ELISA Antibody Quantification from Dried Blood Spots

Microtitre plates (Immulon 4HBX, Thermo) were coated with recombinant MSP-1₁₉.GST (to which antibodies are predominantly of the IgG1 subclass [36 (link)]) or MSP-2.GST (to which antibodies are predominantly IgG3 [36 (link)]) and blocked with 1% (w/v) skimmed milk powder. Samples were assayed as described previously[36 (link),37 (link)] except that coating antigens, test samples and secondary antibody conjugate were each added in a total volume of 50 μl per well. Ten microliters of the antibody-containing eluate of each spot were added to individual wells of the coated and blocked microtitre plate together with 40 μl blocking buffer to give a final concentration of 1:1,000 with respect to the corresponding plasma sample. Each plate included a five-fold dilution series (1:50 to 1:156,250 final dilutions) of a standard African hyperimmune plasma pool. Bound antibodies were detected with either rabbit anti-human-IgG -HRP (Dako, Ely, UK), or sheep-anti-human IgG1 or IgG3-HRPconjugates (The Binding Site, Birmigham, UK) secondary antibodies and developed with o-phenylenediamine-H₂O₂.
A titration curve was fitted to the ODs obtained for the standard plasma dilutions by least squares minimisation using a three variable sigmoid model and the solver add-in in Excel (Microsoft), assuming an arbitrary value of 1000 Units/ml of antibody against each antigen in the standard pool. OD values for the spot extracts were converted to units/ml using this fitted curve.
Recoveries for blood spots were estimated as follows (full details in Additional file 1): serum or plasma ODs were converted to concentrations as above, the concentrations were multiplied by a recovery factor and then converted back to 'corrected' ODs – the ODs which would have been obtained if the serum or plasma had been more dilute. The value of the recovery factor was then optimized by weighted least squares minimisation comparing the actual ODs for the blood spots and the corrected OD values for serum or plasma, using the solver add-in in Excel™.