TEACHER GUIDE FOR ELISA REAGENT PREPARATION

I. Antibody Preparation
To prepare antibodies for ~50 groups (200 students), follow the steps below. Prepare the solutions on
ice. You will need 3 different colored tubes, one for each primary antibody and a clear tube for the
secondary antibody.
Step 1: 1% PBS milk (500 ml)
1. Dissolve 5.0 g nonfat dry milk (any grocery store brand) in 500 ml 1x PBS.
2. Prepare 50 1ml aliquots in colored (choose a color) microfuge tubes (negative control).
Step 2: Primary (1) Antibody (Sigma: Anti-Bovine Serum Albumin B-1520) (1/10,000 dilution) (130
ml)
1. Add 13.0 l Rabbit anti-BSA antibody to 130 ml of 1% PBS milk.
2. Prepare the following aliquots:
a. 50 1ml aliquots in colored (choose a second color) microfuge tubes (Positive control)
b. 50 1ml aliquots in colored (choose a third color) microfuge tubes (Patient A serum)
3. Use 25 ml of this antibody dilution to prepare the 1/20,000 dilution for Patient B tubes.
Step 3: Primary (1) Antibody (1/20,000 dilution) (50 ml)
1. Add 25 ml 1 Antibody (1/10,000 dilution) to 25 ml of 1% PBS milk.
2. Prepare 50 1ml aliquots in colored (choose fourth color) microfuge tubes (Patient B serum).
Step 4: Secondary (2) Antibody (Sigma: A-6154) Anti Rabbit IgG(Whole molecule) peroxidase
Conjugate (1/500 or 1/1,000 dilution) (200 ml)
1. Add 200 l Goat anti-rabbit IgG antibody to 200ml of 1% PBS milk. (for 1:1000, 100 l for 1:500)
2. Prepare ~100 2ml aliquots in 2ml clear microfuge tubes (2 Ab)
Step 5: Store the aliquots into separate ziplock bags, 10 per baggie. Label the bags with the contents,
date prepared, who prepared them and ELISA Kit. Store tubes in the -20C freezer.

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

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II. ELISA Strip Preparation:

For a class of ~ 40 students, prepare 4 ELISA plates (48 8-well strips total) according to the steps below.
If more strips are needed, adjust the following volumes accordingly. If there are excess strips, clearly
label them with the date prepared, who prepared them and store them in the 4C refrigerator.

Step 1: Prepare 1% Bovine Serum Albumin (BSA: Sigma A2153-50G) (20 ml per plate)
a) Dissolve 0.8 g BSA in 80 ml Carbonate Buffer (pH 9.5).
Step 2: Coat the antigen (BSA) to the ELISA strips
a) Pipette 200 l 1% BSA into the eight wells of each removable strip of the ELISA plates.
b) Incubate the plates at room temperature for 1 hour.
Step 3: Prepare 1x PBS (wash solution)
a) Add 100 ml 10x PBS to 900 ml distilled water.
b) Transfer to a squirt bottle.
Step 4: Prepare 1% PBS milk (blocking solution)
a) Dissolve 5g of nonfat dry milk in 500 ml 1x PBS.
b) Transfer to a squirt bottle.
Step 5: Coat the antigen (BSA) to the ELISA strips
c) Pipette 200 l 1% BSA into the eight wells of each removable strip of the ELISA plates.
d) Incubate the plates at room temperature for 1 hour.
Step 6: Block non-specific binding of antibodies with 1% PBS milk
a) Empty the contents of the plates into the sink by turning upside-down and flicking until
no more liquid leaves the plates.
b) Fill each well to the top with 1% PBS milk using a squirt bottle. Let sit for at least 5 min.
c) Empty the contents again by turning upside-down and flicking. Blot on a paper towel
before washing.
Step 7: Wash ELISA strips with 1x PBS
a) Fill each well to the top with 1x PBS using a squirt bottle.
b) Empty the contents into the sink by turning upside-down and flicking. Blot onto a paper
towel until no more liquid leaves the wells. Repeat this wash step 3 more times.
c) Allow plates to air dry.
Step 8: Storage of ELISA strips
a) With a sharpie, add a black mark to one side of each strip. (This mark will enable the
student to determine what well will be well #1).
b) Cover the plates of strips with a plate sealer.
c) Label the plates with the date prepared, who prepared them, and ELISA Kit.
d) Store in the 4C refrigerator until needed.
This simulated HIV ELISA was developed with the support of the Southern CA Biotechnology Center @ Miramar College

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III. SOP for Carbonate Buffer, pH 9.5 (1 L)

Step 1: Dissolve the following in approximately 900 ml of distilled water:
15mM Na2CO3 (FW 106.0 g/mole)
35mM NaHCO3 (FW 84.01 g/mole)
Step 2: Adjust the pH to 9.5 if necessary.
Step 3: Bring the final volume to 1L with distilled water.

IV. SOP for 10x Phosphate Buffered Saline (PBS), pH 7.4 (1 L)

Step 1: Dissolve the following in approximately 800 ml of distilled water:
0.038M NaH2PO4 H2O (FW=137.99 g/mole)
0.162M Na2HPO4 7H2O (FW=268.07 g/mole)
1.5M NaCl (FW 58.44 g/mole)
Step 2: Adjust the pH to 7.4 with HCl or NaOH as needed.
Step 3: Bring the final volume to 1 L with distilled water.

V. SOP for TMB Substrate Preparation

TMB substrate can be bought (see supply list on p. 4,). *CAUTION: TMB is a known toxin! Handle
carefully. If the solution comes into contact with skin, wash the area immediately and thoroughly.
Each group of 4 students will need ~4 ml of TMB. Therefore each group will be given 2 amber
microfuge tubes with 2 ml TMB. For a class of 40 students, prepare 44, 2ml aliquots in amber tubes.
The use of amber/brown tubes is essential since TMB is light-sensitive.
ELISA Reagent Ordering Information
Item

Company

Catalog #

Amount

Anti-Bovine Albumin antibody produced in Rabbit

Anti-Rabbit IgG - Peroxidase antibody produced in
Goat

Sigma-Aldrich

B1520

2 ml

A6154

1 ml

3,3,5,5-Tetramethylbenzidine (TMB Substrate)

Corning 96-well stripwell plates
Elisa Strips Costar EIA/RIA Strip Plates Hi Bind
BSA
Milk, any nonfat dried milk
Amber and Clear tubes

Sigma-Aldrich
Cell Signaling
Technology, Inc.
Sigma-Aldrich
Corning
Sigma-Aldrich
Any vendor
Any vendor

7004L
CLS2592
2580
A-2153

500mL
25 each
100/case
100g

4 Types of colored tubes

Reagents for Carbonate Buffer, see recipe on p.3
Reagents for Phosphate Buffer, see recipe on p.3

Any vendor
Any vendor
Any vendor

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

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

Page 4 of 4