Lab 29: Quantitative ELISA

ELISA (Enzyme-linked immunosorbent assay)= an immunodiagnostic test used to measure specific antigens or antibodies in an unknown sample 

-test is often used to detect the presence or even specific levels of substances such as drugs, hormones, foods, and pathogens

-test is relatively inexpensive, simple, and easily interpreted 

-each test is run in a small plastic well on a microtiter plate= plate with multiple wells for test

-test works bc of antigen-antibody specificity 

-Testing for Unknown Antigen= the unknown antigen will adhere to its specific antibody in the test well 

-Testing for Unknown Antibodies= the unknown antibody will adhere to its specific antigen in the test well 

*in both types of tests an antibody- enzyme conjugate is added

Antibody-enzyme conjugate= another antibody that is linked to an enzyme 

-this enzyme catalyzes a color change rxn in the presence of its substrate, a chromagen 

-chromogen= chemical that can undergo a color change rxn 

-2 Types of Info Obtained from ELISA

1. Qualitative ELISA test=  a color change is enough to confirm a positive result 

Ex. Pregnancy test, or peanut contamination in food- these are yes/no results

2.  Quantitative ELISA test=  the level of the substance is also assessed 

-to do this, the intensity of the color change is read with a spectrophotometer=gives an absorbance reading which is proportional to the amount of antigen or antibody

Ie. High absorbance= high amount of Ag or Ab 

-the results are then compared to absorbance readings from known amounts of Ag or Ab

- we then plot these known values on a graph called a standard curve to be able to determine the level of unknown from the graph

-2 Types of ELISA tests: Direct and Indirect

1. Direct ELISA= “sandwich assay”= tests for an unknown Antigen 

a. well is coated with known Antibody for the specific antigen to be tested 

b. sample is added with unknown antigen, and if present antigen will bind to Antibody 

c. Wash= any unbound material is then washed away 

d.  Antibody-enzyme conjugate is added

e. Wash- unbound material is again washed away 

f. Chromogen added 

color change= positive test= yes Ag 

no color change= negative= no Ag

-sandwich assay- bc it creates a symmetrical grouping of Ab-Ag-Ab

Ex. Direct ELISA= pregnancy test, influenza test 

2. Indirect ELISA= tests for an unknown Antibody 

a. Well coated with known Antigen for the specific antibody to be tested

b. Sample is added – if antibody present will bind to Ag

c. Wash- unbound material is washed away 

d. Antibody-enzyme conjugate added

-this is actually an anti-antibody, bc it adheres to the specific class of Ab that we are testing for 

e. Wash- unbound material washed away

f. Chromogen added

positive= color change

negative= no color change 

ex. Indirect ELISA= HIV antibodies-> bc HIV antigens hide in infected cells and can be harder to find in serum samples 

In this lab- we do indirect ELISA test for salivary IgA antibodies 

IgA= predominant secretory Ab (ie. Found in secretions-saliva, tears)

• IgA can inhibit bacteria that contribute to dental caries= cavities
• Prediction= increased IgAs= decreased cavities bc it inhibits bacteria that cause cavities

*Note- that for our indirect ELISA- the test wells have anti-human IgA adhered to them – this is our Ag

-this is known as a capture antibody- acts like an Ag for Ab- we got by injecting into goats and collecting the Abs made by goat

Brief Outline of our Steps- we do a Indirect Quantitative ELISA for Salivary IgA

1. Add saliva sample which contains IgA to test wells with anti-human IgA adhered to them 
2. Wash
3. Add anti-human IgA Ab conjugated to the enzyme horseradish peroxidase (HRP)
4. Wash
5. Add TMB chromogen= HRP enzyme substrate-

ie if HRP is bound and present TMB chromogen will cause it to change color

Color change should occur- then we place microtiter plate into the ELISA plate reader= spectrophotometer=determines the absorbance readings 

Determine amount of salivary IgA- we do this by creating a standard curve of absorbance readings of known concentrations of human IgA (ie. We graph standards of known concentrations and their  known absorbance’s)

For Graph= makes it a quantitative

X=concentration of IgA standards

Y= absorbance (from spectrophotometer)