Fig. 1: Mass spectrometry enabled conversion to absolute levels of ELISA antibodies—MASCALE—method outline.

Step 1: Identification and synthesis of proteotypic peptide(s) representative of human IgG. Peptides selected are unique to human IgG, have favorable mass spectrometry characteristics and are detectable from tryptic digests. Step 2: Construction of calibration curve(s) by mass spectrometry for peptide(s) selected in Step 1. Peptide calibration curves establish the linear relationship between peptide quantity and mass spectrometric signal expressed as peak area ratio. Steps 1 and 2 apply to all ELISAs where human IgG is to be quantified via the MASCALE method. Step 3: Binding of ELISA reference standard samples to coated antigen. The binding of the reference standard dilution curve to coated antigen is performed using the same experimental procedure and format as done for sample analysis, followed by a wash step to remove unbound antibodies and other serum components. Step 4: Sample denaturation and tryptic digestion. Antigen-antibody complexes are denatured and digested releasing target peptide(s). Step 5: Quantitation of target peptide(s) by mass spectrometry. Tryptic digests are subjected to targeted quantitative mass spectrometry and peptide quantity in the sample is determined using the peptide calibration curve generated in Step 2. Step 6: Generation of MASCALE conversion formula. The linear relationship between total log₁₀ IgG (peptide) quantity per ELISA well and assigned log₁₀ arbitrary unit is established. Step 7: Comparison of immune responses. Reportable values generated from samples assessed in the ELISA are converted from concentrations in arbitrary units to absolute units after applying the corresponding conversion formula from Step 6 and adjusting for sample dilution scheme of the respective ELISA.