Enzyme Linked Immuno Sorbent Assay

Enzyme Linked Immuno Sorbent Assay

EL S

: IMMUNO ASSAY

IMMUNO : Linked with Immunology.

ASSAY : Determination of Content. : ESTIMATION OF ANTIGENS OR ANTIBODIES.

I S A : ENZYME LINKED
TO VISUALIZE THE Ag-Ab BINDING IN THE TEST, A MARKER IS TAGGED WITH THE Ag / Ab ALREADY PRESENT IN THE KIT.
Polystyrene White / Colored Latex Particle. Charcoal Particle. R.B.C. or Erythrocytes. Enzyme-Substrate: Color Reaction.

Gold Sol Particle.

Radio Active Isotope. Toluedene Red Particle.

: ENZYME IMMUNO ASSAYS

Homogenous EIA
SEPARATION
OF BOUND AND FREE (UNBOUND) FRACTIONS OF THE LABELLED IMMUNOCOMPONENTS NOT NECESSARY. WASHING STEP IS NOT REQUIRED.

Heterogenous EIA
SEPARATION OF THE BOUND AND FREE (UNBOUND) FRACTIONS
OF THE LABELLED IMMUNOCOMPONENTS IS NECESSARY. WASHING STEP IS PRESENT .

 DIRECT ELISA INDIRECT ELISA

SANDWICH ELISA
COMPETITIVE ELISA

ELISA / EIA Estimations are of 3 Types :

QUALITATIVE ASSAYS QUANTITATIVE ASSAYS SEMI - QUANTITATIVE ASSAYS

 Qualitative

e.g. hCG Requires Contrals, No graph required

Quantitative e.g. Thyroid estimation Require Standards/Calibrators, To Plot a graph Semi-Quantitative e.g. dsDNA, HIV Require Controls, Calculation based, Graph may or may not be required

ANTIGEN

Single Ag

Multiple Ag

Epitope

ANTIBODY

IgG

IgM

IgG

IgE

IgG + IgM

IgG1 IgG2 IgG3

ELISA

Enzyme Linked

Immuno

Sorbent

Assay

Marker for visualising end result

Ag-Ab Complex

Solid Surface

Estm of Ag/Ab

Solid Surface on which the Ag / Ab is coated through passive adsorption

Microwell - polystyrene
Breakable / Non - Breakable Wells

Tube - polypropylene Bead - polystyrene Cuvettes

 Antigen

Antibody

 Concentration of a substance on a surface Adsorbate is the substance which is adsorbed on a surface ( Sorbent )

 Active Adsorption
Passive Adsorption
Proteins e.g. Antigens & Antibodies adsorb passively to Solid Surfaces, such as Plastic Polymers. The Solid Surface is termed as Solid Phase.

 Passive Adsorption consists of primarily Hydrophobic interactions or Ionic interactions between the Proteins / Biomolecules and the Sorbent ( Solid Phase )
Based on the principle of Static Electricity

 Low Binding
Surfaces that are Hydrophilic and Neutrally Charged are Considered Low Binding Sorbents.

Medium Binding
Non modified Polystyrene surfaces are Hydrophobic in nature and can only bind large bio molecules through passive adsorption. Due to large binding surface area required, binding capacity is not very high.

High Binding
The binding capacity can be increased through Radiation. Radiation breaks the Benzene rings opening up Carboxyl groups for increased Hydrophobic / Ionic interactions. Ideal for small bio molecule immobilization on Sorbents.

 Ploystyrene has a long Carbon Chain with Benzene rings on every other Carbon Polystyrene is a highly Hydrophobic compound The Hydrophobicity is maintained when Polystyrene is moulded into devices ( Microwells ) Carboxyl and Amine groups of Biomolecules can readily be grafted to the hydrophobic surface of Polystyrene Polystyrene can also be modified through Chemical reaction / Radiation to allow Covalent attachment for immobilization of bio molecules

 Non Specific binding of unwanted proteins in available free sites

Blocking Agent : Add immunologically inert proteins so as to block the free sites Blocking agents may be added during coating process; along with sample diluent or in both the steps

BLOCKING AGENTS
Rabbit Serum, Bovine Serum Albumin, Gelatin, Casein etc.

 Immunoassays do not actually measure the analyte. They can only provide a quantitative estimate of concentration by direct comparision with the standard / calibrator used.
The aim of standardization of a laboratory is to improve the accuracy I.e. the results should be as close to the true value as possible.

 Commercially available Capable of being conjugated to an Antigen or Antibody Stability at typical assay temperature : 18C, 20C, 37 40C Stability when stored at 2-8C Easily measurable activity Purity High Substrate Turnover number Specific Activity Unaffected by biological components of the assay

 Horse Radish Peroxidase ( HRP / HRPO )

Calf Intestine Alkaline Phosphatase ( AP ) E. Coli

b - Galactosidase

Parameters Mol.Wt. Size Enzyme Ab Conjugation Ratio Substrate yield

HRPO 40,000 Small 4:1

AP 86,000 Moderate 2:1

B-GLC 300,000 Very Large 1:1

Both Soluble and Insoluble Incompatible with Sodium Azide (Preservative) Sensitive to Temperature

Both Soluble and Insoluble

Both Soluble and Insoluble

Disadvantage

Expensive Large molecule, lower Enzyme-Ab Conjugation ratio. Inactivated by chelating agents (EDTA), Acidic pH, Inorganic Phosphates (Wash buffers, Diluents)

Advantage

High Signal ratio

Higher Stability

Enhanced reaction rate in presence of Alcohol

 Washing is actually a dilution process to optimally dilute the original solution without stripping off the bound / capture protein This optimal dilution step requires 3 - 5 cycles Less than 3 cycles leaves behind residual proteins in the wells

 Composition of the wash buffer The dispensing mechanism that is used to fill up the wells with the wash solution

Aspiration of wash buffer from the wells

Aspiration strength and Dispensing strength of the washer Repeated washing to ensure complete removal of unbound material from the wells

 Wash buffer should be added into the wells gently. A gentle flow will remove unbound components / loosely bound free proteins without stripping the specific immobilized biomolecules (Capture)
Vigorous addition of the wash solution will strip off either the capture biomolecule or inactivate the enzyme

It is important to ensure a constant fluid dispensing pressure in each well for precision of the assay

 The volume of wash solution dispensed per well should be high enough to cover the entire surface area coated with Antigen / Antibody
The entire well must be filled during washing cycle

Enough care is needed to prevent well to well overflowing of wash solution

 Addition of a Soak Step following the last wash cycle is extremely beneficial in terms of removing the remaining non-bound proteins that are trapped in the well corners This step is particularly useful for infectious disease assays

 Drying Effect Wells left dry / total aspiration / slow aspiration is undesirable due to denaturation effect of the bound protein Position of Needle or Pipette Tips Scratching of well bottom is undesirable as it peels off the bound protein Vacuum Strength Optimization of Vacuum Strength is crucial to maintain low c.v.s. High Vacuum Strength (mmHg) creates shear forces and air currents which denature bound proteins and inactivate enzymes. Low Vacuum Pressure residual wash solution will remain in the wells and suppress enzymatic activity because of detergents.

 During washing, more specifically aspiration step, it is recommend to leave a small amount of wash buffer in the wells. This minute quantity of buffer creates a film on the well and thus prevents denaturation due to drying effect.
Once the wash cycle is over, invert the plate and tap on tissue paper / absorbent paper towel to get rid of the residual wash buffer.

 Number of Wash Cycles is directly proportional to effective removal / dilution of original solution
Follow manufacturers guidelines

 Water is a poor Wash Buffer due to :

Variable pH Water lacks protein buffering capability
( Surface bound proteins need to be protected from denaturation )

Water does not posses detergent properties

 Addition of a week detergent (e.g. Tween 20) in a wash buffer is beneficial and adds to the overall performance of the assay system:
Detergent aids in removal of loosely bound proteins without stripping off the capture Ab / Ag

 During Incubation, Antigens are allowed to react with Antibodies or Enzymes are allowed to react with Substrates The reaction between Antigens and Antibodies depends on :
Time Distribution Temperature pH

 Stationary Incubation
Rotary Incubation
Ensures complete distribution / mixing of reactants Provides additional kinetic energy required to the reaction system

 Stationary Incubation
Mixing takes place through diffusion of reagents Because stationary incubation relies on diffusion of molecules, the role of temperature becomes extremely critical To ensure complete reaction longer incubation time is recommended

 Rotary Incubation
The interaction between Antigen and Antibody relies on their closeness, which is encouraged with the mixing during rotation Ensures complete distribution / mixation of reactants Increases contact between analyte and the Capture / Adsorbed reactant In case of viscous samples, chances of reaction between reactants is more high Rotation allows reaction between Antigen & Antibody to be less dependant upon Temperature Provides additional kinetic energy required to the reaction system

 37C 40C
Water bath Hot plate

18 - 21C 20 - 24C

Dry Incubator
Humidity Controlled

 For all ELISAs the final step is the addition of the Substrate. The Substrate is chosen for its quantitative yield of a Coloured end reaction product.
For colorimetric assays, the rate of colour development is directly / inversely proportional, over a certain range, to the amount of Enzyme Conjugate present.

 Soluble Coloured Reaction Product

Measured Colorimetrically For Quantitative Assays For Sensitive assays where Colorimetric measurement is needed

Insoluble Coloured Reaction Product

Measured Visually or Through Densitometer Suitable for Dot Blot Assays Permanent record

 Ability to produce intense coloured end product

Fast reaction rate or Rate of conversion of Substrate to End Product Ability to produce a broad range of colourded end product in a given time depending upon amount of Conjugate (Analyte) it has reacted with

 TMB (Tetra Methyl Benzidine) OPD (O-phenylenediamine) DAB (Diaminobenzidine)

4CN (4-Chloro-1-Naphthol)
ABTS (Azino-di-ethylbenzthiazoline-sulfonate)

BCIP (5-bromo-4-chloro-3-indolyl phosphate) NBT (Nitroblue tetrazolium) p-NPP (p-nitrophenyl phosphate)

Temperature Reaction time pH Ionic strength Buffer composition Substrate depletion Build up of enzyme / product inhibitors Increasing back reaction as product concentration increases Enzyme denaturation Exposure to light

 To take the O.D. of the final colour solution

Prevent unnecessary long assay procedure

 By destroying the Enzyme Component

Acids, Alkalis are known to chelate Enzymes Stop reaction just by adding Acid / Alkali (Enzyme Specific)

IN HCl, 4N H2SO4 , NaOH

 Enzyme -Substrate reaction producing final colour is sensitive to Temperature and in some cases light and Mixing the end product to make the final colour homogenous

Edge Effect refers to greater development of colour in the outside wells than the inner wells. This may happen due to :
Temperature of Substrate not equal to the well temperature

Proximity of the wells to heat source

Light Mixing

 The coloured end product (before adding acid or after) is always concentrated at the periphery of the wells. Elisa Readers, while estimating colorimetrically, aligns the wells electronically and reads only through the centre of the wells. Precision dramatically improves if plates are physically shaken before reading the O.D. of the final Coloured End Product to ensure complete dispersion of the end product in the well (Homogenous)

 Temperature of Substrate not equal to the well temperature Proximity of the wells to heat source

Light

 By Eye Inspection By Spectrophotometric Reading - Microwells in ELISA Reader

 Mathematical Calculations
Graphical
- Linear vs Linear Graphs e.g. T3, T4, TSH - Linear vs Log Graphs e.g. ToRCH

- Log vs Log Graphs e.g. AFP

- Log vs Logit Graphs e.g. Steroids

 Units of Interpretation should be based on International Reference Only

Always follow the Interpretation Units mentioned by the manufacturer.