【화학】 16-1-1강. radioligand binding assay

16-1-1강. radioligand binding assay

 

추천글 : 【화학】 16-1강. 약리학(PK/PD)  

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1. 용어

2. 프로토콜

3. 데이터 해석

4. 레퍼런스

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1. 용어 

⑴ CPM(counts per minute) : 이론적인 방사선 측정 값

⑵ DPM(disintegrations per minute) : 실제 방사선 측정 값

⑶ SA(specific activity) : radioligand 1몰당 radioactivity

① 예 : Ci/mmol로 나타냄

② 1 큐리(Ci) = 2.22 × 10¹² DPM 

⑷ RAC(radioactive concentration)

① 예 : μCi/ml 또는 mCi/ml 

⑸ Eff(efficiency) : DPM / CPM 

⑹ decay rate constant : t_1/2가 반감기일 때, (-0.693 / t_1/2)를 의미함

⑺ PMT(photomultiplier tube) : 형광 물질 등에서 방사된 광자를 탐지함

⑻ SPA(scintillation proximity assay) 

⑼ YSi(Yttrium silicate) : a rare earth metal based SPA bead

⑽ PVT(polyvinyltoluene) : a plastic based SPA bead

⑾ PE LAS : Perkin Elmer Life and Analytical Sciences

 

 

2. 프로토콜 

[³H]-labeled SPA Beads can be prepared by incubating [³H]-labeled biotin with YSi streptavidin beads and washing them (using centrifugation) to remove any unbound radioactivity. Alternatively, a reaction associated with an assay (e.g. WGA beads, membranes, radioligand) can be used. 

 

⑴ Remove a 300 μl aliquot of [³H]-SPA beads to a 1.5 ml polypropylene tube. 

⑵ Centrifuge the tube for 5 seconds in a microfuge to pellet the [³H]-SPA beads. 

⑶ Remove the supernatant. Dispose of it properly, treating it as potential radioactive waste.

⑷ Add 300 μl of PBS and mix beads. Repeat centrifugation and remove supernatant.

⑸ Resuspend in a final volume of 300 μl PBS.

⑹ For a Microbeta, pipette 25 μl of beads into three different wells of a microplate. Add 175 μl of PBS. Allow the beads to settle overnight.

⑺ Count the microplate and determine the average CPM for the three replicates (example: 52,800 CPM).

① gamma counter 이용

⑻ Add 25 μl of beads to three different scintillation vials containing scintillation cocktail. Count the vials on a liquid scintillation counter, which is capable of returning results in DPM, and determine the average for the three replicates (exmaple: 140,582 DPM).

① gamma counter 이용

⑼ Determine efficiency using the following equation

① Eff = CPM / DPM = 52,800 / 140,582 = 0.38

 

 

3. 데이터 해석 

⑴ 반감기를 고려하여 실험 데이터를 보정해야 함

⑵ 다양한 기질 농도, 억제제 농도에서 실험을 하면서 반응속도론에 근거하여 K_m을 계산할 수 있음

⑶ non-linear curve fitting을 해야 함

⑷ overall binding curve에서 고농도일 때의 linear 구간을 찾으면 specific binding과 non-specific binding을 분리할 수 있음 

 

Figure. 1. overall binding curve에서 specific binding curve와 non-specific binding을 분리한 그래프

 

 

4. 레퍼런스 

⑴ Kahl, S. D., Sittampalam, G. S., & Weidner, J. (2012). Calculations and instrumentation used for radioligand binding assays. Assay Guidance Manual [Internet].

⑵ Davenport, A. P., & Russell, F. D. (1996). Radioligand binding assays: theory and practice. In Current directions in radiopharmaceutical research and development (pp. 169-179). Springer, Dordrecht.

⑶ Jiang, D., Im, H. J., Sun, H., Valdovinos, H. F., England, C. G., Ehlerding, E. B., ... & Cai, W. (2017). Radiolabeled pertuzumab for imaging of human epidermal growth factor receptor 2 expression in ovarian cancer. European journal of nuclear medicine and molecular imaging, 44(8), 1296-1305.

 

입력: 2022.01.15 21:40