Measurement in Science Bioassays and Immunoassays

1. Measurement in ScienceBioassays and Immunoassays Karim Meeran 23 October 2009

2. Assay • Method of measuring concentration of a hormone in body fluid (usually serum). • Previously used “bioassay”

3. Bioassay • Administer extract of pituitary to animal and observe • (eg: LH would very slowly cause testicular enlargement).

4. Bioassay • Cells in vitro • Grow

5. Immunoassay • Because antibody could bind to a particular protein, this could be used to measure the amount of a substance. • Make antibody by giving protein to animal of different species

6. Assay for human adrenomedullin • Give adrenomedullin to rabbits until they make antibodies (few months) • Collect blood from the rabbits • Use that blood to mop up antigen

7. Principles of RIA • Have known amount of radioactive adrenomedullin. • Add unknown amount of unlabelled adrenomedullin • Add measured amount of antibody • Add a second antibody against the first • Mix and spin

8. Principles of RIA • The second antibody may be a mouse anti rabbit antibody. • This will be bound to a solid particle that will sink when centrifuged.

9. Example • Adrenomedullin paper

14. Immunoassays • Method of measuring concentration of a hormone in a body • Two methods: - Competitive Assays - Sandwich Assays • Both work on the principle of enzyme specificity.

15. Competitive assays • For a given peptide (P) we are able to produce antibodies by administering the peptide to a different species. E.g.: human peptide to rabbit. • Let the rabbit make antibodies (Abr), after a couple months remove the blood and it will have Ab for this P

16. Competitive assays • Have Ab to P and we need to be able to accurately measure P. How do we do this: • Make a Radioactive version of the peptide (Phot) and an Abm to the rabbit produce • Abrinto a mouse. But this Abm we also covalently attach to a particle of charcoal so it is heavier.

17. Competitive Assays A mixture of P and Phot

18. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot

19. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot

20. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Abm which binds to the Abr

21. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Abm which binds to the Abr

22. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Abm which binds to the Abr Spin

23. Competitive Assays Following centrifugation, the supernatant and pellet need to be separated.

24. Competitive Assays Following centrifugation, the supernatant and pellet need to be separated.

25. Competitive Assays Following centrifugation, the supernatant and pellet need to be separated.

26. Competitive Assays Count FREE radiation Following centrifugation, the supernatant and pellet need to be separated. Count BOUND radiation.

27. Competitive Assays Count FREE radiation 50% Following centrifugation, the supernatant and pellet need to be separated. Count BOUND radiation. 50%

28. Competitive Assays Count FREE radiation 50% Repeat for different concentrations of P Following centrifugation, the supernatant and pellet need to be separated. Count BOUND radiation. 50%

29. Separating bound from free

31. Bound to antibody + charcoal

32. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot

33. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot

34. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Abm which binds to the Abr Spin

35. Competitive Assays Following centrifugation, the supernatant and pellet need to be separated.

36. Competitive Assays Following centrifugation, the supernatant and pellet need to be separated.

37. Competitive Assays Following centrifugation, the supernatant and pellet need to be separated.

38. Competitive Assays Count FREE radiation Following centrifugation, the supernatant and pellet need to be separated. Count BOUND radiation.

39. Competitive Assays Count FREE radiation 0% Following centrifugation, the supernatant and pellet need to be separated. Count BOUND radiation. 100%

40. Competitive Assays Count FREE radiation 0% Following centrifugation, the supernatant and pellet need to be separated. Repeat for different concentrations of P Count BOUND radiation. 100%

41. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot

42. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot

43. Competitive Assays A mixture of P and Phot Addition of Abrwhich bind specifically to the peptides but cannot discriminate which ones are hot Addition of excess Abm which binds to the Abr Spin

44. Competitive Assays Count FREE radiation 33% Following centrifugation, the supernatant and pellet need to be separated. Repeat for different concentrations of P Count BOUND radiation. 66%

45. Summary Zero blue peptide gives 100% bound radiation. Three blue peptide gives 66% bound radiation. Six blue peptide gives 50% bound radiation. Twelve blue peptides gives 33% bound radiation. This is the basis of how it works but for each assay we do using the antibodies we have we must run a series of these to obtain a reference curve.

47. What is serial dilution?

49. Competitive Assays Set up a series of tubes with known varying concentrations of P (achieved by serial dilution). 1 2 3 4

50. Competitive Assays Set up a series of tubes with known varying concentrations of P (achieved by serial dilution). To all of these we add a set amount of Phot. 1 2 3 4