Qualitative ELISA (Time: 20 minutes)

1

Entered testing area wearing closed-toed shoes, then donned proper PPE: glasses/safety glasses/goggles and gloves (lab coat is optional)

2

Labeled a 12-well microplate strip:

● the first three wells with + for the positive controls

● the next three wells with a – for the negative control

● the next three wells with an S to indicate the sample.

3

Transferred 50 µl of purified antigen (AG) into each well.

*Judge verified competitor added AG to all 9 wells and (i) used a 20-200 µl micropipet set to 50 µl, (ii) used a proper pipet tip, and (iii) transferred liquid without air bubbles or losing sample in the transfer process

4

Incubated the samples at room temperature for 2 min

5

Followed wash protocol:

a. Tipped each microplate strip upside-down onto a short stack of paper towels and gently tapped strip a few times to drain the wells while making sure to avoid splashing sample back into wells

b. Discarded the wet paper towels.

c. Used a transfer pipet (same transfer pipet can be used) to fill each well with wash buffer, taking care not to touch the well or spill the buffer into neighboring wells

6

Repeated step #5

7

Transferred 50 µl of the positive control (+) into the three + wells.

*Judge verified competitor added + to only the first 3 wells labeled + and (i) used a 20-200 µl micropipet set to 50 µl, (ii) used a proper pipet tip, and (iii) transferred liquid without air bubbles or losing sample in the transfer process

8

Transferred 50 µl of the negative control (-) into the three – wells.

*Judge verified competitor added - to only the 3 wells labeled - and (i) used a 20-200 µl micropipet set to 50 µl, (ii) used a proper pipet tip, and (iii) transferred liquid without air bubbles or losing sample in the transfer process

9

Transferred 50 µl of the sample (S) into the corresponding three wells.

*Judge verified competitor added S to only the 3 wells labeled S and (i) used a 20-200 µl micropipet set to 50 µl, (ii) used a proper pipet tip, and (iii) transferred liquid without air bubbles or losing sample in the transfer process.

10

Incubated the samples at room temperature for 2 minutes

11

Performed wash protocol (step 5) two times

12

Transferred 50 µl of enzyme-linked antibody (ELA) into each well.

*Judge verified competitor added ELA to all 9 wells and (i) used a 20-200 µl micropipet set to 50 µl, (ii) used a proper pipet tip, and (iii) transferred liquid without air bubbles or losing sample in the transfer process

13

Incubated the samples at room temperature for 2 minutes.

14

Performed wash protocol (step 5) three times

15

Transferred 50 µl of enzyme substrate (SUB) into each well.

*Judge verified competitor added SUB to all 9 wells and (i) used a 20-200 µl micropipet set to 50 µl, (ii) used a proper pipet tip, and (iii) transferred liquid without air bubbles or losing sample in the transfer process

16

16. Cleaned work area:

a. Disposed of used pipet tips, transfer pipets, and paper towels into waste receptacle, cleaned area of any spilled liquid, returned micropipette to rack (if available).

b. Cleaned work area with surface disinfectant.

c. Removed PPE.

d. Washed hands or used alcohol-based hand-rub for hand hygiene. *Judge verified steps 16a-d were performed in the order written here.

17

17. Observed and reported results.

*Judge verified (+) and S wells were blue, (-) was colorless; competitor confirmed sample was positive