indirect elisa

BY: SAI MANOGNA (MSIWM014)

ELISA stands for Enzyme Linked Immunosorbent Antigen assay. ELISA is an ANTIGEN-ANTIBODY response.
In 1971, ELISA was founded. By Peter Perlmann and Eva Engvall at Stockholm university in Sweden. This is a plate-based assay technique used to detect and quantify substances such as peptides, proteins, antibodies and hormones. An antibody conjugated enzyme interacts with a colorless substrate to create a colored substance. This substrate is considered as a chromogenic substrate. A variety of enzymes has been used in ELISA, such as alkaline phosphatase, beta galactosidase and horse-radish peroxidase. Some substrates such as ortho-phenylenediamine dihydrochloride (for peroxidase), p-nitrophenyl phosphate (for alkaline phosphatase), which are hydrolysed by the enzyme referred to above, are used to generate a coloured end product.

In ELISA, an antigen must be immobilised on a solid surface and complicated with an antibody associated with an enzyme. Detection is achieved through an incubation with a substrate to create a measurable product by measuring the conjugated enzymes activity. A highly specific antimicrobial interaction is the most important aspect of the detection strategy.

Principle : Usually an ELISA test is performed in a multi-well plate of 96 or 384 well plates. The multi-well plate provides the stable surface for the antigen to stabilise. The immobilisation of the analyte makes it possible to isolate the antigen from the other components in the sample. This function makes ELISA one of the easiest experiments to conduct simultaneously in the multiple samples.

Types of ELISA :

ELISA assays can be found in various formats, based on the binding interactions between antigen-antibody.

They are :

Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

Direct Elisa : the antigen is immobilised and detected on the surface of the multi-well plate with an antigen specific antibody, directly conjugated to HRP or other detection molecules.

Indirect ELISA : With an indirect ELISA, an antibody can be detected or quantitatively determined.

1. An antigen-coated microtiter well is applied to the serum or any other biological sample containing primary antibody (Ab1) and allowed to react with antigen attached to the well.
2. The presence of an antibody bound to the antigen is identified after washing away any free Ab1 by adding an enzyme-conjugated secondary anti-isotype antibody (Ab2), which binds to the primary antibody.
3. Any free Ab2 antibody is then washed away, and an enzyme substrate is added.
4. Specialized spectrophotometric plate readers calculate the amount of colored reaction product that forms and can measure the absorbance of all the wells of a 96-well plate in seconds.
4. This method of choice is for detecting the presence of serum antibodies against Human immunodeficiency virus (HIV), the causative agent of AIDS.

Reaction combinant envelope and core HIV proteins are adsorbed as solid-phase antigens to microtiter wells in this assay. HIV infected individuals can develop serum antibodies to these viral proteins in the epitopes. HIV serum antibodies will usually be detected by indirect ELISA within 6 weeks of infection.

Sandwich ELISA : The antigen can be detected or measured using sandwich ELISA

1. In this procedure, the antibody is immobilised on a microtiter well (rather than the antigen).
2. An antigen containing a sample is added and allowed to react antibody being immobilised.
3. A second enzyme linked antibody specific to different epitopes on the antigen is added after the well is washed and allowed to react with the bound antigen.
4. After washing, substrate is added after any free second antibody is removed and the coloured reaction product is detected.

Competitive ELISA : Competitive ELISA is another variation for measuring the amounts of antigen in the sample.

1. In this procedure, antibodies are first incubated in a solution with an antigen-containing sample.
2. To an antigen-coated microtiter well, the antigen-antibody mixture is then added.
3. The more antigen present in the sample, the less free antibody will be available for binding to the well coated with antigen
4. The addition of an enzyme-conjugated secondary antibody (Ab2) unique to the primary antibody isotype can be used to calculate the quantity of primary antibody bound to the well. (as well as in Indirect ELISA).
5. Nevertheless, in the competitive assay, the higher the concentration antigen, the lower the absorbance i.e present in the original sample.

Chemiluminescence : During certain chemical reactions, the measurement of light emitted by the chemiluminescence provides a convenient and highly sensitive alternative to absorption measurements in ELISA that use chemiluminescence. For Example, the oxidation of the luminol compound by H2O2 and the horse-radish peroxidase (HRP) enzyme produces light.

Enhanced sensitivity is the value of chemiluminescence assays over chromogenic ones. In general, by switching from a chromogenic to luxogenic substrate and with the addition of enhancing agents, more than 200 times, the detection limit can be increased at least tenfold. In fact as few as 5moles (5 attomoles) of the target antigen have been detected under ideal conditions.

ELISPOT Assay : ELISPOT Assay is the modification of the ELISA assay. This assay makes it possible to quantitatively determine the number of cells present in a population that contain antigen-specific antibodies or an antigen for which a specific antibody is present.

1. In this method, the plates are coated with the antigen recognised by the antibody of interest or with the antigen-specific antibody which is being tested for production. These specific antigen and antibody are known as capture molecules.

2. The coated plates are then supplemented with a suspension of the cell population under examination and incubated.

3. The cells settled on the plate surface. The secreted molecules that react with the captured molecules, will create a ring of antigen-antibody complexes around each cell that produces molecules of their interest.

4. The plate is then washed, added and allowed to bind an enzyme-linked antibody specific to the secreted antigen or specific to the species ( eg; goat anti-rabbit) of the secreted antibody.

5. Subsequent production of the assay shows the location of the each antigen or antibody producing cell as a point of colour or light by introducing an appropriate chromogenic or chemiluminescence-producing substrate.

Advantages and Disadvantages :

ELISA	Advantages	Disadvantages
Direct ELISA	– Saves time and reagents (Short protocol) – No cross reactivity from secondary antibodies	– In sample all proteins bind to the surface – Primary antibody must be labelled individually. – No signal amplification – Low flexibility
Indirect ELISA	– Has signal amplification – High flexibility	– Long protocol compared to direct ELISA – Potential cross reactivity from secondary antibody.
Sandwich ELISA	– High specificity – Suitable for complex samples. – High flexibility and sensitivity	– Finding two antibodies to the same target that identify different epitopes and function well together even at difficult times.
Competitive ELISA	– Suitable for small antigens.	– Depends on base ELISA selected.