LABELED IMMUNOASSAYS PART 2 Enzyme Linked Immunosorbent Assay (ELISA) Lab. 3

Labeled Immunoassays The basic underlying principles of indicator labeled immunoassays are the same There are differences with respect to the detail of the protocols The designation given to each test differs according to the label used to detect the antigen/ antibody complexes 2

Enzyme Linked Immunosorbent Assay (ELISA) One of many assays collectively called enzyme immunoassays (EIA) Can be used to detect both antibody and antigen Very Sensitive (ng & pg/mL), Relies on Monoclonal Abs An enzyme is used as an indicator molecule The enzyme does not provide detection directly but through the break-down of a substrate 3

Enzymes used in ELISA Enzymes used as labels for immunoassay are chosen according to the following criteria:  Turnover number  Sensitivity  Ease and speed of detection  Stability  Absence of interfering factors in patient samples  Availability and cost of enzyme and substrate 4

Enzymes used in ELISA The most commonly used enzymes are:  Horseradish peroxidase (HRP) and  Alkaline phosphatase (AP) Each has a high turnover number (rapid conversion of substrate to a product) resulting in high sensitivity Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options These include beta-galactosidase, acetylcholinesterase and catalase 5

Enzyme Substrates A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate Substrates for AP and HRP, depending upon the plate-reading equipment available and the level of sensitivity required in the ELISA An array of, and substrates is available for use with either enzyme ChromogenicColorspectrophotometer FluorogenicFluoroescence fluorometer Chemiluminescentlight luminometer Chemiluminescent and chemifluorescent substrates provide a stronger signal than Chromogenic substrates Steadily gaining in popularity because of their: sensitivity (less than 1 pg/ml “ ”), large linear range for detection and excellent antibody conservation 6

Enzyme Substrates 1. Chromogenic substrates A common ELISA substrate for HRP is: Tetramethylbenzidine (TMB), TMB is oxidized to yield a blue product that is water-soluble and absorbs light at 650 nm The reaction can be halted by addition of acid or another stop reagent Using sulfuric acid turns TMB yellow color which may be read at 450 nm 7

Enzyme Substrates The most common chromogenic substrate for alkaline phosphatase is: p-nitrophenyl phosphate (PNPP) PNPP yields a yellow reaction product that is water-soluble and absorbs light at 405 nm 8

Enzyme Substrates 2. Chemiluminescen substartes Luminol is one of the most widely used chemiluminescent reagents and its oxidation by peroxide results in creation of an excited state product called 3-aminophthalate This product decays to a lower energy state by releasing photons of light Using chemiluminescence allows multiple exposures to be performed to obtain the best image Sensitivity: picogram or femtogram level (1.0 × grams) 9

Enzyme Substrates Fluorogenic substartes A Fluorogenic Substrate is a nonfluorescent material that is acted upon by an enzyme to produce a fluorescent compound Different substartes are also available for AP and HRP enzymes p-Hydroxphenylpropionic acid (HPPA) is used for HRP enzyme 10

Linkage of Enzyme The enzyme label is linked to antibody or analyte by several means 1. Glutaraldehyde is often used as a crosslinker to join amino groups of the enzyme and the molecule to be labeled 2. Maleimide derivatives are also used to attach the enzyme label  The heterobifunctional cross-linker 11

Linkage of Enzyme 3. Treatment with periodate Glycoproteins such as horseradish peroxidase can be activated for conjugation by treatment with periodate This provides a mild and efficient way of generating reactive aldehyde groups for subsequent conjugation with amine- or hydrazide- containing molecules 12

Linkage of Enzyme 4. The use of biotin- avidin binding The secondary Ab is labeled with biotin An enzyme-avidin conjugate is added 13

Coating of Microplate A key feature of the solid-phase ELISA is that antigens or antibodies can be attached to surfaces easily by passive adsorption This process is commonly called coating Most proteins adsorb to plastic surfaces, probably as a result of hydrophobic interactions between nonpolar protein substructures and the plastic matrix 14

Coating of Microplate Since most of proteins' hydrophilic residues are at the outside and most of the hydrophobic residues orientated towards the inside Partial denaturation of some proteins results in exposure of hydrophobic regions and ensures firmer interaction with the plastic This can be achieved by exposing proteins to low pH or mild detergent 15

ELISA Protocol 1- Coating antibody or antigen onto the microplate  Dilute the protein to be coated in a buffer such as PBS or Carbonate-Bicarbonate and add 100 μl of this solution per well  Incubate for hours at room temperature or 4°C  Block unoccupied sites with a blocking agent ( μl/well)  Store plate at 4°C with a desiccant for future use 16 Analyte = antibody Analyte = antigen Coated with Antibody when analysing antigen Coated with Antigen when analysing antibody Incubate, wash

ELISA Protocol 2- Blockage of free binding sites  Block the unoccupied sites on the surface of the well to reduce the amount of nonspecific binding of proteins  A variety of blocking buffers ranging from nonfat milk to highly purified proteins have been used to block unreacted sites  The blocking buffer should improve the sensitivity of the assay by reducing the background interference 17 Analyte = antibody Analyte = antigen Incubate, wash

3. Add sample. Incubate, Wash Analyte = antibody Analyte = antigen ELISA Protocol Add conjugate. Incubate, Wash Analyte = antibody Analyte = antigen E E

5. Add substrate 6. Incubate, stop, measure color change Colourless ENZYME OD Concentration 19 ELISA Protocol

Types of Enzyme Immunoassay  Three main types: 1. Competitive ELISA 2. Indirect ELISA 3. Sandwich ELISA 20

1- Competitive ELISA Enzyme activity is inversely proportional to the concentration of the test substance A sensitivity of nanograms (10 -9 g)/ml can be achieved This method can be used for measurement of small molecules that are relatively pure such as insulin, and estrogen 21

Competition ELISA to detect Antigens (Antibody-coated plate) Anti-analyte 2. Analyte-E + sample 1. Anti-analyte Low [analyte] High [analyte] 1- Competitive ELISA E E E E E E E 3. Wash 2. Analyte- E + sample 1. Anti-analyte Low [analyte] High [analyte] 4. Substrate 3. Wash 2. Analyte-E + sample 1. Analyte Low [analyte] High [analyte]

2- Indirect ELISA Indirect ELISA is more sensitive than the competitive Much assays are capable of detecting concentrations of less than 1 pg/ml ( g)/ml. 23

Screening of hybridoma supernatants Detecting clinically important antibodies (eg. Autoantibodies) 1. Antigen Indirect ELISA 2. Sample (human) antibody 1. Antigen E E E EE E 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody 1. Antigen E E 4. Substrate 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody 1. Antigen

3- Sandwich ELISA If the antibody is bound to the solid phase, these assays are called sandwich immunoassays Antigens captured in these assays must have multiple epitopes Used for detection of hormones, drugs, tumor antigens, cytokines 25

1. Anti-analyte Sandwich ELISA 2. Sample 1. Anti-analyte EE 3. Anti-analyte-enzyme 2. Sample 1. Anti-analyte 4. Substrate 3. Anti-analyte-enzyme 2. Sample 1. Anti-analyte

Lateral Flow Immunoassay (LFIA) (Immunochromatography System Assays) (Membrane based immunodiagnostic assay) 27

Principle Are based on the migration of nano or micro particles on strips for analytes detection in several areas In principle, any coloured particle can be used, however latex (blue colour) or nanometer sized particles of gold (red colour) are most commonly used The technology is based on a series of capillary beds, such as pieces of porous paper These elements has the capacity to transport fluid (e.g., urine, plasma,..) spontaneously 28

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Control Immoblized Ab support Sample pad Principle Monoclonal Ab labeled with colored particles 30 While the sample fluid start moving it dissolves particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure Immoblized Ab

Apply sample solution, upon application of sample biochemicals dissolve Immobilised Antibody area Control area Negative: no antigen Positive: antigen present 31 Principle

Uses Human and animal diagnostics Point of Care testing (PoCT) Forensics Environmental testing Advantages Rapid test (<15 minutes) Reliable and easy-to-use (no special equipment required nor trained staff) Semi-quantitative or quantitative results Non-refrigerated storage 32

Detection of HBsAg Lateral Flow Immunoassay (LFIA) 33

Principle Atlas HBsAg One Step Hepatitis B Surface Antigen Test cassette (Serum/Plasma) is a rapid chromatographic immunoassay for the qualitative detection of Hepatitis B Surface Antigen in serum or plasma The membrane is precoated with anti-HBsAg antibodies on the test line region of the test During testing, the serum or plasma specimen reacts with the particle coated with anti-HBsAg antibody The mixture migrates upward on the membrane chromatographically by capillary action to react with anti-HBsAg antibodies on the membrane and generate a colored line The presence of this colored line in the test region indicates a positive result, while its absence indicates a negative result 34

Quality Control A procedural control is included in the test A red line appearing in the control region (C) is the internal procedural control It confirms sufficient specimen volume and correct procedural technique (membrane wicking has occurred) 35

Limitations LINEAR HBsAg cannot detect less than 1 ng/mL of HBsAg in specimens If the test result is negative and clinical symptoms persist, additional follow-up testing using other clinical methods is suggested A negative result at any time does not preclude the possibility of Hepatitis B infection 36

Procedure 1. Allow the test strip to equilibrate to room temperature prior to testing 2. With arrows pointing towards the specimen, immerse the test strip vertically in the serum or plasma for at least 5 seconds Do not pass the max. line on the test strip when immersing the strip 3. Place the test strip on a non-absorbent flat surface 4. Start the timer and wait for the red line (s) to appear 5. The result should be read at 15 minutes It is important that the background is clear before the result is read 37

Interpretation Positive 38 Control line Test line NegativeInvalid