生物技術應用-醫療診斷之革新

台北醫學大學呂思潔

Tel: 27361661-3911Email:cmbsycl@tmu.edu.tw

醫療診斷

Sensitivity (敏感性) -what is the minimal amount of substrate that can be detected?

Specifisity (特異性) -what percentage of assays correctly distinguish normal from cancer-containing samples?

生物技術

1.單株抗體(Monoclonal antibody)2. 核酸探針(Nucleic acid probe)3.聚合鋂鏈鎖反應 (PCR)4.基因晶片 (Genechip)5.磁共振造影（MRI）

單株抗體 (monoclonal antibody, mAb)

Dr. Georges Kohler & Cesar Milstein

1984 Nobel Prize in Medicine

細胞融合瘤技術 (Hybridoma Technique)

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 43.15

Advantages of Monoclonal Ab

MonospecificHomogeneous bindingHigh concentration

專一性高 避免交叉反應

單株抗體之應用

Purification of ProteinsIdentification & Isolation of Lymphocyte Subpopulation & ClonesTumor Detection & ImagingTumor killingDiagnostic Reagents

September 2005, Volume 23 No 9

ImmunoassayEIA (Enzyme-Linked Immunosorbent Assays)

Radioimmunoassay (RIA)

Sandwich assay

Highly versatile, sensitive, and quantitative technique that requires little equipment & for which critical reagent are readily available

If the patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate.

Positive ELISA Test

Partially purified, inactivated HIV antigens pre-coated onto an ELISA plate

Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies.

Multichannel pipette

Diagnostic ReagentsDetect pregnancyDiagnose infectious protozoan, bacterial, & viral pathogensMonitoring therapeutic drug levelsDetecting heart damageMatching histocompatibility antigensDetecting diabetesDetecting tumor cellsInexpensive & convenient

Pregnancy Tests

Checks a woman’s urine for human Chorionic Gonadotropin (hCG) (人類絨毛膜親生殖腺素)

A hormone produced by the placenta after egg fertilizationThe confidentiality offered by these tests has made them the most popular of all types of home diagnosis kits

Identification & Isolation of Lymphocyte Subpopulation & Clones

Analysis of hematological malignancies

(Provide immunophenotypic data and information on cellular morphology)

Monitoring of disease

Evaluation of tumor response to therapy

p. 34-41

張子文 教授

清華大學生命科學系

發明第一個上市的過敏抗體藥物Xolair (中和 IgE)

科學人 2005 9

Herceptin

2005, 12,21

生物技術

1.單株抗體(monoclonal antibody)

2. 核酸探針(nucleic acid probe)

原位雜交技術(in situ hybridization)

瑩光原位雜交技術 (FISH)

染色體之分析

以一小段DNA為探針與分裂中之染色體,或休止期之細胞核,進行雜交(hybridization),探針將附著在DNA序列相同之處

瑩光原位雜交技術 (FISH)之應用

1.確定一新發掘成功的基因為處染色體之何處

2.染色體分析時,遇有無法清晰辨識之異常,可借探針之已知來源屬性,而加以確認

3.基因醫學診斷之研究-分析基因套數之放大,減少,或轉位

4. 避開傳統染色體分析之細胞培養

生物技術

1.單株抗體(monoclonal antibody)

2. 核酸探針(nucleic acid probe)

3.聚合鋂鏈鎖反應 (PCR)

PCR (Polymerase Chain Reaction)聚合鋂鏈鎖反應

“Jurassic Park”

Amplified DNA in vitroA fast, inexpensive technique for making an unlimited number of copies of any piece of DNA. Sometimes called "molecular photocopying," PCR has had an immense impact on biology and medicine, especially genetic research.

Dr. Kary B. Mullis, 1993 Nobel Price

for contributions to the developments of methods within DNA-based chemistry" "for his invention of the polymerase chain reaction (PCR) method"

"for his fundamental contributions to the establishment of oligonucleiotide-based, site-directed mutagenesis and its development for protein studies"

Kary B. Mullis Michael Smith

USA Canada

La Jolla, CA, USA

University of British Columbia Vancouver, Canada

1944 -

The Nobel Prize in Chemistry 1993

The PCR method – a copying machine for DNA molecules

230

1073741824=109

PCR1. Denature double-stranded DNA

2. Primer annealing

3. ElongationTaq DNA polymerase (Thermus aquaticus)

YellowStone Park

PCR-PE9600, PE-2400

5

680 bp

330 bp

LambdaKappa

500 bp

Figure 8. PCR amplification of human light chain gene.

Research applicationsPCR is used to amplify human-specific DNA sequencesDetection mutations using PCR amplificationAnalysis of RAS gene point mutation by PCRDiagnosis of retinoblastoma (視網膜胚細胞膜)PCR amplification is used for mornitoring cancer therapyPCR amplification is used to detect bacterial and viral infectionDetection of human immunodeficiency virusDetection of hepatitis B virusDetection and typing of genital human papillomavirusesDetection of human cytomegalovirusPCR amplification of enterovirusesPCR amplification is used for sex determination of parental cellsDetection of X-linked disorder in malePCR methods permit linkage analysis using single sperm cellsPCR techniques are used in studies of molecular evolution

WHO collaborative multi-center research project on Severe Acute Respiratory Syndrome (SARS) diagnosis

- Severe Acute Respiratory Syndrome (SARS): Laboratory diagnostic tests - Sampling for Severe Acute Respiratory Syndrome (SARS) diagnostic tests- Recommendations for laboratories testing by PCR for presence of SARS coronavirus -RNA- Update 31 - Coronavirus never before seen in humans is the cause of SARS- PCR primers for SARS developed by WHO Network Laboratories- Multi-centre Collaborative Network: Laboratories testing for SARS

http://www.who.int/csr/sars/project/en/

Recommendations for laboratories testing by PCR for presence of SARS coronavirus -RNA

29 April 2003- The PCR procedure should include appropriate controls in each run, which

should yield the expected results. They should include:1 negative control for the extraction procedure and 1 water control for the PCR run 1 positive control for extraction and PCR run the patient sample spiked with a weak positive control to detect PCR inhibitory substances (inhibition control)

- If a positive PCR result has been obtained, it should be confirmed by: repeating the PCR starting from the original sample OR having the same sample tested in a second laboratory.

Amplifying a second genome region will increase test specificity.A test kit with positive and negative control has been made available through

the WHO collaborative multicentre research project on SARS diagnosis.

The PCR method already of great use

DNA from fossil remains can be mass-produced by PCR. Researchers have succeeded in amplifying genetic material from insects trapped in amber that have been extinct for more than 20 million years.

The PCR method already of great use

In PCR the police have a new and very reliable fingerprinting method, since the DNA content can be analysed from a single drop of blood or a single hair found at the site of a crime.

DNA 親子鑑定

1994 年，美國血液庫組織公佈的親子鑑定實驗室標準，已納入聚合脢連鎖反應( PCR ) 的規範，我國內政部亦已通過DNA 採樣條例

聚合酵素連鎖反應-短段片重複序列基因(PCR-STR)準確度 99.9%

Queenvictoria

Albert

Alice Louis

Alexandra CzarNicholas IIof Russia

Alexis

犀牛是個大近視眼，但是聽覺與嗅覺靈敏。為了消暑、降體溫、趕走身上寄生蟲，牠常在泥漿裡打滾。犀牛頭的前端長有一個或兩個角，通常後面的角較小，犀牛角稀有珍貴，是歹徒覬覦的目標，也是促使犀牛瀕臨滅絕的原因。

你是否看得出這些雕刻品是由大大小小的犀牛角所製成？下圖是幾年前海關查獲的走私品，經DNA鑑定後證實，這些成品均是由犀牛角雕刻而成。

生物技術

1.單株抗體(monoclonal antibody)

2. 核酸探針(nucleic acid probe)

3.聚合鋂鏈鎖反應 (PCR)

4. 基因晶片 (Genechip)

生物晶片功能多變

基因表現

癌症分類

新藥開發

疾病檢驗: 肺癌食品生技

農業發展

DNA CHIPS in handheld housings can sense the on/off state of up to 400,000 genes in a tissue sample.

Scientific American February 2001 33

生物技術

1.單株抗體(Monoclonal antibody)2. 核酸探針(Nucleic acid probe)3.聚合鋂鏈鎖反應 (PCR)4.基因晶片 (Genechip)5.磁共振造影（MRI）

2003年諾貝爾生醫獎：MRI的發明美國化學教授羅特博（Paul C. Lauterbur）和英國物理教授曼斯菲德（Peter Mansfield），共獲本年度諾貝爾生醫獎

磁共振造影（Magnetic Resonance Imaging, MRI）

To see is to believe

一張圖片勝過千言萬語

安全性高

不需穿刺血管

可檢查全身各部位

這是目前最為昂貴的醫學成像系統，但是不僅不像X-光電腦斷層掃描一樣，沒有輻射傷害的問題，而且可以掃描任何方向的截面，同時也可以獲得解剖與功能影像，是目前神經認知科學有關腦功能研究的利器。

Magnetic Resonance Imaging磁共振成像的臨床應用是醫學影像學中的一場革命，是繼CT等影像檢查手段後又一新的斷層成像方法，與CT相比，MRI具有高组織分辨力和無放射損傷等優點。

透過在人體內佔了三分之二重量的水分子，分析其中氫原子共振所放射出來的電波，可顯示三維影像，使得人體內的各個器官組織的病程變化無所遁形。

在全世界每年有6000多萬例檢查和研究採用MRI技術

成本雖高 回收快 醫院莫不視為金雞母

這些在體外對身體內器官與組織進行探查的醫療技術，由於不需有儀器伸入體內、沒有侵入性，已是越來越普遍、安全的診斷與追蹤利器

ReferencesmAb天下文化“諾貝爾的榮耀-生理醫學桂冠”, p.127-134

• PCR天下文化“諾貝爾的榮耀-化學桂冠”, p.56-64

Gene chip科學發展 2004,9月 381期 “基因晶片與生物醫學” p 64-69

科學發展 2004,6月 378期MRI

科學發展 2004,10月 382期 “透視人體的利器-電腦斷層掃瞄系統” p 56-61