Determination of parallelism and nonparallelism in bioassay dilution curves

Abstract

There is a lack of consensus among investigators who use a variety of immunoassay techniques (e.g., enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay) regarding the protocols for describing and forming standard reference or calibration curves and interpolating serum antibody concentrations. This confounds the issue of detecting the presence or absence of parallelism between standard reference serum and serially diluted serum sample curves. These curves must be parallel to support the assumption that the antibody-binding characteristics are similar enough to allow the determination of antibody levels in the diluted serum sample. There is no universal and widely adopted strategy for assessing parallelism in bioassays, and without an assurance of parallelism, investigators are not able to calculate reliable estimates for antibody concentrations in serum samples. Furthermore, single-point (dilution) serum assays do not provide information related to parallelism and nonparallelism, and this, too, may lead to considerable error when calculating antibody concentrations. When assay methodology, technique, and precision improve to the extent that standard reference serum and serially diluted serum sample curves are fit with little error, standard analysis of variance techniques are overly sensitive to negligible departures from parallelism. We present a series of guidelines that compose a protocol for assessing parallelism between bioassay dilution curves that are applicable to data derived from ELISAs. These criteria should be applicable, with minor modifications, to most immunoassay experimental situations and, most importantly, are not dependent on the mathematical model used to form the standard reference curve. These guidelines have evolved in our laboratories over the past 4 years during the performance of thousands of ELISAs for antibodies to the capsular polysaccharides of Neisseria meningitidis groups A and C and Haemophilus influenzae type b.

Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma

“…The mean percent change in dilution-corrected concentration between contiguous dilutions was 13.6 and 11.1% for PfHRP2 and P. falciparum pLDH, respectively, as determined over 66 independent runs. These data are within the acceptance criteria of +/-20% (38). However, at concentrations close to the ULOQ, the percent change showed an overestimation greater than 20% for both PfHRP2 and P. falciparum pLDH (Table 2).…”

Avidity of Serogroup A Meningococcal IgG Antibodies after Immunization with Different Doses of a Tetravalent A/C/Y/W135 Polysaccharide Vaccine

“…IgG antibody concentrations were calculated using a 4-parameter logistic curve-fitting analysis [32] against the standard reference curve. Multiple data points obtained from dilutions that yielded OD values in the linear portion of the curve were averaged, and IgG geometric mean concentrations (GMC) were calculated from wells where no thiocyanate was added.…”

Accurate measurement of extended half‐life and unmodified factor VIII low levels with one‐stage FVIII assays is dependent on the matrix of calibration curves

“…The samples and the calibration curves were considered parallel over the range of the assay when the intra-assay CV was <20%. 10 Three automated dilution methods were compared. In the first method, the samples were prediluted two-and fourfold with FVIIIdeficient plasma (Siemens) as recommended by Cinotti et al 6 In the second method, the samples were prediluted with diluent buffer.…”