ELISA Techniques: Direct, Indirect, Sandwich, Competitive PDF

ELISA Wittaya Chaiwangyen, Ph.D. Division of Biochemistry School of Medical Sciences University of Phayao Definition of Antibodies  Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system...

ELISA Wittaya Chaiwangyen, Ph.D. Division of Biochemistry School of Medical Sciences University of Phayao Definition of Antibodies  Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. 1 ใช้จับก ับ anti แตก า คงง ต่  Definitions- cont  Antigens งแปลกปล A substance that when introduced into the body stimulates the production of an antibody - protein/glycoprotein ง ก ระะ นให้ วง การส าง ่ 3 - polysaccharide ↳ อ งขนาดให Immunoassay ก ษาภ -จับ นของ Ag บ Ab เ อ ระะ / A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample กั กั ศึ พื่ ร่ ที่ ตุ้ สิ่ บู สิ ต้ ร้  ่วนของ ง Antigen- กร ะ ุ้นใ Antibod Antigenic determinant ↓ ม ี6 -7 & ที่ ส่ ตุ ห้  chai ·variable delight / chain heavy · constan 1Antige 3 Spitope Mouse immunoglobulin classes and subclasses: - Classes: IgG, IgM - Subclasses:IgG1, IgG2a, IgG2b, IgG3 Rat immunoglobulin classes and subclasses: - Classes: IgG, IgM - Subclasses IgG1, IgG2a, IgG2b , IgG2c Human immunoglobulin classes and subclasses - Classes: IgG, IgM, IgA, IgD , dg ↑ - Subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2  =5 โมเลกูล อ EC2 โมเลกูล อ we epsilon delfa miU gamma alpha จน. Antige * 2 เ กส านร รถไ เพราะะขนาด เ กส ระบ บท ลา งแปลกป wur ส า งห งจวกได บAg คร ้งแรกใน วิต ne nt BPhil, ly ท ลงสสาร ก บ B ce พบ ท สสาร น ด histimin ก จัด แ / ผ่ ั้ รั ทั้ จั ตั ที่ พ้ ล็ ล็ ำ ร้ กั สิ่ ภู ต่ ลั ต่ อั ชี ำ มิ  & กระะท ป / ป / ที ำ  Antibody Production ทา สง ท สนใจ ฉืดใน ต  Specific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat or rabbit for small quantities of antibody, or goat, sheep, or horse for large quantities of antibody. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen. polycional Ab = Ag ท่ ภาชน กแ หลาย spi top&ท ใ ไ A ntib สั ที่ ที่ นั ำ ฉี ว์ ห้ ต่ ด้  B - lymphocyte เ ็นเซล ส วง Antibody - ัว ก กร ะ นจะะเป ่ยน ~ถ ปเ น & Antibody Production-cont ส าง Antibod  To obtain antibody that is specific for a single antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies m Antibod ผ lymphocyte - fuse( รวม 1 บ r cell =hybridomas 1. y 2. เ มจเ นวนไ ไม จ ด แพ ราะเ B- cancer - Cell) (. ไ monoclonal l antibod 5 เ บได ้ 1 fpitop ล์ ป็ กั ม่ จ็ ป็ ตุ้ ลิ ร้ ำ ด้ ถู กั ป็ ร้ ลี พิ่ รู ด้  ก Ay- เ บ ↳- lymphocyte- cancer cel เ ↳ท ให้cell membrane ขอ ง 2 cellfuse รวม = hybridoma cel ที อี ที่ ติ ก็ ำ  ใ - ่ม ้ Antigen 1 fpitope ไ ปใ ส่ e า น น แส ดง า า9 A กระ นใ ถ้ มี ที ตุ้ ช้ จั ว่ กั หลาย Epitop 1 Epitope  จะ tag บ หรือ enz สี กั  The difference between a primary antibody and a secondary antibody A primary antibody binds directly to a particular antigen, whereas a secondary antibody doesn’t bind to the target antigen. Instead, it binds to the primary antibody. เพราะ ผ ต วิ A b กับ 2 At จากคน ละ spec During antibody production, the target antigen and host species used to produce the primary antibodies are different from those used to make the secondary antibodies. ลิ  =primary Antibody ค ง แรกส าง ด Ab จา กห ฉืดใส่ hos t ค ้ง สองสร าง าน Ab ของห ฉี ต้ ล้ รั้ รั ร้  Ag/ดฉลา ก วย Enzy Enzyme Linked Immunosorbent Assay (ELISA) ELISA is a plate-based assay technique. Along with the enzyme-labelling of antigens or antibodies, the technique involves following three principles in combination which make it one of the most specific and sensitive than other immunoassays to detect the biological molecule: An immune reaction i.e. antigen-antibody reaction. Enzymatic chemical reaction i.e. enzyme catalyses the formation of colored (chromogenic) product from colorless substrate. เป ่ยนสสาร ง น ไ ม Signal detection and Quantification i.e. detection and measurement of color intensity of the colored products generated by the enzyme and added substrate. ั้ ถ้ ต้ ที่ ลี ตั  Types of ELISA Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA  1. Direct ELISA 1. Any coat ใน 16 Well plate 2. ใ ่ Ab- จ เพระะ และ ด tag e ( อ ง นางส่ ่วน 3. เ ม substra - Generally, an antigen is immobilized to a multiwell plate. The antigen is then detected by an antibody directly conjugated to an enzyme such as horse radish peroxidase (HRP). ด Antige Application:This ELISA technique is typically used when the immune response to an antigen needs to be analyzed. อ : เเ วเพ ราะ ้นต - Faster than other ELISA techniques as fewer steps are required ที่ ที่ ข้ วั ร็ ต้ ล้ ตั ดี ำ ขั ต็ ส่  2. Indirect ELISA ใช้หา Antibody ใ ใน วอย - Detects the presence of antibody in a sample - Add the antigen ! !มอเตอรเค ่ไม -The unlabeled primary antibody present3.in ว นท เ ม sample ่ม - the sample bind specifically to the antigen ตัด tag zy ท en after addition of sample -The solution is washed to remove unbound antibodies and then enzyme conjugated secondary antibodies (typically polyclonal) are added ี่ ที่ ที รื่ ร้ ตั ต็  2. Indirect ELISA -The substrate for enzyme is added to quantify the primary antibody through a color change -The concentration of primary antibody present in the serum directly correlates with the intensity of the color Ab เยอะ - ีเข ส PrAb cons. ใน sampl Application: Suitable for determining total antibody concentration in samples  1. Coat plate วย A 3. Sandwich ELISA ท อ เเพาะ บ Ag A อง 2. block เ อ อง น non-specific binding ว 3. เ ม สง องการตร 4. ว 5. เ ม secondary Ab ท fag enz ใช้ตรว จหา A - Identifya specific sample antigen - Microtiter plate are coated with the antibodies - Non-specific binding sites are blocked using bovine serum albumin - The antigen containing sample is applied to the wells - A specific primary antibody is then added after washing - Enzyme linked secondary antibody is added that binds primary antibody - Unbound antibody-enzyme conjugates are washed off - The substrate for enzyme is introduced to quantify the antigens ที่ ที่ กั ื่ ด้ กั ที่ต้ ี่ ต็ ต้ ล้ ป้ ำ ต็ พื ด้  Both monoclonal and polyclonal antibodies can be used for sandwich ELISAs. However, a polyclonal antibody is often used as / the capture antibody in order to pull down the greatest amount of antigen possible ( antibod primary เ อให้ บ ใ ้ไ บ Ag แ ว อยใ &.specific secondary Al secondary Ab betag enzyme ห อ3 tenzym ↳3 จ เพาะผ 7 ก ช้ พื่ ล้ ค่ ห้ จั รื กั ำ Sandwich ELISA ส A cone. เยอะ - ีเข  4.Competitive ELISA 1. Coat plate วย Al 2. เ ม Ag= ่ องการท พ อม บ A y ดากฐฐวน - Commonly used when the antigen is small and has only one tag - enzy m epitope, or antibody binding site ตรว จหา Ag ขนาด เ ก 1 Spitop - Labeling purified antigen instead of the antibody - - Unlabeled antigen from samples and the labeled antigen compete for binding to the capture antibody -A decrease in signal from the purified antigen indicates the - presence of the antigen in samples when compared to assay wells with labeled antigen alone ที่ ล็ ต้ ร้ ติ กั ด้  Competitive and Inhibition ELISA The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal Ag ใน วอ าง อยย -7 ส ค.เ มสี 9 (แ + ปร เมก บ Ag conc A ป มา อ วาณเยอะ~ ับกับ ได้เยอ ตั ข้ ย่ ริ น้  Advantages and disadvantages of each ELISA type Advantages Disadvantages ่ ด า Sampl ไ e cors 1. High background. วยประห ดเวลา &น 1. Simple protocol, time-saving, and reagents 2. No signal amplification, since only a primary saving. antibody is used and a secondary antibody is Direct ELISA 2. No cross-reactivity from secondary not needed. ิ antibody. ไม่เ ดป ก ยา น ดข 3. Low flexibility, since the primary antibody must be labeled. 1. Signal amplification, since one or more เส secondary antibodies can be used to bind to 1. Complex protocol and longer compared with Indirect the primary antibody. เ ม ญญาณไ (เพรจะ ELISA 2. High flexibility, since the same secondary direct ELISA. บ อน , ใ เวลา 2. Cross-reactivity from secondary antibody. antibody can be used for various primary เ ดการ ไข น น Ag โ 2 :A antibodies. ดหย ุ่ 1. High flexibility. ดหยุ่ 1. The antigen of interest must be large enough 2. High sensitivity (2.5 times than Direct and so that two different antibodies can bind to it at Indirect ELISA) ด ่ค.เซ มข different epitopes. 19 องให ใ คลาชนิดม ้ Sandwich 3. High specificity, since different antibodies 2. It's sometimes difficult to find two different ELISA bind to the same antigen for detection. จจ เน antibodies that recognize different epitopes on 4. Suitable for complex (or crude/impure) the antigen of interest and cooperate well in a ิท samples (not require purification ) sandwich format. เหมาะ บ samplc ไ บ 1. High flexibility. บ 2. High sensitivity. ·um: v sample e coMc. 1. Relatively complex protocol. เพราะะ อ งเต - Competitive 3. Best for small antigens (low ไม่ องบ ส ELISA 2. Needs the use of inhibitor antigen. inhibitor ส concentrations) and no sample processing is required (crude or impure samples) ↓Ag มาตรฐาน) ง่ สี ถ้ ธี มีซั ด้ ยื ที่ ที วั ยื ห้ ญ่ ทีี่ ซั พิ กิ สุ ต้ ชั ม่ ต้ จั ิ่ ำ กั จั มี กิ ช้ พ่ ซ้ ต้ สั ซ้ ธิ์ ริ ผิ กั ริ ฏิ ยั  General Procedure of ELISA 1 ใ polystyrene e plat า ↳> block องก non- Specific binding า +IMG Ab า เ ม substra · ่าน พ ล้ ล้ ล้ ว่ ต้ ช้ ป้  ELISA Data Interpretation The ELISA assay yields three different types of data output: หาป มา Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples. เ ง ณภาพเ ร ไ 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. -สีเป ยน- ไ ไ เป ย น : ส พี สี ชิ ม่ คุ ริ ลี่ ลี่ An example of an ELISA experiment  Before starting the work read kit instruction carefully  1- The 96 well plate is labeled carefully and the first wells are used to draw the standard curve 2/3 ซ  The sample is added to plate in duplicate or triplicate and then the mean result is calculated  The quality control sample which is provided with the kit is treated as the test samples  Results  After reading the results the standard curve is drawn were the concentration is blotted on เ ค. มน ศาก. ดกลนแ the X-axis and the absorbance on the Y-axis Absorption (nm) Concentration (ng/ml) ื่ ดู ย้ Results-cont  The standards concentrations is specified on the x-axis and the reading of each standard is specified on the y-axis and the standard curve is drawn  The quality control sample concentration is determined from the standard curve and if the result is in the range given by the kit manufacturer the results could be accepted  Blocking Buffer Overview: Blocking buffers are used in various applications to decrease non-specific signaling generated by non-specific binding of proteins or peptides ประโยช Additional benefits of blocking include: Prevent nonspecific binding อง -specifi c bindi น ho Reduce ELISA background signal backgroun on Block nonspecific binding to adsorbed proteins อง น protei ่นn Stabilize adsorbed protein for better interactions protein ทมา บด ง วม ป้ ป้ จั ที่ อื ตั กั กั  Two types of blocking reagent Protein - Bovine serum albumin (BSA - Non fat dry milk - Serum - Fish gelatin Non-ionic Detergent (washing solution) - Tween 20 +wash buffer  Secondary Antibody Selection - Specifically detect the species that produced the primary antibody - When using a polyclonal antibody produced by rabbit you will select an anti-rabbit secondary antibody that was raised in an alternate host species such as goat or donkey - Polyclonal antibodies are sufficient for most needs - Monoclonal antibodies are more difficult to produce but are highly specific and yield more consistent results over time  Streptavidin-biotin conjugated นกันเป็น hon-corale nt แ ง ~ ใน ง The affinity of streptavidin for biotin is the strongest non-covalent biological interaction Streptavidin-biotin conjugates are extremely popular in techniques such as Western blotting, ELISA and flow cytometry จั ซ็ สิ่ ที่ มี  AVIDIN-BIOTIN COMPLEX (ABC system) Avidin / Streptavidin เพ ม ญญ าณ > ใ (nayme เ ย =สีช Biotin Vitami ท Biotin is small (MW=224) vitamin molecule and it can be chemically attached to proteins, antibodies. Avidin and streptavidin are proteins that have nearly identical functions in binding very strong and specifically to the biotin molecule. ิ่ ส่ สั  Detection Systems - Enzyme conjugated secondary is most likely the best choice 1. Peroxidase 2. Alkaline phosphatase 3. ß-D-galactosidase คง วท te m mp ต า - stability at typical assay temperatures: 4°C, 25°C, and 37°C - greater than six months shelf life when stored at 4°C - capable of being conjugated to an antigen or antibody - inexpensive - easily measurable activity - high substrate turnover number substrate เป ่ยนเป็น produ ct - unaffected by biological components of the assay ไม่รบกว วนป ว่ ี่ ตั ฏิ กิ ลี่  Horse radish Peroxidase (HRP) - a small molecule (MW ~40,000) that can usually be conjugated to an penzyme antibody in a 4:1 ratio Ab ↳ - Peroxidase is very inexpensive compared to alkaline phosphatase - Peroxidase is that it is incompatible with many preservatives, such as ไม่นียมเ ม สา ร sodium azide - Peroxidase enzyme substrates are acted on by the enzyme and - undergo a measurable color change or emit light in the presence of an enzyme-antibody complex bound to specific analytes. Soluble peroxidase substrates for ELISA include TMBE and ABTS TMB (3,3',5,5' tetramethylbenzidine) is a blue colour compound measurable at 650 nm ห ดป มา าว and used in endpoint assays by stopping the reaction with 1M phosphoric acid. A yellow reaction product is formed upon acidification that is measurable at 450 nm ให้สา าร เขียวอมน ABTS (2,2'-azino-di [3-ethylbenzthiazoline] sulfonate) is a blue-green compound measurable at 405 to 410 nm. Its reaction rate is suitable for endpoint assays and is easily stopped with 1% SDS (sodium dodecyl sulfate) ห ดป ติ สี ฟ้ ธิ กั ยุ นุ ฏิ ฏิ กิ กิ  Alkaline Phosphatase - Alkaline phosphatase (ALP) enzyme substrates are used in colorimetric assays. - approximately double the size of peroxidase (MW ~86,000) - lower enzyme to antibody conjugation ratio - slightly more expensive than peroxidase, but is considered to be more stable EDTA - inactivated by chelating agents, acidic pH (< 4.5), or inorganic phosphates - chelators (EDTA) and acidic pH are typically used as convenient and inexpensive stopping reagents for alkaline phosphatase reactions - pNPP is a soluble alkaline phosphatase substrate for ELISA p-NPP (p-nitrophenylphosphate) yellow color measurable at 405 to 410 nm and stopped with 1N NaOH. It is not recommended for kinetic analysis  Beta Galactosidase - Four subunits combined have a molecular weight of greater than 300,000 - Enhanced reaction rate in the presence of alcohols, which lends itself as a suitable enzyme for assays performed on hydrophobic membrane surfaces - BGAL substrates are specifically acted on by the enzyme ß-galactosidase and undergo a measurable color change, or white to blue color change, in the presence of antibody- enzyme binding target proteins or cells. substrate ONPG is a soluble BGAL substrate for ELISA - ortho-Nitrophenyl-β-galactoside (ONPG): galactosidase hydrolyzes the substrate produc ONPG molecule into galactose and ortho-nitrophenol. ↳สเห The latter compound has a yellow color and measurable at 420 nm wavelength by stopping the reaction with 1M Sodium Carbonate. &US MX ลื  Peroxidase Chemiluminescent Substrate Chemiluminescent HRP substrates are extremely sensitive, nonradioactive, enhanced luminol-based chemiluminescent substrates for the detection of horseradish peroxidase (HRP). Chemiluminescent HRP substrates are designed for both western blotting (WB) and enzyme immunoassay (ELISA) use นเ เ นแส ห็ ห็  Fluorescence fluorophore https://www.youtube.com/watch?v=ZtQBSRdpF-c