| | Re: quantification of poliovirus in solution
We propose a indirect immune-enzymatic assay method (sandwich) on microplate (MPT), which can be employed for direct search and identification of the poliovirus, both from cell culture and stool or other biological fluids, inclusive the water.
Sample extracted from specimens (culture or biological fluids) were dispense in coated MTP well with polyclonal antibody versus poliovirus (1,2 and 3). Possible intact virus in sample, form immune complex with polyclonal antibody absorbed on surface well. After wash, were dispensed first antibody from mouse against structural protein VP1 present on every enteroviruses. The formed sandwich were identified by second antibody from goat versus mouse IgG conjugated with alkaline phospatase. State of the reaction, were detected through specific substrate-stopping unit, realized by my self, presented at the Eurospital firm – Trieste (Italy), and Abs of the coloured reaction, read at 510 nm, were calculated employing calibrated standard curve which use a mix of the poliovirus types.
Coated plate : distribute, in plate BIOHIT- high bond, 100 µl in coating solution carbonate/bicarbonate buffer 0.1 M at pH 9.6, containing 15.0 µg/ml polyclonal antiserum anti intact polioviruses type 1,2 and 3, IgG isotypic from bovine or horse, and incubate at room temperature for 16 hrs. Wash the wells, twice with washing solution, and therefore distribute 200 µl of blocking solution (5% fetal calf serum [FCS], 0.5% Tween-20 in PBS 0.01 M at pH 7.2 contained 0.01 % thimerosal) and incubate for 1 hr. at 37 °C. Wash one time the plate with washing solution, and preserve dry at 4 °C in plastic bag containing a drying.
Diluting specimen solution : sodium phosphate buffer 0.1 M at pH 7.2 contained 0.1 % sucrose, or trehalose or arginine hydrochloride to stabilize antigenic surface of the virus, and 0.025 % of formaldehyde for virus inactivation.
Mix polioviruses antigen : mix of the polioviruses type 1,2, and 3 (Chemicon – Temicula CA – USA) were diluted in PBS (0.1 M at pH 7.2 contained 0.1 % sucrose, or trehalose or arginine hydrochloride, and 0.01 % thimerosal as preservative) for obtained different concentrations of the viral proteins 0.03, 3.0 and 30 μg/ml, after formaldehyde inactivation, and store at -20 °C.
Washing solution : 0.05% Tween-20 in PBS 0.01 M at pH 7.2, contained 0.01 % thimerosal
Fist antibody : mouse monoclonal antibody versus VP1 in blocking solution opportunely diluted.
Second antibody : antibody anti mouse IgG from goat conjugated with alkaline phosphatase (Sigma) in blocking solution, opportunely diluted.
Substrate : dissolve in 100 ml of carbonate buffer 0.05 M at pH 10.15, 0.215 g disodium phenylphosphate, 0.09 g 4-aminoantipyrine.
Stopping solution : dissolve in 200 ml dH2O, 2.6 g boric acid, and 0.38 g of potassium ferricyanide.
Cell culture : centrifuge cellular suspension at 300 rpm for 1 h, and mix surnatant with equal volume of diethyl ether at 4 °C, and incubate for 2 hrs in ice bath for to separate organic from aqueous phase. The aqueous phase were treated for to remove, under empty, ether residue, dilute 1:4 in 15 % (v/v) Tween-20 in PBS, and 100 µl of diluted were add to MTP wells coated polyclonal antiserum.
Stool specimen : collection stool specimen in clean and dry containers, without detergents. Necessary quantity of feces for the test is 4.0 - 8.0 gr. or 3 - 5 ml, and if specimen are not examined within 48 hrs these they to be preserved at -20 °C. A 10 % (wt/v) fecal suspension was made in diluting specimen solution, and vortex mix. The suspension was allowed to stand for 10 min. at 37 °C, centrifuge at 10900 rpm for 5 min., and then 100 µl of clear supernatant was added to MTP wells coated polyclonal antiserum.
Together at duplicate processed sample, distribute, as the sample, 100 µl of different concentrations positive standards antigen, for to realize standard curve, and incubate for 16 hrs at 4 °C or at 37 °C for 1-2 hrs. Wash the plate 4 times with washing solution, and therefore distribute 100 µl of First antibody and incubate for 1 h at 37 °C. Wash as above and dispense 100 µl of Second antibody and incubate for 1 h at 37 °C. Wash again as above, and add 100 µl of the substrate and incubate at 37 °C for 30 min. Stop the reaction dispensing 100 µl of the stopping solution and read Abs at 510 nm versus 630 nm reference. Calculate using the standard curve.