Colorimetric SEAP Assay Protocol (Roden/Bossis)

Purpose:  An alternative method for detection secreted alkaline phosphatase.  Although it is somewhat less sensitive than the standard chemiluminescent method described in the main papillomavirus neutralization assay protocol, colorimetric detection is much less expensive and can be performed using a conventional ELISA plate spectrophotometer.

 Reagents

1. Diethanolamine 99% - Acros Organics-Cat#113920025
2. P-Nitrophenyl phosphate tablets (PNP)- Sigma, Cat# N-2765
3. CHAPS, Sigma Ultra, Cat# C5070-1G

Preparation of working solutions

1. Dilute 99% Diethanolamine (9.5M) to 2M with water. We usually make like a 1 liter batch.  Cover with aluminum foil and keep at 4°C.  It is not necessary to adjust the pH.  To the 2M diethanolamine solution add 1mM MgCl₂and 0.5mM ZnCl₂.  0.5M ZnCl₂stock solution may be cloudy so resuspend before using.
2. To make the coloring substrate, we take 20ml of the diethanolamine/Mg/Zn solution (above), then add 1 tablet of PNP and leave at room temperature protected from light for 20 mins or so with occasional mixing.
3. Make 15%  CHAPS stock in water and from there make 0.05% CHAPS in PBS as the working solution.

Procedure

1. To the 96 well plate, add 20ul of 0.05% CHAPS in PBS.
2. To this add 40ul of supernatant from the neutralization plate (virus+antibody+cells).  Although it is not strictly necessary to heat-inactivate the supernatants prior to testing (see Great Escape kit protocol for details), heat inactivation may reduce the background somewhat.
3. Add 200ul of coloring substrate per well. Incubate at room temperature in the dark for 2 to 4 hours. Read absorbance at 405nm.

Notes:  the signal to noise ratio for the colorimetric method is 100- to 1000-fold lower than for the standard Great Escape SEAP Chemiluminescence (BD Clontech) method.  Usually three to ten fold more pseudovirions must be used in the colorimetric method compared to the chemiluminescent assay.  A sufficient amount of pseudovirus should be used to generate a signal five to fifteen fold over the background.  If a pseudovirus stock with a good particle to infectivity ratio is used, the assay will still be operating under conditions of antibody excess and hence the 50% neutralization cutoffs will be similar for both detection methods.  For a review of the important concept of antibody excess see Klasse (2002) JGV 83:2091.