면역분석학 Immunoassay

Bioinorganic and Immunoassay Group, Seoul Women’s University Immunoassay - I Bioinorganic and Immunoassay Group, Seoul Women’s University

Immunoassay (IA) 면 역 분 석 학 담당교수: 이 인 숙 강의자료: 프린트 물, 지정도서 자료 연구실: 제1과 305 (5657) / 제1과 304 (7715) 홈페이지: https://swubig.weebly.com/ 시험 2차례 실시 + 발표 단 휴대전화가 울리면 다음시간에 퀴즈 기타 궁금한 사항은 email (irpaeng@swu.ac.kr) 을 통해 문의

Immunoassay for Beginners Immunoassays use reagents to generate a signal from minute amounts of target analyte in a sample.

Basic principle of IA Clip / analyte / Antigen Reagents : (1) Magnet / Binder / Antibody (2) A signal from the captured material / Signal generator Immunoassays derive their unique specificity, sensitivity, and flexibility from three important properties of antibodies: Their ability to bind to an extremely wide range of natural and man-made chemicals, biomolecules, cells, and viruses. Exceptional specificity for the substance to which each antibody binds. The strength of the binding between an antibody and its target.

Immunoassay Based on Specific Interaction between Ag (analyte) and  Ab (binding Protein, Binder) cf. hand in glove

Simplicity Sensitivity Specificity Based on Specific Interaction between Ag (analyte) and  Ab (binding Protein, Binder)  Immune Reaction Ag + Ab ↔ Ag / Ab  Binding Reaction Analyte + Binding Protein ↔ Analyte / Binding protein  Label Radioisotope ; 125I, 14C Enzyme ; HRP, MDH, G6PDH Fluorophore ; fluorescein Luminescent species ; luminol, AEQ Particle ; latex Metal ion ; Au3+ ng ~pg range / upto atto M IA Simplicity Specificity Sensitivity

Comparison Instrumental analysis : Immunoassay Disadvantages of Instrumental analysis expensive instruments need experts need pre-treatment of samples long experimental time needed Advantages of IA relatively cheap and very simple treatment minimal training no pre-treatment of samples fast screening test biosensor

Advantages of Immunoassay Minimal training No pre- treatment Relatively cheap fast Simple treatment 습식분석 기기분석 면역분석 현장측정용 Home test 용

Expand the area of immunoassay agglutination reaction membrane chromatography PCR CE Affinity With other methods

Applications of immunoassay drugs antigens antibodies hormones toxins insecticides viruses clinical environmental industrial aggricultural Research/diagnosis

현대의학이 당면한 의료환경의 급속한 변화 생명과학의 혁신적 발달 인구의 급속한 노령화 의료비용의 사회 경제적 부담 증대

(Genomics & Proteomics) 미래 임상 진단기술 수요의 대폭적 증가 미주, 유럽 등 선진국 인구의 노령화 중국, 인도 등 아시아 신흥공업국 고도의 경제성장 삶의 질 개선 욕구 의료보호 필요 증대 분자생물학의 발전 (Genomics & Proteomics) 대량의 유전자 및 생화학 정보 요구 임상진단시험 시장의 대폭적 확대 임상진단기술의 고도화 양질의 의료서비스 요구 의료기술의 혁신

다양한 모양의 임신진단 키트 : 소변속의 hCG 검출

< 임신진단 키트의 면역크로마토그래피 스트립 모식도>

Exponential growth of IA In the range of applications In the number of novel and ingenious assay designs

Antigen (analyte) For the functional sense, Antigen : complete -------- Immunogen         incomplete ------- hapten Immunogen       : immunogenicity + specificity hapten               : specificity Immunogenicity  : the ability to induce an immune response specificity  : the ability to react with these lymphocytes and antibody 

Antigenic determinants ( = epitope ) 1) Direct Immunogen 2) Indirect Immunogen ( = hapten ) carrier protein : BSA (Bovine Serum Albumin)                KLH (Keyhloe limpet hemocyanin)                 OVA (ovalbumin) Antigenic determinants  ( = epitope ) large molecule : Immunometric assay small molecule : Competitive assay

Bovine Serum Albumin (BSA) * 583 aa - Mw ; 66,463 Da * 104 reactive groups - ε-amino group of lysine residues (59) - α-amono group (1) - hydroxyphenyl groups of tyrosine residues (21) - sulphydryl groups of cystine residues (6) - imidazole groups of histidine residues (7)

Antigenic determinants ( = epitope ) 항체와 반응할 수 있는 site Ag surface 에 존재 한 Ag 에 여러 종류의 epitope 가 가능 ( multivalent / multispecific ) large molecule : Immunometric assay small molecule : Competitive assay

Antibody (Binder, Binding Protein) PAb : polyclonal Ab MAb : monoclonal Ab Mw = 150,000 Da 25,000 Da/ each Paratope ( = Ab binding site )

- The variable domains each have three hypervariable loops (CDRs) which bind to antigen. CDR : complementarity-determining regions 1 domain : ~107 aa

Fragmentation of IgG

Antibody fragments

Ag / Ab reaction IAs involve a binding reaction between an analyte and at least one Ab This reaction takes from a few seconds to many hours to achieve equilibrium depending on a range of different factors ex) pH, ionic strength, temperature, material matrix

Law of Mass action affinity : single binding interaction   affinity : single binding interaction avidity : the summation of all of the individual interactions

development of analytical kit immunoaffinity purification   KA ( M-1 ) Specificity Affinity PAb usually high affinity 1010-12 ↓ ↑ low affinity 105 MAb 106-9 Thus, High affinity Ab development of analytical kit Low affinity Ab immunoaffinity purification

IA classification Test sample : Ag ( analyte) Ab ( binder, binding protein) (2) Assay system label principle separation End-point detection RIA EIA FIA CLIA BLIA etc. Competitive IA = reagent limited Non-competitive IA = Immunomeric IA = reagent excess Heterogeneous assay Homogeneous assay Visual IA Instrumental IA

Various Micro-Well Plates 96 Wells 384 Wells 1536 Wells

High-Throughput Well Sensor

EIA, Competitive, Heterogenous, Enzyme-Linked Immunosorbent Assay (ELISA ) Limited E E E E E E Enzyme activity Antigen-Enzyme conjugate > Free antigen Limited [Antigen] E E Antigen-Enzyme conjugate < Free antigen

EIA, Non-competitive, Heterogenous Immunometric assay (= Sandwich assay) E E E E Washing Enzyme activity E Excess Washing [Antigen] Washing E Excess

EIA, Competitive, Homogenous, Enzyme-Multiplied immunoassay technique (EMIT) Enzyme activity Limited E E [Antigen] Antigen-Enzyme conjugate < Free antigen E E × % Inhibition Limited [Antigen] Antigen-Enzyme conjugate > Free antigen

Enzyme-Linked Immunosorbent Assay ELISA EMIT Enzyme-Linked Immunosorbent Assay Emzyme Multiplied Immunoassay Technique Heterogeneous assay Homogeneous assay Separation required No separation needed Slower than EMIT Faster than ELISA Sensitivity greater than EMIT Sensitivity less than EMIT Measured macromolecules (Ag, Ab) Measured small molecules (Haptens) Solid phase assay Liquid phase assay

FIGURE 1. Immunometric immunoassay

FIGURE 2. Immunometric assay for antibody testing.

FIGURE 3. Enzyme immmunoassay for detection of antibodies (ELISA).

FIGURE 4. Competitive immunoassay (solid phase separation).

FIGURE 5. Example of homogeneous immunoassay.