Ligand binding test

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The protein complex Arp 2/3 exists due to affine bonds between the subunits.

A ligand binding assay ( English ligand binding assay , LBA) is a method of Biochemistry and Pharmacology to measure the affinity of binding of two molecules to each other.

principle

Binding of tacrine (blue) to cholinesterase .

The tendency of two molecules to bind to one another is known as affinity. Any molecule can bind to any other with the appropriate affinity, which can be determined in a ligand binding test. The binding partners of a bond can e.g. B. be a ligand and a receptor . Small molecules are usually bound in a recess in a protein , as the enlarged contact area enables a more affine binding. The receptor is immobilized first . This is followed by a measurement of the ligand concentration at maximum binding of the ligand (C B max , synonymous with maximum saturation) and the binding of the ligand at different concentrations of C ligand . With a graph of the saturation (the proportion of bound ligand C bound ligand / C B max ) over the concentration of the ligand C ligand , the dissociation constant can be read off at a saturation proportion of 0.5. The dissociation constant corresponds to the ligand concentration of half-maximal binding. The maximum ligand binding B max and the dissociation constant K d can also be determined using a Scatchard diagram . The dissociation constants of high affinity bonds are around 10 −9 M −1 (e.g. hormones at their receptors, stable protein complexes , antigen - antibody bonds). Changes in the function of a receptor after ligand binding are not recorded by ligand binding tests.

Label Free Ligand Binding Assays

In contrast to the ligand binding tests in the following section, surface plasmon resonance spectroscopy , bio-layer interferometry and the measurement of changes in impedance measure the change in layer thickness without using a marker , but usually require higher concentrations of ligands. Different variants have been developed for high throughput screening . The affinity can be determined with isothermal titration calorimetry , provided that the binding is not driven exclusively by entropy (in the case of an isenthalpic binding). No markings are required here either, and short-term bonds are not recorded.

Label-based ligand binding tests

Ligand binding test with 18 F-FDG in vivo by PET scan

Filter membranes

The filter test (synonymous membrane binding test ) is achieved by adding a marked possible binding partner to a molecule immobilized on a surface (e.g. filter paper , PVDF membranes, polyethyleneimine- coated glass fiber filters ) . The label can be radioactive , a reporter enzyme , biotin , a fluorophore , or an oligonucleotide . After several washing steps of the coated surface, the amount of remaining bound molecules is determined. Processes without washing steps are sometimes referred to as mix-and-measure .

First, the equilibrium constant is measured at various concentrations of the ligand labeled with the reporter. Here, the labeled molecule is incubated with the immobilized binding partner until chemical equilibrium is reached . The membrane is then washed and dried, and the amount of bound reporter molecule is determined. After a predetermined number of washes, the amount of the reporter molecules is measured on the membrane again, which information on the leaching of reporter molecules are ( English off-rate ). Alternatively, the washout can also be determined by determining the time course of the amount of reporter molecules.

In the case of radioligands, the specific activity is determined as radioactivity divided by the amount of substance used (in Ci / mmol). The amount of substance or concentration of the radioligand is determined and communicated by the manufacturer. The actually effective concentration ( picomolar ) can be determined by the following equation:

Size exclusion chromatography

If the receptor and ligand have different molar masses , the binding partners bound to one another can also be isolated by size exclusion chromatography and the amount can be determined using the label.

ELISA

Immunoprecipitation

Fluorescence polarization

Fluorescence resonance energy transfer

PET scan

By positron emission tomography , the affinity may in vivo be determined.

history

The first ligand binding test, a radioimmunoassay , was developed in 1960 by Rosalyn Sussman Yalow and Solomon Aaron Berson , for which they received the 1977 Nobel Prize in Physiology or Medicine . Ligand binding tests were originally used in the protein characterization of hormones , neurotransmitters, and their receptors. In medical diagnostics of breast cancer , a variant was used, is measured in the cell extracts bound to radiolabeled estradiol, after adsorption unbound estradiol on dextran -coated activated carbon .

literature

Individual evidence

  1. ↑ GS Sittampalam, SD Kahl, WP Janzen: High-throughput screening: advances in assay technologies. In: Current opinion in chemical biology. Volume 1, Number 3, October 1997, pp. 384-391, ISSN  1367-5931 . PMID 9667878 .
  2. ↑ a b L.A. de Jong, DR Uges, JP Franke, R. Bischoff: Receptor-ligand binding assays: technologies and applications. In: Journal of chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. Volume 829, Number 1-2, December 2005, pp. 1-25, ISSN  1570-0232 . doi : 10.1016 / j.jchromb.2005.10.002 . PMID 16253574 .
  3. ↑ D. DeSimone, JY Shih, HC Gunn, V. Patel, L. Uy, TM Thway: Method transfer for ligand-binding assays: recommendations for best practice. In: Bioanalysis . Volume 3, Number 18, September 2011, pp. 2143-2152, ISSN  1757-6199 . doi : 10.4155 / bio.11.199 . PMID 21942524 .
  4. ^ TD Pollard: A guide to simple and informative binding assays. In: Molecular biology of the cell. Volume 21, Number 23, December 2010, pp. 4061-4067, ISSN  1939-4586 . doi : 10.1091 / mbc.E10-08-0683 . PMID 21115850 . PMC 2993736 (free full text).
  5. ^ Stefan Offermanns, Walter Rosenthal: Encyclopedia of molecular pharmacology . 2nd edition, Springer 2008. ISBN 9783540389163 . P. 585.
  6. ↑ D. DeSimone, JY Shih, HC Gunn, V. Patel, L. Uy, TM Thway: Method transfer for ligand-binding assays: recommendations for best practice. In: Bioanalysis. Volume 3, Number 18, September 2011, pp. 2143-2152, ISSN  1757-6199 . doi : 10.4155 / bio.11.199 . PMID 21942524 .
  7. ↑ YM Wang, V. Jawa, M. Ma: Immunogenicity and PK / PD evaluation in biotherapeutic drug development: scientific considerations for bioanalytical methods and data analysis. In: Bioanalysis. Volume 6, Number 1, January 2014, pp. 79-87, ISSN  1757-6199 . doi : 10.4155 / bio.13.302 . PMID 24341496 .
  8. ^ SS Leung, EA Dreher: Automate it: ligand-binding assay productivity in a discovery bioanalytical setting. In: Bioanalysis. Volume 5, Number 14, July 2013, pp. 1775-1782, ISSN  1757-6199 . doi : 10.4155 / bio.13.149 . PMID 23862709 .
  9. ^ Anthony P. Davenport: Receptor Binding Techniques. Humana 2005. ISBN 1-58829-420-X . Pp. 18f., 101f., 121f., 203f.
  10. ↑ GE Abraham: Radioimmunoassay of steroids in biological fluids. In: Journal of steroid biochemistry. Volume 6, Numbers 3-4, 1975 Mar-Apr, pp. 261-270, ISSN  0022-4731 . PMID 1102794 .
  11. ^ A b Steven D. Kahl, G. Sitta Sittampalam, Jeffrey Weidner: Calculations and Instrumentation used for Radioligand Binding Assays . In: Assay Guidance Manual . May 1, 2012, pp. 1–21. Retrieved March 20, 2014.
  12. ↑ RS YALOW, SA BERSON: Immunoassay of endogenous plasma insulin in man. In: The Journal of clinical investigation. Volume 39, July 1960, pp. 1157-1175, ISSN  0021-9738 . doi : 10.1172 / JCI104130 . PMID 13846364 . PMC 441860 (free full text).
  13. ↑ WL McGuire: Estrogen receptors in human breast cancer. In: The Journal of clinical investigation. Volume 52, Number 1, January 1973, pp. 73-77, ISSN  0021-9738 . doi : 10.1172 / JCI107175 . PMID 4345203 . PMC 302228 (free full text).