Enzyme-Linked

Immunosorbent Assay
Erna Sulistyowati
Program Studi Pendidikan Dokter
Fak Kedokteran Univ Islam Malang
2012

Introduction



ELISA
Tipe
Applikasi
Prinsip

2

What is ELISA?
• Teknik biokimia yang
mengg prinsip imunologi
utk mendeteksi
keberadaan antibodi/Ab
atau antigen/Ag pd
sampel.
• Digunakn utk deteksi HIV,
Hep,dll.
• Dilakukan pd sumuran/well
plastik 8 cm x 12 cm msg2
matrix 8 x 12 dr 96
sumuran, dg diameter 1
cm tinggi& 0.7 cm

3

Types of ELISA • Qualitative ELISA – Positive or Negative results • Quantitative ELISA – optical density or fluorescent units of the sample is interpolated into a standard curve. 4 . which is typically a serial dilution of the target.

Sandwich ELISA (also called direct ELISA) 3.Competitive ELISA 2.ELISA technique The technique is divided into: 1.Indirect ELISA .

g. HIV. common. Lyme Disease. GMO. STD etc • Detections of antigens e. pregnancy hormones. mad cow disease • Detection of antibodies in blood sample for past exposure to disease e. drug allergen.g. walnuts.APPLICATIONS OF ELISA • Serum Antibody Concentrations • Detecting potential food allergens (milk. bird flu.tracking the spread of disease e.g. trichinosis. peanuts. HIV. bird flu 6 . colds. cholera. almonds and eggs) • Disease outbreaks.

g. This is one (e.Basic principles of ELISA • the antibodies fixed to a solid surface. βgalactosidase) that produces a colored product from a colorless substrate. • a preparation of the same antibodies coupled to an enzyme. such as the inner surface of a test tube. 7 ..

Competitive ELISA • Ag yg sdh dilabel/ditandai berkompetisi dg Ag yg tdk dilabel utk mengikat Ab primer semakin tinggi konsentrasi Ag pd sampel. .semakin sdkt Ag terlabel yg bersisa.

.

Sandwich ELISA • The ELISA plate is coated with Antibody to detect specific antigen .

• Block any non specific binding sites on the surface • Apply the antigen-containing sample to the plate. .Sandwich ELISA • Prepare a surface to which a known quantity of capture antibody is bound.

Sandwich ELISA-Cont  Wash the plate.  Apply enzyme linked primary antibodies as detection antibodies which also bind specifically to the antigen. so that the unbound antibodyenzyme conjugates are removed. so that unbound antigen is removed.  Wash the plate. .

• Measure the absorbency of the plate wells to determine the presence and quantity of antigen .Sandwich ELISA-Cont • Apply a chemical which is converted by the enzyme into a coloured product.

Sandwich ELISA .

Indirect ELISA • The protein antigen to be tested for is added to each well of ELISA plate. where it is given time to adhere to the plastic through charge interactions • A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen .

which will bind to the antibody bound to the test antigen in the well.Indirect ELISA-Cont  Then the serum is added. This secondary antibody often has an enzyme attached to it . a secondary antibody is added. which contains a mixture of the serum antibodies.  Afterwards. of unknown concentration. some of which may bind specifically to the test antigen that is coating the well.

this substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody. Often a spectrometer is used to give quantitative values for colour strength . the stronger the colour change. Often.  The higher the concentration of the primary antibody that was present in the serum. which strongly implies that the donor has had an immune reaction to the test antigen.Indirect ELISA-Cont  A substrate for this enzyme is then added.

Results-cont • The quality control sample concentration is determined from the standard curve and if the result is in the range given by the kit manufacturer the results could be accepted .

Indirect ELISA .

An example of an ELISA experiment • Before starting the work read kit instruction carefully • 1.The 96 well plate is labeled carefully and the first wells are used to draw the standard curve .

An example of an ELISA experiment-Cont • The sample is added to plate in duplicate or triplicate and then the mean result is calculated • The quality control sample which is provided with the kit is treated as the test samples .

Results • After reading the results the standard curve is drawn were the concentration is blotted on the Xaxis and the absorbance on the Yaxis Absorptio n nm Concentration ng/ml .

Results-cont • This standard curve is used to determine the unknown concentration of each sample by finding the opposite concentration to the absorbance Absorptio n nm Concentration ng/ml .

Results-cont • The standards concentrations is specified on the x-axis and the reading of each standard is specified on the y-axis and the standard curve is drawn .

Methodolog y 25 .

the half of the shared sample (750μl) was placed in the group’s tube. • The “bodily fluid” was then transferred into the tube of another group.Methods • A yellow tube and plastic transfer was labeled. 26 . The samples were then mixed and after which.

Methods • The sharing protocol was repeated twice. On the first 3 wells. 27 . it was labeled as “+” while for the next 3 it was labeled as “-”. • The 12-well strip was then labeled. • The remaining wells were labeled with two of the member’s initials.

28 . • 50μl of the group’s sample was transferred into the the appropriately initialed three wells.Methods • A fresh pipette tip was used to transfer 50μl of the positive control into the “+” wells (the same was done for the “-” wells).

29 . • The microplate strip was then tapped onto the paper towels provided.Methods • There was a 5 minute waiting period so as to allow the proteins in the samples to bind to the plastic wells. • The well was then filled with wash buffer.

• Washing of the wells was again performed.Methods • A 50μl of primary antibody was then transferred into the 12 wells of the microplate strip. 30 . • The fluids was then allowed to stand for another five minutes so as to allow the antibody to bind.

31 .Methods • A 50μl of secondary antibody was then transferred into the 12 wells of the microplate strip. • A 50μl of enzyme substrate was then transferred into the 12 wells of the microplate strip. • A 5 minute waiting period and washing of the microplate wells was again performed (2x).

• The results were then observed and recorded.Methods • The enzyme substrate was let to stand for five minutes. 32 .

com/en/a543-direct-enzymelinked-immunosorbent-assay-elisa • ELISA • http://www.ualberta.swf .Useful sites • http://www.html • http://www.biology.com/webcontent/a nimations/content/ELISA.ca/facilities/mu ltimedia/uploads/procedures/elisasound.edumediasciences.sumanasinc.