ServicesProtocols

Method

Enzyme Linked Immunosorbent Assay

 

Principle

A polystyrene microplate is coated with an antigen. In a second step a specific antibody (monoclonal AB to be tested ) is applied and biochemically attaches to the antigen if it recognises the right epitope. In a third step, an Alkaline-Phosphatase labelled secondary antibody is applied which binds to the primary antibody. The antigen- antibody complex is made visible using a chromogen/substrate solution which is capable of promoting a colour reaction.

Absorbance is read in a Elisa reader.

 

Sample

• antigen corresponding to the produced antibody
• culture supernatant; (Produced Mouse Monoclonal Antibodies from our Facility)
• Premium bleeds
• bleeds

 

Reagents/Material

• F96 Maxisorp, Nunc- Immunoplate, Nunc
• donkey anti-mouse IgG (H+L), alkaline phosphatase labelled, Jackson (715-055-150)
• 0.1N Sodium Carbonate buffer (NaHCO3), self made
• 1 x PBS, self made
• 1 x PBS/0.05%Tween, self made
• 3% BSA/PBS solution, self made
• dH2O Sigma Fast™, p-nitrophenylphosphate (PNPP) tablet sets, Sigma (N-2770)
• 5ml polystyrene tubes, Falcon
• 50ml Falcon tube

 

Equipment

• Elisa reader, TECAN
• pipettes

 

Procedure

use working sheet

1. coat immunoplate with 0.2µg of antigen per well, solving the antigen in Sodium Carbonate buffer. Use a volume of 50µl per well.

2. incubate 1h at RT (overnight incubation at RT is possible)

3. wash plates 3 x 5minutes with 1xPBS/0.05%Tween by filling up the wells to the top

4. block plates with 3% BSA/PBS solution for 1h at RT, using 200µl per well (overnight blocking is not recommended)

5. wash 2x with 1xPBS/0.05%Tween

6. wash 1x with 1xPBS only.

7. incubate with 50µl with culture supernatant/bleeds/controls for 1h at RT

8. wash 3x10minutes with 1xPBS/0.05%Tween

9. prepare secondary Antibody 1:5000 in PBS, use 50µl per well

10. incubate 1h at RT

11. wash 3x10minutes with 1xPBS/0.05%Tween

12. prepare substrate, using 50µl per well (1tablet set in 20ml dH2O yields 1.0mg/ml PNPP and 0.2MTris buffer)

13. add substrate and incubate 20minutes

14. read absorbance at 405nm and evaluate mean

15. Mark positive and negative control

16. highlight positive clones and label the sheet

 

Additional comments  

	Bleeds/Supernatants	Bleeds/Supernatants	Bleeds/Supernatants
Elisa using bleeds	Bleeds 1:500 and 1:2500	Premium bleed 1:500 and 1:2500	none
Elisa using supernatants	Supernatants no dilution	Premium bleed 1:500	Last bleed 1:500
Elisa subcloning	Supernatants no dilution	none	none

reconstitution of AB resuspend in 0.5ml of 50%Glycerol/dH2O 

 

 Interpretation

	1:500	1:2500
Elisa using bleeds	Abs. >0.1 and >neg.control mouse responding	Abs. >0.3 mouse ready for fusion
	Negative Control
Elisa using supernatants	Abs. >neg.contr. positive clone to pick
Elisa subcloning	Abs. > 0.1 positive clone to pick