TEACHER GUIDE FOR SCBC- ELISA KIT

Kit Non-consumables (return to Miramar College):

10 P-200 micropipettes
10 Boxes of P-200 tips
10 Microfuge racks
20 Squirt bottles for 1x PBS
10 Pans for washing

10 Waste buckets for tips

1L 1x PBS bottle for dilution
Icebox
10x PBS Buffer (1 L)

Kit Consumables: Dispose of properly, see instructions below or return any unused consumables:
Positive control tubes (1 mL)
Secondary antibody tubes (2 mL)
Negative control tubes (1 mL)
TMB Substrate tubes (2 mL)
Patient A serum tubes (1 mL)
ELISA strips (1 per student)
Patient B serum tubes (1 mL)
ELISA Lab Preparation:
The ELISA Kit is designed for students working individually in groups of 4. Each student will prepare
their own 8-well ELISA strip but they will share the reagents and equipment amongst their group.
Divide the number of students by 4. This will be the number of required set ups (X).
You will need the following consumable reagents: X Positive Control tubes, X Negative Control tubes,
X Patient A tubes, X Patient B tubes, 2X Secondary Antibody tubes and 2X Substrate tubes and 4X
Coated Strips for each group.
Prior to the lab, the 10x PBS will need to be diluted to 1x PBS:
1.
2.
3.
4.

To the included 1L bottle, add 100 mL of 10x PBS

Add 900 mL of distilled water (if unavailable, tap water can be used instead)
Transfer the 1x PBS to the squirt bottles.
This will need to be done several times as the students will use significant amounts of 1x
PBS for washing.

Storage of Reagents:
a) The microfuge tubes with the reagents will be transported on ice. Positive, Negative, Patient
A, Patient B, and the Secondary Antibody samples should then be placed in a -20C freezer
until use.
b) The ELISA strips and the TMB substrate should be placed in a 4C refrigerator until use.
c) 1X PBS (Buffer) store at room temperature

This simulated HIV ELISA was developed with the support of the Southern CA Biotechnology Center @ Miramar College

Page 1 of 2

Day of ELISA Lab:

1. Lab Station Setup (for each group of 4):

4 ELISA strips (1 per student)

1 P200 micropipette and tips
1 microfuge rack

1 waste bucket for tips

1 pan for washing
2 squirt bottles for 1X PBS Buffer

2. Consumable Reagents in Microfuge Tubes (Keep frozen until use). Immediately before the lab period,
remove the samples and allow them to thaw. Mix the tube contents by gently inverting.

1 positive control

1 Patient B serum

1 negative control

2 Secondary antibody

1 Patient A serum

2 TMB substrate

If the blue color does not fully develop during the class period, the ELISA strips can be kept overnight at room
temperature for later observation.
CAUTION: TMB is a known toxic!
1. Handle carefully. If the solution comes into contact with skin, wash the area immediately.
2. Discard ELISA strips in container provided.
Post Lab Clean-up

All students should wash their hands immediately after using the TMB.

Discard all of the tubes in the trash.

Rinse the pans and squirt bottles.

Please, check the kit contents prior to returning the kit to be sure all of the pipettors and supplies are
there. Thank you!

This simulated HIV ELISA was developed with the support of the Southern CA Biotechnology Center @ Miramar College

Page 2 of 2