Automatic Measurement of AFP-L3% by Using Clinical Automatic Analyzer “LiBASys” 본 과 이 외 의 자 료 도 포 함 되 었 습 니 다 생화학계 이은자

Marker of diagnosis,prognosis of HCC(HepatocellularCa rcinoma) HCC 의 진단 : 방사선 학적 진단, 혈청학적 진 단 HCC 의 혈청학적 검사 법 : 민감도, 특이도가 낮 음 What’s AFP Benign Liver Disease AFP- L1 HCC AFP- L3 Ｆｕｃ Ｆｕｃ α １ ↓ ６ Sugar Chain of AFP

AFP 의 structural heterogenueity AFP 는 분 자 량 인 종 양 태 아 당 단 백 으 로 분 자 당 한 개 의 Asparagin-linked oligosaccharide 를 포 함 하 는 데 양성 간질환에서 악성종양으로 진행함에 따라 변화함 AFP-L3 가 HCC 에 가 장 특 이 한 subtype 으 로 sugar chain 의 말단환원기에 additional fucose residue 를 갖 는 다고 밝 혀 짐

Fractionation of AFP-L3 by LCA affinity electrophoresis Agalose gel, which contains LCA Ｌ３Ｌ３ Ｌ１Ｌ１ （－） （＋） （－） （＋） Pre electrophoresis Post electrophoresis LCA means lens culinaris agglutinin and it has an affinity for sugar chain of AFP-L3, therefor, there are differences of migration speed of AFP by different affinity to LCA.

Utility of LBA method 1. 항원항체반응이 빠르게 일어남 2.solid phase 에서와 같은 non- specific reation 이 일어나지 않음 3. 충분한 양의 ab 공급으로 prozone 현상이일어나지않음 4. Ab 와 column 의 elutionpattern 을선택 함에따라 2 가지 ag 을동시에측정 할수있음 Investigation of LBA (Liquid-phase Binding Assay) method 항원항체반응이 액체상에 서 실행되며 결합및 비결합 반응물의 분리 또한 액체상 에서 column 에 의해 빠르 게 일어남 AｇAｇ AｇAｇ AｇAｇ AｇAｇ AｇAｇ AｇAｇ Antigen Labeled antibody Immune complex ＋

Application of LBA method for measurement of AFP-L3% LCA A4-4 AFP 2) The two anti-human AFP monoclonal antibodies, which are not inhibited by LCA are also employed. By using combination of these three monoclonal antibodies and LCA, the reagent, which can simultaneously measure AFP and AFP-L3% is developed. 1) The anti-human AFP monoclonal antibody Clone No. A4-4, which is inhibited to bind to AFP-L3, under existing LCA is employed. Epitope of A4-4

3) A4-4 is labeled with octamer of sulfated tyrosine, other antibodies are labeled with POD (peroxidase) and pentamer of sulfated tyrosine, respectively. LCA AFP POD YS8 YS5 Application of LBA method for measurement of AFP-L3%

4) When the three labeled antibodies and LCA react, two kind of immune complexes of AFP are formed because of the difference of affinity to LCA. YS5 POD AFP- L3 Complex 1 YS8 YS5 POD AFP- L1 Complex 2 5) AFP-L3 forms Complex 1, which has 5 sulfate residues, since AFP-L3 is not bound to A4-4 due to LCA binding. 6) AFP-L1 forms Complex 2, which has13 (8+5) sulfate residues. Since AFP- L1 is LCA non-reactive, it can be bound to all antibodies. Application of LBA method for measurement of AFP-L3%

두가지 면역복합물이 서로 다른 sulfated residue 를 가지므로 ION Exchange column 을 사용하여 분리함 Column Reaction mixture are introduced into column Complex 2 Column 8) The obtained two fractions are mixed with fluorescence substrate, and activity of labeled- enzyme is measured as intensity of fluorescence. Complex 2 Complex 1 Fractionation for Complex2 (by 3M NaCl) Complex 1 Fractionation for complex 1 (by 0.9M NaCl) Column Wash out the surplus components Application of LBA method for measurement of AFP-L3%

Reation flow Setting reagent & sample 1 st Reaction 2 nd Reaction Column separation Elution of complex1 Elution of complex 2 Enzyme Reaction Fluoroscent detection

Utility of LBA method for measurement of AFP-L3% Immune reaction 2 Washing 2 Blotting Immune reaction 1 Washing 1 Washing 3 Coloring reaction Densitometry Electrophoresis Preparation Immune reaction1 Immune reaction2 Fractionation Result is printed out every three minutes Measurement Start 1hour 2hour 3hour 4hour 5hour 50 Tests 1st data is printed out Measurement time is shortened, and complicated handling is not necessary.

Automatic measurement by new method has been desired. Problems of electorophoretic method 3. 검사요원의숙련도에따 른 차이 4. 자동화에의적용이 어려움 2. 다량의검체측정불가 는 1. 조작이어렵고시간걸 림 1. 측정시간이 짧아짐 2. 조작이 쉽고다량의 검체처리와 자동화가능 3. 한시약으로 AFP 와 AFP- L3 를 동시에 측정 Utility of LBA method for measurement of AFP-L3%

Clinical Automatic Analyzer “ ＬｉＢＡＳｙｓ ” ←Over view of LiBASys

Characteristics of LiBASys 1)Compact Packaging width 540mm height 1150mm length 620mm weight 130kg 1150mm 620mm 540mm

2)The way of reaction mixture inlet and fraction outlet are reversed by special probe, which is directly connected to column. →It is hard to stop up a filter in the column.1 Sucking up reaction mixture Pushing out fraction Ion exchange column Characteristics of LiBASys

3)Immune reaction, which needs low temperature and enzyme reaction, which needs high temperature can be performed, since there are two separate reaction area for cooling and heating. For immune reaction: 8 ℃ Characteristics of LiBASys For enzyme reaction: 37 ℃

Name for Part of LiBASys 1)Over View PrinterKey board and Display Analysis Unit Pure Water Tank and Drain Tank Main SwitchElution Buffer A, B, C Sleep Switch

2)Part of Turntable-1 Column and Column Holder Display and Key Board Column Probe Sample Probe Name for Part of LiBASys

3)Part of Turntable-2 Optical Unit Sample Disk Outside Cuvette Reagents holder Inside Cuvette Name for Part of LiBASys

Outline for measurement of LiBASys 1)Sampling Dispense the sample (15uL) to inside cuvette Sample Cup Sample Probe

2)Dispense the reagents Ｒ１ 100uL, Ｒ２ 10uL are dispensed to inside cuvette according to the reaction sequence and they react under 8 ℃. R1 R2 Sample Probe Outline for measurement of LiBASys

3)Sucking up the reaction mixture The reaction mixture is sucked up to column (80uL) and surplus antibody is washed out the from the column. Column ProbeCuvette Inside Outline for measurement of LiBASys

4)Fractionation Complex1 and ２ are fractionated to outside cuvette. Cuvette Outside Column Probe Outline for measurement of LiBASys

5)Measurement of Fluorescence Complex1 and ２ are preheated and the substrate is added, then fluorescence intensity are measured by optical unit. Three minutes later, the results are printed out. Optical Unit Substrate Outline for measurement of LiBASys

우리 Lab 의 AFP(ECL & LBA) & AFP-L3 의 비 교 ECL : Electrochemiluminiscence assay ELECSYS ( B.M. Germany) LBA : Liquid Binding Assay LiBASYS (WAKO Japan) 검 체 수 : 204 건 (AFP 와 AFP-L3 동시 order) 기 간 : ~

Comparison of ECL AFP and LBA AFP

Correlation between LibaSys AFP and AFP-L3 (r=0.2851)

Clinical Significances of AFP-L3% 1. 양성간질환과 악성간질환의 구별 2. AFP-L3% 는 biological malignancies of HCC 를 의미함. 3. 화상진단보다 조기에 HCC 발견 4. 치료후 효과에 대한 평가 및 예후

Total AFP AFP-L3 참 고 치 <20ng/ml <10% 측 정 범 위 0.8~1000ng/ml 0.5~99.5 Processing speed 50 sample/4hr 의 뢰 건 수 (2000.8~ ) 497 건 running cost 원 (VAT 포 함 ) 일 반 수 가 원