1. Practical Course ELISA
Experiment to prove a
HIV infection
proof of anti-HIV antibodies in blood

2. Preparations by the teacher

3. 1. Preparation of buffer solutions
PBS-buffer (100 ml)
90 ml H2Odest ml PBS (10x)
PBS-buffer is needed for rehydration of hydrophilic antigens and antibodies.
Washing buffer (900 ml)
805 ml H2Odest ml PBS (10x) + 4,5 ml Tween 20
washing buffer is needed to dilute the antibody solution and for washing the microtiter plates.
Tween 20 contains BSA to block free solid phase surface.

4. ! 2. Rehydration open the stopper of antibody and antigen vessels!
pipette 0,5 ml diluted PSB-buffer to the antigen and to the primary and secondary antibodies!
close the vessels with the stopper and shake them for about 2 minutes! (stock solution)
All stock solutions are fifty fold concentrated!

5. 3. Labelling Label two polyethylene bottles with: antigen
primary antibody
secondary antibody

6. Preparations by the pupils 8 teams
The chemicals suffice for three courses with eight teams each! (3 x 160 µl stock solution = 480 µl; 500 µl (total volume)

7. Overview of the tubes green “AG“ antigene 800 µl orange “sAB“
tube color
labelling
contents
quantity
green
“AG“
antigene
800 µl
orange
“sAB“
secondary enzyme linked antibody
brown
“SUB“
substrate
violet
“pAB“
primary antibody
120 µl
blue
“WB“
washing buffer
yellow
- -
Still empty!

8. 1. Preparation of samples P1 – P8
- label the 8 yellow tubes with your group number!
- pipet 60 µl primary antibody solution (pAB, positive
result of the test) or
60 µl washing buffer (WB, negative result of the test) into each of the 8 yellow tubes. Write down your set- ups! (P1 – P8 are serum samples)!
- distribute 1 tube each of to the eight teams! !

9. Complete overview of the tubes (test beginning)
tube colour
labelling
contents
quantity
green
“AG“
antigen
800 µl
orange
“sAB“
secundary enzyme linked antibody
brown
“SUB“
substrate
violet
“pAB“
primary antibody
120 µl
blue
“WB“
washing buffer
yellow
P1-P8
“blood serum” sample 1-8
60 µl each

10. 2. Execution of the test

11. 2.1 Antigen binding on microtiter plate
- label a microtiter plate according to the following picture!
P1 P2 P3 P4 P5 P6 P7 P8
- pipet 50 µl antigen solution (AG, green tube) into each
of the 12 wells!
- incubate for 5 minutes!

12. Antibody detection basic principle
Antigens bind to solid phase

13. 2.2 Washing the microtiter plates
tap the plates on tissues!
pipet 250 µl washing buffer into each well!
Attention! During pipeting the washing buffer should not spill over into other wells!
tap plates on new tissues!

14. Antibody detection basic principle
antigens bind to solid phase
free plastic surface is blocked

15. 2.3 Preparation of controls and samples
pAb
positive control: pipet 50 µl primary antibodies (pAB, violet tube) into 2 with “ + ” labelled wells!
negative control: pipet 50 µl washing buffer (WB, blue tube) into 2 with “ - ” labelled wells! WB
P1 P2 P3 P4 P5 P6 P7 P8

16. 2.3 Preparing of controls and samples
samples1-8: pipet 50 µl „serum samples of the test person“ P1 – P8 into the corresponding labelled 8 wells!
Use a new tip for each well !
P1 P2 P3 P4 P5 P6 P7 P8
incubate for 5 minutes!

17. Antibody detection basic principle
antigens bind to solid phase
free plastic surface is blocked
add serum to be tested for the
presence of antibodies

18. 2.4 Washing the microtiter plates (see 2.2 )
empty
+ 250 µl WB
empty

19. 2.5 Addition of secondary antibody
pipet 50 µl secondary antibody solution (sAB, orange tube) in each of the 12 wells!
incubate for 5 minutes!

20. Antibody detection basic principle
antigens bind to solid phase
free plastic surface is blocked
add serum to be tested for the
presence of antibodies
enzyme-linked antibodies bind to the epitope of eventually present antibody

21. 2.6 Wash microtiter plates twice (see 2.2 )
empty
+ 250 µl WB
empty

22. 2.7 Addition of the substrate
pipet 50 µl substrate solution (SUB, brown tube) into each of the 12 wells!
incubate for 5 minutes!

23. Antibody detection basic principle
antigens bind to solid phase
free plastic surface is blocked
add serum to be tested for the
presence of antibodies
enzyme-linked antibodies bind to the epitope of eventually present antibody
proof of infection by enzymatic reaction

24. 3. Evaluation
Positive samples are blue-coloured, negative samples stay colourless (controls)
Check your test results (P1 – P8) in the team!
e.g.:
P P2 P3 P P5 P6 P7 P8