ELISA Summary

ELISA = Enzyme linked immunosorbant assay

Types of ELISA are:

• Direct - (antigen-coated plate; screening antibody)

• Indirect - Used to detect an antibody against a known antigen/ (antigen-coated plate; screening antigen/antibody)

• Continuous - Used to detect how much of an antigen is present/ (antibody-coated plate; screening antigen)

• Sandwich - Used to detect an antigen of interest/(screening antibody)

Cotinine

• The amount of cotinine is related to exposure to nicotine, so this helps to distinguish between active and passive smokers

• Cotinine can also be measured using non-invasive samples e.g. saliva

• The amount of cotinine in a saliva sample reflects levels in the blood and thus serves as a reasonable surrogate sample matrix for blood

IMPORTANT FACT!

In a competitive ELISA, the higher the final absorbance, the LESS antigen there is in the sample, because MORE primary antibody is bound to the antigen on the plate

C1V1=C2V2

An important equation which helped in the practical to identify volume or concentration

C1 = Starting concentration/Stock concentration (e.g. ng/ml , g/L )

V1 = Initial Volume (e.g. ml , L , ul )

C2 = Final concentration

V2 = Final Total Volume

EXAMPLE Question

You have a 200 ng/mL (C1) concentrated stock solution in water and you need to prepare 0.5 mL (V2) of a 10 ng/mL (C2) solution. How much concentrated stock do you need and how much water?

In this question we need to calculate the inital volume needed “as we INITIALLY need to prepare” before getting the final solutions.

1. Use the equation C1V1=C2V2

   • C1 = 200

   • V1 = ??

   • C2 = 10

   • V2 = 0.5

2. Rearranging the equation to find V1

   • V1 = (C2xV2)/ C1

   • V1 = (10X0.5)/200

3. Calculate the answer for V1

   • V1 = 0.025

4. USE THE CORRECT UNITS

   • 0.025ml this is in ml because in the question they all values are in ml

   • To convert into a specific unit e.g. microlitres we need to mulitply value by 1000

   • 0.025ml x 1000 = 25ul (microlitres)

EXTRA: This answer highlights you need 25ul of stock solution but if you need to dilute with water to create a total volume of 500ul how much water will be needed?

Total volume of whole sol. - volume of stock = volume of water needed

• 500 - 25 = 475ul of water is needed to dilute

Results from the practical