Equipment & Materials
Determination of IgG Concentration by ELISA

Microbiology 542 -- Eric Martz


The "ACID" solution is sufficiently concentrated to damage whatever it touches (eyes, skin, clothing). Wear goggles and handle with care. Rinse immediately should it get on your skin or clothing. It is phosphoric acid, so it is nontoxic when diluted. (Phosphoric acid is a major ingredient in soft drinks.)

The "SUB" (substrate/peroxide) solution contains organic chemicals and peroxide which are skin irritants and suspected carcinogens. Wear goggles, handle with care, and rinse any spills immediately.


P1000's, P200's, P20's, blue and yellow tips.

100 or so test tubes (half a dozen per pair) for initial unknown dilutions.

ELISA reader programmed for 450 nm, subtraction of average of wells A1-3, program "3-TMB 450".

Quantities are estimated assuming 15 pairs of students. Each student does one plate at a time. Each plate has one standard curve plus 3 unknowns or 4 sets, each set being 5 serial dilutions in triplicate (15 wells), + 6 wells of controls, total 126 wells/pair, about 2,000 wells/class.

30 well holders (Dynatech, 800-336-4543, Removawell Strip Holders, Cat# 011-010-6604, 10/box)

2,000 wells (Dynatech Immulon Removawells, Cat# 011-010-6302, 320 strips/box, 12 wells/strip = 3,840 wells/box.)

"COAT": Need 100 ml distributed as 10 tubes of 10 ml each. Normal rabbit serum diluted 1/5,000 in PBS with sodium azide, 0.1%. It is convenient to add a small amount of phenol red to make the solution colored so it is obvious which wells have received it. Dilute 0.5% phenol red stock 1/1,000 into the COAT.

"PBST": 400 ml distributed as 25 ml/pair in small flasks. PBS (MMBLS has 10X stock made up) with 0.5% Tween 20 detergent. This is for dilution of competitors and must contain Tween to prevent competitors from binding to any residual bare well surface.

"STD": Need 20 ml, distributed as 10 tubes of 2 ml each. Normal rabbit serum diluted 1/2,500 (half-maximal inhibition occurs at about 1/12,000). Based on prerun results, we'll call this 1/2,500 dilution 2.9 µg IgG/ml. (Concentration of IgG to be determined by pre-run ELISA vs. authentic IgG, namely Sigma I-8140 rabbit IgG technical grade, 99% pure by SDS-PAGE. Note that this comes in 0.1% azide, but when diluted about 100-fold the resulting azide concentration or 0.001% is no longer inhibitory. I50 occurs at about 0.5 µg IgG/ml, which is a 22,500-fold dilution of the 11.25 mg/ml stock; thus the azide is no problem.)

Unknowns: samples saved by each pair of students containing starting rabbit serum, dialyzed salt precipitate, DEAE peak, and peaks from Sephadex column run.

"HRPC": Need 100 ml, distributed as 10 tubes of 10 ml each. HRP-conjugated goat anti-rabbit IgG (H+L), 1/5,000 in PBS with 0.5% Tween 20.  The Tween is essential to prevent the conjugate from binding directly to any uncoated plastic. Here again, addition of phenol red is convenient. (No azide! It inactivates the enzyme!) [Jackson Immunoresearch product 111-035-006]

"SUB": Need 100 ml, distributed as 10 tubes of 10 ml each. TMB + peroxide substrate mixture. Mix equal volumes of TMB and peroxide stocks on the same day as the lab. [Kirkegaard and Perry 50-76-01 "TMB Peroxidase Substrate" solution and 50-65-00 "Peroxidase Solution B", 800-638-3167. TMB is 3,3',5,5'-tetramethylbenzidine, 0.4 g/l, in 26% N,N-dimethylformamide. The peroxide is 0.02% in citric acid, pH ?.]

"ACID": Need 100 ml, distributed as 10 tubes of 10 ml each. 1 Molar phosphoric acid. Concentrated stock is 85% with a density of approx. 1.7 g/ml, formula weight 98. Thus for each liter, 98 g of phosphoric acid is needed, which is 68 ml. To make 200 ml, mix 14 ml concentrated phosphoric acid with 200 ml water.