Enzyme-Linked Immunosorbent Assay

INTRODUCTION

Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. ELISA is so named because the technique involves the use of an immunosorbent, an absorbing material specific for one of the components of the reaction, the antigen or antibody. ELISA is usually done using 96-well microtitre plate suitable for automation

What is ELISA?
Technique used to detect (assay) specific molecules (e.g. proteins & carbohydrates) in samples. Immunological technique: uses antibodies. Quantitative. Very sensitive. Commonly used in medicine and scientific research.

PRINCIPLE
Substrate
Coloured product

Secondary antibody

Primary antibody

Different antigens in sample

The technique is divided into :

1- Competitive ELISA 2- Sandwich ELISA (also called direct ELISA) 3- Indirect ELISA

COMPETITIVE 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. Advantage: The ability to use crude or impure samples and still selectively bind any antigen that may be present. Application: Used for the detection of human T-cell leukemialymphoma virus type III (HTLV-III).

INDIRECT ELISA

COLOR OF SOLUTION & COLOR OF SOLUTION ARE COMPLEMENTARY COLOR OF FILTER WAVELENGTH COLOR OF
SOLUTION VIOLET 420 BROWN

BLUE

470

YELLOWISH BROWN

GREEN

520

PINK

YELLOW

580

PURPLE

RED

680

GREEN/BLUE

INDIRECT METHOD

This method is largely used to measure antibodies in almost all human infections Eg : HIV antibody detection In patients with AIDS, the human immuno deficiency virus(HIV) produces specific antibody. To detect the HIV antibody, indirect ELISA method is used

SANDWICH METHOD

Eg : Assay of thyroid hormone (T4)

Reaction :
HRP

H2O2
Diamino oxidized Benzidine DAB (colorless) (brown color)

H2O + [O]

MULTIPLE & PORTABLE ELISA

This new technique uses a solid phase made up of an immunosorbent polystyrene rod with 8-12 protruding ogives.

The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwells of standard microplates pre-filled with reagents.

The advantages of this technique are as follows:

The ogives can each be sensitized to a different reagent, allowing the simultaneous detection of different antibodies and/or different antigens for multitarget assays The sample volume can be increased to improve the test sensitivity in clinical (saliva, urine), food (bulk milk, pooled eggs) and environmental (water) samples One ogive is left unsensitized to measure the nonspecific reactions of the sample

The use of laboratory supplies for dispensing sample aliquots, washing solution and reagents in microwells is not required

APPLICATIONS OF ELISA

Serum Antibody Concentrations

Detecting potential food allergens (milk, peanuts, walnuts, almonds and eggs) Disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc

19

 Detections

of antigens e.g. pregnancy hormones, drug allergen, , mad cow disease

human

chorionic gonadotropin (HCG), the commonly measured protein which indicates pregnancy. A mixture of purified HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only HCG with linked enzyme will bind.

 The

more HCG which is present in the test sample, the less enzyme linked HCG will bind. substrate the enzyme acts on is then added, and the amount of product measured, such as a change in color of the solution.

The

Detection of antibodies in blood sample for past exposure to disease e.g. Lyme Disease, trichinosis, HIV, bird flu ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.

To monitor diabetes, glucose concentrations will be checked. Bacterial left-over in milk can be determined with an ELISA test. ELISA assays are as well employed in foodstuff protection through signifying the occurrence of salmonella ELISA assays can also be utilised to diagnose several cancers (eg: bladder cancer)

ADVANTAGES OF ELISA

ELISA tests are generally relatively accurate tests. Highly sensitive and specific Antigens of very low or unknown concentration can be detected since capture antibody only grabs specific antigen Generally safe: do not require radioactive substances, contains diluted sulfuric acid Used in wide variety of tests

DISADVANTAGES

Only monoclonal antibodies can be used as matched pairs

Monoclonal antibodies can cost more than polyclonal antibodies

Monoclonal antibodies are more difficult to find Negative controls may indicate positive results if blocking solution is ineffective [secondary Ab or antigen can bind to open sites in well] Enzyme/substrate reaction is short term so microwells must be read as soon as possible