HPV 16 Capture ELISA
Materials
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H16.V5 ascites
HPV16 virus like particles(VLPs) purified from Sf9 cells
Carbonate buffer (0.1M pH 9.5)
Development buffer (0.1M carbonate)
Goat serum
Alkaline Phosphatase linked goat-anti-human IgG (Perkin Elmer,
NEF801001EA)
Phosphate buffered saline (PBS)
Tween-20 (Fisher Scientific)
96 well plate (Immulon 2, Fisher Scientific)
Sigma 104 Phosphatase substrate (Paranitrophenolphosphate)
Plate Reader (BioTech Instuments Inc. ELx808)
The day before:
1) Coat the plate with 1:2500 dilution of H16.V5 ascites in carbonate
buffer, using 50 l/well. Incubate overnight at 40C.
2) Dilute human serum (1:100) in cluster tubes using 5 l into 500 l
blocking buffer (PBS, Tween 0.05%, goat serum (5%)). Incubate at 40C
overnight.
Day of assay:
3) Wash the plates 4X with PBS and remove excess liquid by rapping on
paper towels.
4) Add 200 l blocking buffer Incubate at rt for 2 hr or at 40C for as long
as several days.
5) Dilute VLPs (purified from SF9 cells) in blocking buffer at a
concentration determined by optimization experiments (approx.
1:100). Shake out the plate and add 50 l of diluted VLPs to half of the
wells and blocking reagent without VLPs to the rest of the plate.
Incubate at rt0C for 1 hr on a plate shaker (medium-low).
6) Wash the plate and add diluted human sera in blocking reagent (50 l)
to 3 wells with VLPs and 3 without. Incubate at rt0C for 1 hr on a plate
shaker (medium).
7) Wash the plates and add 50 l goat-antihuman IgG-alkaline
phosphatase diluted in blocking reagent at 1:1000 to all of the wells.
Incubate at rt0C for 1 hr on a plate shaker (medium).
8) Wash the plates. Add phosphatase substrate (4.3 mg/ml, Sigma 104
substrate) in reaction buffer (0.1M CO3, 10mM MgCl2, pH 9.5) to each
well (100 l/well). Incubate at rt for 30 min.
9) Read immediately at 405 nm on microtiter plate reader (alternatively,
stop the reaction by the addition of 50 l/well of 1.5 M NaOH and then
read plate).
10)The ELISA values are calculated as the average of the natural log of
the wells with antigen minus the average of the natural log of the wells
without antigen. A coefficient of variation of greater than 20% among
the triplicate values with antigen or the triplicate values without
antigen indicates that the serum should be retested.
11)When screening a cutoff point should be calculated as 2 SD above the
mean women with no sexual partners who tested negative for HPV
DNA by PCR. Using this method the cutpoint is generally between 0.2
and 0.3.