Definitions Antibodies (also known as immunoglobulinsabbreviated Ig) are gamma globulin proteins thatare found in blood and are used by the immunesystem to identify and neutralize foreign objects,such as bacteria and viruses.
Definitions- cont AntigensA substance that when introduced into the bodystimulates the production of an antibody ImmunoassayA laboratory technique that makes use of thebinding between an antigen and itshomologous antibody in order to identify andquantify the specific antigen or antibody in asample
Definitions- cont AnalyteThe sample being analyzed and inimmunoasssays the analyte is either Antibodyor Antigen
Antigen Is present naturally in the body like hormones Is manufactured in special disease status forexample human chorionic gonadotrophinhormone (HCG) which is normally producedby cells of the placenta in pregnancy is found inthe body in some types of cancer Is not present in the body in normal conditionlike drugs
Introduction The Antibody: An immunoglobulin, aspecialized immune protein, produced becauseof the introduction of an antigen into the body,and which possesses the remarkable ability tocombine with the very antigen that triggeredits production (specific affinity) The antibody recognises and bind to theantigenic determinant region of the antigen
Antibody Production Specific antibodies are produced by injecting anantigen into a mammal, such as a mouse, rat orrabbit for small quantities of antibody, or goat,sheep, or horse for large quantities of antibody.Blood isolated from these animals containspolyclonal antibodies—multiple antibodies thatbind to the same antigen—in the serum, whichcan now be called antiserum.
Antibody Production-cont To obtain antibody that is specific for a singleantigen, antibody-secreting lymphocytes areisolated from the animal and immortalized byfusing them with a cancer cell line.The fusedcells are called hybridomas, and will continuallygrow and secrete antibody in culture. Singlehybridoma cells are isolated by dilution cloningto generate cell clones that all produce thesame antibody; these antibodies are calledmonoclonal antibodies
ELISA techniqueIs a biochemical technique used mainly inimmunology to detect the presence of anantibody or an antigen in a sample. The technique is divided into1- Competitive ELISA2- Sandwich ELISA (also called direct ELISA)3- Indirect ELISA
Competitive ELISA The labelled antigen competes for primaryantibody binding sites with the sample antigen(unlabeled).The more antigen in the sample,the less labelled antigen is retained in the welland the weaker the signal).
Sandwich ELISA The ELISA plate is coated with Antibody todetect specific antigen
Sandwich ELISA Prepare a surface to which a known quantityof capture antibody is bound. Block any non specific binding sites on thesurface Apply the antigen-containing sample to theplate.
Sandwich ELISA-Cont Wash the plate, so that unbound antigen isremoved. Apply enzyme linked primary antibodies asdetection antibodies which also bind specificallyto the antigen. Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
Sandwich ELISA-Cont Apply a chemical which is converted by theenzyme into a coloured product. Measure the absorbency of the plate wells todetermine the presence and quantity ofantigen
Indirect ELISA The protein antigen to be tested for is addedto each well of ELISA plate, where it is giventime to adhere to the plastic through chargeinteractions A solution of non-reacting protein is added toblock any plastic surface in the well thatremains uncoated by the protein antigen
Indirect ELISA-Cont Then the serum is added, which contains amixture of the serum antibodies, of unknownconcentration, some of which may bindspecifically to the test antigen that is coatingthe well. Afterwards, a secondary antibody is added,which will bind to the antibody bound to thetest antigen in the well.This secondaryantibody often has an enzyme attached to it
Indirect ELISA-Cont A substrate for this enzyme is then added. Often,this substrate changes colour upon reaction withthe enzyme.The colour change shows thatsecondary antibody has bound to primaryantibody, which strongly implies that the donorhas had an immune reaction to the test antigen. The higher the concentration of the primaryantibody that was present in the serum, thestronger the colour change. Often a spectrometeris used to give quantitative values for colourstrength
An example of an ELISA experiment Before starting the work read kit instructioncarefully 1-The 96 well plate is labeled carefully and thefirst wells are used to draw the standard curve
An example of an ELISA experiment-Cont The sample is added to plate in duplicate ortriplicate and then the mean result is calculated The quality control sample which is providedwith the kit is treated as the test samples
Results After reading the results the standard curve isdrawn were the concentration is blotted onthe X-axis and the absorbance on theY-axisConcentration ng/mlAbsorptionnm
Results-cont The standards concentrations is specified onthe x-axis and the reading of each standard isspecified on the y-axis and the standard curveis drawn
Results-cont This standard curve is used to determine theunknown concentration of each sample byfinding the opposite concentration to theabsorbanceConcentration ng/mlAbsorptionnm
Results-cont The quality control sample concentration isdetermined from the standard curve and if theresult is in the range given by the kitmanufacturer the results could be accepted