hypertensive rat (SHR) hearts were compared to the same parameters in sarcolemma from normotensive rat (WKY) hearts. Sarcolemma prepared from SHR heart contained significantly less ouabain-inhibitable ATPase activity than sarcolemma from WKY heart. No significant differences in sarcolemmal protein content or recovery were noted between the two groups. The numbers of phosphorylation sites and ouabain binding sites were lower for SHR hearts than for WKY hearts. The K D values for ouabain binding were the same (0.30 fiM) in cardiac sarcolemma of SHR and WKY. The 1^ values for inhibition by ouabain of Na + ,K + -ATPase were also the same for both groups (SHR = 49 JXM; WKY = 44 /xM). These data suggest that the decrease of cardiac sarcolemmal Na + ,K + -ATPase activity in SHR hearts is due to a decrease in the number of active sites. (Hypertension 5: 682-688, 1983)

show abstract

A simple method for the determination of affinity and binding site concentration in receptor binding studies

Cited by 136 publications

References 9 publications

Development and Preclinical Testing of a High-Affinity Single-Chain Antibody against (+)-Methamphetamine

Development and Preclinical Testing of a High-Affinity Single-Chain Antibody against (+)-Methamphetamine

Interaction of the Growth Hormone-Releasing Peptides Ghrelin and Growth Hormone-Releasing Peptide-6 with the Motilin Receptor in the Rabbit Gastric Antrum

Decrease in Na+,K+-ATPase activity and [3H]ouabain binding sites in sarcolemma prepared from hearts of spontaneously hypertensive rats.

Contact Info

Product

Resources

About