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Hbv
- 1. 亠仗舒亳 于亳 丕弌-324: .舒亟舒仄亞, .弌舒仍亢舒于仍舒仆
- 2. 勵 亠亶仆 弍亳亠仆 仆 42 仆仄 仄亢亶 舒亟仆舒 弍勵勵勵仍 仆 7-8 仆仄 亰亰舒舒仆舒亶,仂 亟舒于舒 仍亳仗亳亟 仂亞仂仆 亞舒亟舒亞亞亳亶仆 亞唏唏亞 弍ﾑ HBsAg(HB surface antigen)仆 亞于舒仆 唏唏 弍勵 弍勵亳亶 仆从仍亠仂亳亟 仂亞亟仂亞. 丐仂仄 仄仂仍亠从仍 LHBsAg(L-large) 仆亟 仄仂仍亠从仍 MHBsAg(M-medium) 亳亢亳亞 仄仂仍亠从仍 SHBsAg(s-small) 舒仗亳亟 仆 22-25仆仄 亟亳舒仄亠亶 亞 仄 弍勵亶
- 3. 勵 preS1,preS2-HBV 亞舒亟仆舒 弍舒亶仍舒亟舒亞 舒亞 S-亞舒亟舒亞亞亳亶仆 舒亞 HB-弍唏唏仄亳亶仆 舒亞 丱-亳亶仆 仄仂仍亠从仍 丐-亠仄亳仆舒仍 仗仂亠亶仆 RT-亞(于 舒仆从亳舒亳) LHBs,SHBs,MNBs- 舒仆亳亞亠仆亳亶 亞于舒仆 唏唏 仆从仍亠仂亳亟
- 4. 于亳仆 亞亠仆仂仄 320 bp 舒亶 丱-亳亶仆 仂 舒仍舒亞 仆从仍亠仂亳亟 丱-亳亶仆 仂 舒仍舒亞 仆: 勵仆 舒亞亳舒亞 弍ﾑ 唏唏亞 亞舒(negativ-minusstrang-) 仆从仍亠仂亳亟.5` 唏亞亞唏仍亟 从仂于舒仍亠仆 仂仍弍仂仂 弍勵亳亶 -亞亳亶仆 仄仂仍亠从仍舒亶. 丐舒仍 舒亳舒亞 弍ﾑ 亠亞 亞舒 (positiv-plusstrange+) 仆从仍亠仂亳亟 仂亞仂仆仂.3` 唏亞亞唏仍亟 从仂于舒仍亠仆 弍 仂仍弍仂仂 -亞亳亶仆 仄仂仍亠从仍舒亶.
- 5. 亠仗舒亳 于亳亳亶仆 亞亠仆仂仄 仆 亟舒于舒亞亟舒仆 亟 仍弍舒仍舒亞亟舒 亠从于亠仆(direct repeats DR1 DR2)-亳亶仆 亞亶 弌亠从于亠仆 仆 弍勵亟 11bp 仆从仍亠仂亳亟亶. HBV-仆 亞亠仆仂仄亟 亟舒于舒亞亟舒仆 仍弍舒仍舒亞亟舒 唏仍唏唏 亟舒舒舒仍舒仍 唏唏 仂仂仂仆亟仂仂 舒亟亳仍亞勵亶 亞舒舒 亞勵勵亟亳亶亞 仍弍舒仍舒仆 仆亳亶仍亞亢勵勵仍亟 仂仂仍仂仆仂. 1. 亳仆 亞舒亟舒亞亞仆 舒亞 弍仂仍仂 SHBsAg,MHBsAg,LHBsAg 舒仆亳亞亠仆 从仂亟仍仂亞 唏仍唏唏 亟舒舒舒仍舒仍 2. 舒仗亳亟 弍仂仍仂仆 弍唏唏仄亳亶仆 舒亞 仄唏仆 HBcAg 舒仆亳亞亠仆 亟亞 仆磪 仂仍弍仂亞亟仂仆 HBeAg 舒仆亳亞亠仆 从仂亟仍仂亞 唏仍唏唏 亟舒舒舒仍舒仍 3. 舒亞 亰舒亟仍舒亞 唏仍唏唏 亟舒舒舒仍舒仍 4. 丱-舒亞 弍ﾑ 舒于亟仆 舒亞 从仂亟仍仂亞 唏仍唏唏 亟舒舒舒仍舒仍 仆从仍亠仂亳亟仆 4 亟舒舒舒仍舒仍 仆 弍亳亠 弍亳亠仆 勵仍 舒仄舒舒舒仆 仂亟仂仂亶 亞 仆从仍仂亳亟亞 从仂亟仍仂亟仂亞 舒舒 亟舒于舒亞亟舒仆 仍弍舒仍舒亞亟舒 唏仍唏唏 亟舒舒舒仍仍亞 仆亳亶亟 仆 于亳仆 亞亠仆仂仄 亞仆.
- 6. 亠仗舒亳亳亶仆 于亳仆 亞亠仆仂仄仆 仂亞仂 ORF-2 亳仄 Pre- S1,Pre-S2,S 亞亠仆仂仄 ORF-1 亳仄亟 从舒仗亳亟亳亶仆 弍ﾑ 亞亠仆仂仄 ORF-3 亳仄亟 弌 亞亠仆仂仄 ORF-4 亳仄亟 丱 亞亠仆仂仄
- 7. 亠仗舒亳仆 于亳仆 弍唏唏仄亳亶仆 舒仆亳亞亠仆 (HBcAg) 亠仗舒亳仆 于亳仆 从舒仗亳亟亟 弍唏唏仄亳亶仆 舒仆亳亞亠仆亳亶 舒亞 舒亞仍舒亞亟舒仆舒. 22kD 仄仂仍亠从仍 亢亳仆亶. 亳仆 亠仗仍亳从舒仆 磦舒亟 仍亞仆亳亶 亳亶仆 从亳仆舒亰舒 亠仄亠仆亳亶仆 勵亶仍仍仍 亠亳仆 仂仂亢亳 仗仂亠亞 亳仄亢勵勵仍亟亞. 仆仍仂亞 仆 仍亞仆亳亶 亳亶仆 亞亳亶仆 弍勵仍亞亶 仂仍弍仂亞亟仂仆,仍亞仆亳亶 亳亶仆 亟仄仆 仄亟仍仍 仍弍舒仍舒亞亟舒仆 亰唏唏于唏仍唏亞亟唏 亞亞 于亳仆 弍勵亳亶仆 舒亞 勵勵 弍仂仍亞仂仆 于亳亞舒亢 于亳亳仂仆 弍勵仍亟 仗仂亠 仂仂仍亟仂亞.
- 8. 亠仗舒亳仆 于亳仆 亳仍 舒仆亳亞亠仆(HBeAg) HBcAg 舒仆亳亞亠仆亳亶 舒仄亳仆勵亳仍 亳仆亠亰仍亞亟 磦舒亟 舒仄亳仆 唏亞亞唏仍亳亶仆 亞 29 舒仄亳仆勵亳仍 仆 HBeAg 亳仆亠亰仍亞亟 仍仍 弍仂仍仂仆 于亳亟舒亞. HBeAg 舒仆亳亞亠仆 亳仆亠亰仍亞亟 仍仍 弍仂仍仂仆 pre C 亞 弍仂仍 仂亞于仂仂亶 亳亞仆舒仍仗亠仗亳亟 弍唏亞唏唏亟 仍亞仆亳亶 亳亶仆 亠亳从仍亳亶仆 仆亟仂仗仍舒亰仄亳亶仆 仄亠仄弍舒仆 亞舒亟舒亞亟 舒亞 亳仆亠亰仍亞亟仆 舒 仗仂亠亞 仆唏唏仍亟勵勵仍仆. 弌亳仆亠亰仍亞亟仆 舒亞 仂仍亟亢仆 舒仗仗舒舒亞 亟舒仄亢亳仆 仍亞仆亳亶 亳亶仆 亞舒亟舒亞亟 亰唏唏于唏仍唏亞亟唏亢 亠-舒仆亳亞亠仆亳亶亞 仂亟仂仂亶仍仆仂.
- 9. 亠仗舒亳仆 于亳仆 亞舒亟舒亞亞仆 舒仆亳亞亠仆 HBsAg 仂亞亠仗舒亟仆舒 于亳仆 亞舒亟仆舒 弍勵勵勵仍亳亶仆 仄亠仄弍舒仆亟 弍勵 磿亞舒舒舒亶 亞于舒仆 唏于唏仄唏 亞仍亳从仂仗仂亠亶仆 舒亞仍舒亞亟舒仆舒.勳勵仆亳亶亞 亞亠仗舒亳仆 于亳仆 亞舒亟舒亞亞仆 舒仆亳亞亠仆 HBsAg 亞仆. HBsAg-舒仆亳亞亠仆亳亶亞 亳亢亳亞 仄仂仍亠从仍 舒仆亳亞亠仆 (SHBsAg) 仆亟 亰亞亳亶仆 仄仂仍亠从仍 舒仆亳亞亠仆(MHBsAg) 丐仂仄 仄仂仍亠从仍 舒仆亳亞亠仆 (LHBsAg) 仄仆 仆仍仆.
- 10. SHBsAg 于亳仆 舒仍亟于舒仆 勵仆亟仆 弍勵亟仍 226 舒仄亳仆勵仍 弍勵仍亟仆 24kD (p24) 仄仂仍亠从仍 亢亳仆亶 舒仆亳亞亠仆. 仄亳仆勵仍亳亶仆 146 亟 弍舒亶仍舒亟舒 舒仗舒舒亞亳仆亳亶 勵仍亟亞亟仍 (Asn146)亞仍ﾐ歳笑経湖勤湖 27从D(gp27)仄仂仍亠从仍 亢亳仆亶 亞仍亳从仂仗仂亠亶仆 弍仂仍亟仂亞.
- 11. MHBsAg 33从D 仄仂仍亠从仍 亢亳仆亶 亞亳亶亞 pre S2-HBsAg 亞亢 仆仍亟亞.
- 12. LHBsAg 39kD 仄仂仍亠从仍 亢亳仆亶 亞亳亶亞 pre S1-HBsAg 亞亢 仆仍仆.
- 13. 仂仍亳仄亠舒亰舒(P 舒亞) -于亳仆 丱-亳亶仆 舒仍 舒亞亳舒亞 亅亠亞 亞舒 仆从仍亠仂亳亟仆 3` 唏亞亞唏仍亟 从仂于舒仍亠仆 弍 仂仍弍仂仂仆亟 弍舒亶仍舒 90从D 仄仂仍亠从仍 亢亳仆亶 -舒亞 于亳仆 舒仍亟于舒仆 仆亞亢 弍仂仍亟仂亞.
- 14. -舒亞 17kD 仄仂仍亠从仍 亢亳仆亶 亞仂仄仂亟亳仄亠 弍勵亶 仂亞亠仗舒亟仆舒 于亳仆 唏唏仍亟 亳仍亟亞. 亅仍亞仆亳亶 亳亶仆 舒于亟舒 (Hepato zellular karzinom) 勵勵 舒仍亞舒舒仆 弍仂仍仆仂. -舒亞 仍亞仆亳亶 亳亶仆 仗仂仄仂亠 弍仂仍仂仆 于亳仆 仗仂仄仂亞 亟舒仄 亳亟于亢勵勵仍亞 (transaktivator) 弍仂仍亟仂亞.
- 15. 亟亠仆亳仆仄仂仆仂仂舒仆 亳从仍亞 亰仂亳仍舒亞 亞亳亶仆 舒仆舒从亳于舒仂仆 (CREB) 亞亶 仆亞亟仆 丐丐 弍仂从仆 舒亞 (TBP) 仗仂仄仂亠亞 亳亟于亢勵勵仍亟亞.丐 亳亟于亢亳亟 舒亞 唏唏亳亶仆 从舒弍仂从亳仍 唏亞亞唏仍亳亶仆 亟仂仄亠仆舒舒 舒于亟舒 亟舒舒仆亞亶仍舒亞 舒亞 53-舒亶 仂仍弍仂亞亟仂仂亟 亳亶仆 舒仆舒从亳于舒仂 勵亶仍仍仍 舒舒仆舒.丱于 弍勵 仆 于亳 唏唏仍唏亞亟唏仆 丱-亳亶仆 亳仆亠亰 亠仗亠舒亳亶仆 仄亠-舒舒 亰舒舒亞亟舒亞勵亶 弍仂仍 53 舒亞 仄亞亞 丱 舒亞仍舒仆 亳亶仆 舒仗仂仗仂亰亞 亟舒亞舒仆舒.亅仆 勵亠亟 仍亞仆亳亶 亳亶仆 于舒舒亞亟仍仆 亰仂亳仍亞舒 舒仍亟舒亞亟舒亢 礌舒仍亞勵亶 仂仍亳仆仂仂 仍亞仆亳亶 舒于亟舒 勵勵仆.
- 16. 丐舒舒仍
- 17. HBV 亠仗仍亳从舒亳: 亅亰仆 弍亳亠亟 仆于 亅亰仆 弍亳亠亟 仆于: 舒仆亠舒仍: 亟舒舒 舒亳舒 亳亶, 勵勵仍 仄仆亟亳亶仆 舒亢亳仍亳亟 唏唏仄亞亳亶 仍亞亳亶仆 亰舒仄: 弍亳亠 勵仆仍亞, 亳亢亳仍 勵亶仆勵勵亟 唏唏仄亞亳亶 亠亳仆舒舒仍 (于亠亳从舒仍): 亢亳亟 仆 (HBeAg+) 勵勵亟 唏唏仄亞亳亶
- 18. HBV 亠仗仍亳从舒亳: 丐舒舒 丶舒舒 亟舒仄亢亳 弌亟舒仆 仆亟仂亠仍 亳亳亶仆 亰舒亶 (Space of Disse) 亅亅 丐丶丐
- 19. HBV 亠仗仍亳从舒亳: 亅仍亞仆亳亶 亶 仂仍弍仂亞亟仂 亠仗舒仂亳仆 仂仍仂仆 唏仍亳亶仆 亠亠仗仂仂亶 仂仍弍仂亞亟仂亢 弍仂仍仂 ﾐ 亞亢 勵亰亢 弍舒亶仆舒. LHBsAg (preS1) 亠仗舒亳仆 仍舒- 仗仂亠仂亞仍亳从舒仆 (仄亳亳仆) 亳仗亳亟亳亶仆 仍亳亞舒仆亟 丱仂仍亠仂仍仆 亠亠仗仂 亳亞舒仆亟 弌亠亳仆 仗仂亠舒亰舒 舒舒仍舒亞 舒从仂 亳仍亞仍亳从仂仗仂亠亶仆 仆仆亠从亳仆 V
- 20. HBV 亠仗仍亳从舒亳: 亅 亟仂仂 仂仂 亅亳亶仆 亞舒亟仆舒 弍勵勵勵仍 舒 舒仗亳亟舒舒 亰舒亟仍舒 (仍亞仆亳亶 亳亶仆 亞亳亶仆 仂仂仍仂仂仂亶) 丼唏仍唏唏 亞亠仆仂仄 弍亳亳仍 亞舒舒 亟舒仄亢亳亢 唏唏仄唏仆亟 仄仗仂亳仆 留, 硫 亠亠仗仂仂亶 仂仍弍仂亞亟仂 rcDNA (舒仍 舒亞亳舒, 亠亞 亞舒) cccDNA (covalently closed circular DNA) 勵勵仆. (仗亳仂仄 弍舒 亳仆亠亞舒亳仍舒亞亟舒仆)
- 21. HBV 亠仗仍亳从舒亳: 亠仆亳亶仆 舒仆从亳仗亳 丱 仗仂仍亳仄亠舒亰舒 II Pre C pgRNA (pregenomic 弍唏唏仄亳亶仆 亟舒仍) - 2,4/2,1kb mRNA - 0,7kb mRNA HBeAg, HBcAg 亳亳仂仆 仂亞仂仍弍仂仍仂亞 亟仄亢亳 亞亟 LHBsAg MHBsAg SHBsAg HBxAg P 舒亞
- 22. 丐仂亟仂仂亶 仆亞仆 弍舒亶仍舒仍亟 亰亞亳亶亞 亠仗仍亳舒从亳于 HBxAg 仍亞仆亳亶 亳亶仆 舒仄亳仆 勵亳仍亟 仂仍弍亳仍亟仂亞 亳仆亠亞舒亳于
- 23. HBV 弌亠仂亳仗 SHBsAg (120-163) 亅亞弍亳亠 亠仆仂仄仆 DR1, DR2, ORF, 亰舒亳仄 亞亳亶仆 亟舒舒舒仍舒仍亟 仄舒亳 勵勵仆 弍舒亶亟舒亞 弍舒 舒 亠唏仆亳亶 亟亠亠仄亳仆舒仆 弍勵仍亞 d 弍亟亠亠仄亳仆舒仆 + q, x, g y 弍亟亠亠仄亳仆舒仆 r 弍亟亠亠仄亳仆舒仆 w 弍亟亠亠仄亳仆舒仆 /2, 3, 4/
- 24. HBV 弌亠仂亳仗仆 舒舒仍
- 25. HBV 舒仍亟于舒 仆: 丱 (仍亞 舒仍亟于舒仍仍舒)= 10^10 IU/ml 丐亞亳亟- 10^6 (1仄仍- HBsAg) 舒仍舒舒亶 10^2 (1仄仍- HBsAg) /亞勵亶/ 亳亠亳亶仆 亳仆亞仆 亟 HBV 勵于亳仆: 哦仆亟唏: , 亳亶仍亟, 舒仆 勵勵亟 仆亟: 勵亳亶仆 亳仆亞仆, 勵仆亳亶 亳仆亞仆, 勵仍 舒亞舒: , 弍舒舒, 唏仍, 勵勵, 仆仍亳仄
- 26. 丱舒仍亟于舒仆 磦 丶仂仄仂亞 舒仍亟于舒: 10^6 /1仄仍/ + / 舒亞 舒仍亟于舒: HBsAg 6 舒 弍舒 勵勵仆 亟 仂亟仂仂亶仍仂亞亟仂
- 27. HBV 仂仄仂亞 舒仍亟于舒
- 28. HBV 舒舒亞 舒仍亟于舒
- 29. HBV output
- 30. HBV:舒弍仂舒仂仆 仂仆仂仍仂亞仂仂 1. 亶仍亟 亟仍舒仍: 亅亞 弍亳亠亳亶仆 仄舒从亠 2. 亳仂仍仂亞亳: HBV-DNA assay 亳仆 亠仗仍亳从舒亳亶仆 勵亠亟 PCR 舒舒 仆从仍亠亶仆 勵亳仍 亳仍勵勵仍 3. 亳仂亳仄亳亶仆 勵亰勵勵仍仍勵勵亟 ALT, AST 4. 亳仂仍仂亞亳 亳仆亢亳仍亞 丕亟亳仍舒仆 亞亳亶仍 亳仆亢亳仍亞 丶仆 亟仂仆仂, 仆 亳仍勵勵仍 仂...
- 31. 丕亟亳仍舒仆 亞亳亶仍仍 亟于亞勵亶: HBV- 亳仄仄仆仂亞仍仂弍仍亳仆 仂仍/ HBsAg, HBeAg + 唏唏仆 仆舒亶亟 12-48 舒亞亳亶仆 亟仂仂 舒从亳仆 (sHBs 舒亞) Anti HBs 1980 仂仆亟 亞舒亞舒亢 舒于舒仆. 0,1, 6 舒 5仄亞 舒仍舒舒 仍舒舒仆 40仄亞 舒仍舒舒 仂亞仂亞勵亶. (SHBs-G145R) escape mutation 舒 唏仆亟唏, 舒亞舒仍舒仍舒亶, 舒亳仆亟 亟仂仆仂
- 32. HBV 仄亳仍亞 Adefovir dipivoxil (Hepsera) Lamivudine (Epivir HBV) Interferon alfa (Intron A) 舒舒仍舒舒, 亟舒舒仆 亞舒舒舒舒亶 仂亳 pgRNA inhibitor cccDNA inhibitor
- 33. 亠仗舒亳仆 于亳 Hepatitis D virus / HDV 亳仆亶 亞亠仗舒亳 D
- 34. HDV 弍勵 亳仂亶亟仆 弍勵仍亞 32-36仆仄 亟亳舒仄亠 唏仄弍唏仍唏亞 仍弍亶 - ssRNA 舒亞亳舒亞 1700nt 亠仆仂亳仗亳亶仆 8 唏唏仍 弍勵亞亞亟仆
- 35. MP-HDV SHBeAg MHBsAg LHBsAg HBV 仆 弍勵勵勵仍亳亶亞 舒仆亳亞亠仆亶 仆 舒仄 仍弍舒仍舒亢 弍勵勵勵仍亢亟亞 仂 & 弌仗亠 舒仍亟于舒 HDV 弍勵 SHD 24kD LHD 27kD
- 36. HDV 亠仗仍亳从舒亳
- 37. HDV 舒仍亟于舒仆 舒亶, 舒仍亟于舒 亟舒仄亢亳 亰舒仄 丱舒仍亟于舒仆 舒亶 仂仄仂亞 弍仂仍仂仆 舒舒亞 亞亠仗舒亳舒亶 唏于唏仆 亞亠仗舒亳 于亳 亞 丱舒仍亟于舒 亟舒仄亢亳 亰舒仄 仗舒亠仆亠舒仍 (, 弍仂亳 弍舒亞舒亢, 亰勵勵 舒亳) 弍仍亞亳亶仆 亰舒仄 舒亞 (于亠亳从舒仍) 亞舒舒舒 2-6 亟仂仍仂仂仆 仂仆仂亞
- 38. HDV 舒舒仍
- 39. HDV 仂仆仂亳仍亞仂仂 PCR 亞亠仆仂仄 舒从亠 Anti-HDV Anti-HDV-IgM 仂-舒仍亟于舒 弌仗亠-舒仍亟于舒 aHBc-IgM + - aHDV-IgM + +
- 40. How E.L.I.S.A.s work 丕弌-324 .舒舒亶
- 41. What is an ELISA? ELISA = enzyme-linked immunosorbent assay Enzyme-Linked: enzyme amplifies antigen-antibody interaction Immunosorbent: reaction products adsorbed on container Assay: assessment of potential analyte
- 42. HISTORY Prior to the development of the EIA/ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively-labeled antigens or antibodies. Avramais (1966, 1969) and Pierce (1967) developed methods to chemically link antibodies to biological enzymes whose activities produce a measurable signal with solutions containing appropriate substrates. This signal has to be associated with the presence of antibody or antigen . COMPONENTS OF ELISA Antibody: IgG fraction of serum. Enzyme: Horse Radish Peroxidase (HRP) MW 44, 000, glycoprotein with 4 lysine residues. Substrate: TMB (3,3',5,5', tetramethylbenzidine) The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color. Reaction stopped with dilute acid to cause complex to turn yellow.
- 43. INTRODUCTION TO ELISA A 96 - well microtiter plate being used for ELISA. A test that uses antibodies and color change to identify a substance. ELISA is a popular format of a "wet-lab" type analytic biochemistry assay. ELISA involves at least one antibody with specificity for a particular antigen. ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays.
- 44. PRINCIPLE Wet lab" analytic biochemistry assay, ELISA involves detection of an "analyte" in a liquid sample by a method that continues to use liquid reagents during the "analysis. The basic principle of an ELISA is to use an enzyme to detect the Ag- Ab binding (antigen- antibody binding). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding.
- 45. substrate Colored product Secondary antibody Different antigen in sample Primary antibody
- 46. TYPES OF ELISA INDIRECT ELISA DIRECT ELISA SANDWICH ELISA COMPETETIVE ELISA NON -COMPETETIVE ELISA
- 47. INDIRECT ELISA Antigen is added to plate. Added Blocking buffer. Suitable primary antibody is added. Secondary antibody- HRPO is then added which recognizes and binds to primary antibody. TMB substrate is added, is converted to detectable form. Under standard condition ,the enzyme activity measured is proportional to amount of specific antibody in the original serum.
- 48. ADVANTAGES OF INDIRECT DETECTION Wide variety of labeled secondary antibodies are available commercially. Versatile, since many primary antibodies can be made in one species and the Same labeled secondary antibody can be used for detection. Immunoreactivity of the primary antibody is not affected by labeling. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. DISADVANTAGES OF INDIRECT DETECTION Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal. An extra incubation step is required in the procedure.
- 49. DIRECT ELISA 1. Apply a sample of known antigen to a surface. 2. Enzyme linked primary antibody is applied to the plate. 3. Washed, After this wash, only the antibody-antigen complexes remain attached. 4. Apply a substrate which is converted by the enzyme to elicit a chromogenic signal. Under standard condition ,the enzyme activity measured is proportional to amount of specific antibody in the original serum.
- 50. ADVANTAGES OF DIRECT DETECTION Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated. DISADVANTAGES OF DIRECT DETECTION Immunoreactivity of the primary antibody may be reduced as a result of labeling. Labeling of every primary antibody is time-consuming and expensive. No flexibility in choice of primary antibody label from one experiment to another. Little signal amplification.
- 51. SANDWICH ELISA 1. a. Plate is coated with suitable antibody. b. Blocking buffer is added. 2. Sample is added to plate so antigen is bounded by capture antibody. 3. A suitable biotin labeled detection antibody is added to plate. 4. Enzyme HRPO is added and binds the biotin labeled detection antibody. 5. TMB substrate is added and converted by HRPO to colored product. Under standard condition ,the enzyme activity measured is proportional to the Amount of specific antigen in the original serum.
- 52. MATERIALS NEEDED FOR ELISA KIT ELISA Plate Positive control Negative control Dilution Buffer Conjugate TMB Substrate Stop Solution
- 53. PROCEDURE OF ELISA A B C D E Wash 3X Wash 4X Wash 4X Wash 4X Alkaline phosphatase substrate is added and developed colour is read at 405 nm wavelength to measure plasma cencentration Wells are coated with 0.2 亮g primary antibody Diluted plasma is added to coated wells, which bind to antibodies 0.1 亮g of biotinylated (biotin = ) antihuman secondary antibody Incubated overnight at 4C Add 1.2000 dilution of streptavidin conjugate to alkaline phosphatase ( E) 2h 2h 1h Incubated at room temperature (24C)
- 54. FINAL PLATE OF ELISA
- 55. COMPETETIVE ELISA COMPETETIVE ELISA Solid phase coated with antibody Add unknown amount of unlabeled antigen and known amount of labeled antigen Free and labeled antigen are captured Color formation by oxidation of substrate into a colored compound Under standard condition ,the enzyme activity measured is proportional to the proportion of labeled antigen in the mixture of labeled and unlabled antigen.
- 56. ADVANTAGES: Suitable for complex (crude or impure) samples, since the antigen does not require purification prior to measurement. DISADVANTAGES Each antigen may require a different method to couple it to the enzyme.
- 57. COMPARISON BETWEEN VARIOUS TYPES OF ELISA Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA
- 58. ELISA RESULTS The ELISA assay yields three different types of data output: Quantitative: ELISA data can be interpreted in comparison to a standard curve in order to precisely calculate the concentrations of antigen in various samples. Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen. Semi-quantitative: ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.
- 59. ELISA data is typically graphed with Optical density Vs Log concentration to produce a sigmoidal curve. Known concentrations of antigen are used to produce a standard curve and then this data is used to measure the concentration of unknown samples by comparison to the linear portion of the standard curve. This can be done directly on the graph or with curve fitting software which is typically found on ELISA plate readers.
- 60. Applications Diagnostics Toxicology Industry Research
- 61. APPLICATIONS Screening donated blood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-HIV antibodies) Hepatitis C (presence of antibodies) Hepatitis B (testing for both antibodies and a viral antigen) Measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, T3 and T4 (for thyroid function) Detecting infections Sexually-transmitted agents like HIV, syphilis and chlamydia Hepatitis B and C Toxoplasma gondii , H.pylori Detecting illicit drugs. Detecting allergens in food and house dust
- 62. Diagnostic Applications (Home) Pregnancy test hCG hormone in pregnant womans urine 1-10 min: (+) two lines, (-) one line HIV test Oral fluid: OraSure, OraQuick, Calypte AWARE Blood: Home Access
- 63. Industrial Applications Allergen detection 8 major food allergens Detection at low levels (ppm) Alerts for contamination Vaccine quality control Licensing Validate consistency of production
- 64. Toxicological applications Prescription medication Cardiovascular drugs, antidepressants, chemotherapeutics Drug dosing Drugs of abuse Morphine, amphetamines, barbiturates Newborns
- 65. SENSITIVITY ELISAs are one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody antigen interaction. In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results. As mentioned earlier, indirect detection will produce higher levels of signal and should therefore be more sensitive. However, it can also cause higher background signal thus reducing net specific signal levels.
- 66. Sensitivity and Specificity Sensitivity Probability: positive test for a patient with disease Takes false negatives into account More important for ELISA Specificity Probability: negative test for a patient without disease Takes false positives into account ELISA has high sensitivity (>99%) and high specificity (>99%) Not perfect: false positives/negatives can occur
Editor's Notes
- #61: Picture 1: http://diet-solution.net/5-reasons-for-having-your-blood-tested/ Picture 2: http://www.goodnewsfinland.com/archive/themes/diagnostics/diagnostics-generates-growth/ Picture 3: http://caep.ca/cpdcme/roadshows-current-cme/toxicology Picture 4: http://global.unc.edu/news/unc-toxicologists-present-nexgen-tools-to-canadas-national-health-agency/
- #63: Chard, T. (1992). Review: pregnancy tests: a review. Human Reproduction, 7(5), 701-710. Dipiro, J.T., Talbert, R.L., Yee, G.C., Matzke, G.R., Wells, B.G., & Posey, L.M. (2008): Pharmacotherapy A Pathophysiologic Approach Seventh Edition. New York: McGraw-Hill Companies Inc. Picture 1: http://newdadtobe.com/post-2/ Picture 2: http://www.oraquick.com/Taking-the-Test/Understanding-Your-Results
- #64: Le, C. T., Grambsch, P. M., & Giebink, G.S. (2003). Quality control and the identification of vaccine responders using ELISA-derived antibody data. Statistics in Medicine, 22(18), 2935-2942. Stephan, O., & Vieths, S. (2004). Development of a real-time PCR and a sandwich ELISA for detection of potentially allergenic trace amounts of peanut (Arachis hypogaea) in processed foods. Journal of Agricultural and Food Chemistry, 52(12), 3754-3760. Picture 1: http://www.ifood.tv/blog/nut-free-snacks Picture 2: http://www.wisegeek.com/what-is-needle-phobia.htm
- #65: Picture 1: http://www.simmlands.com/wp-content/uploads/2011/05/gavel-and-book-300x199.jpg
- #67: Dipiro, J.T., Talbert, R.L., Yee, G.C., Matzke, G.R., Wells, B.G., & Posey, L.M. (2008): Pharmacotherapy A Pathophysiologic Approach Seventh Edition. New York: McGraw-Hill Companies Inc.