Optimization of Analytical Procedures Reduces Variability of Results

Towards this end, investigators at the Barcelona Institute for Global Health (Spain) analyzed the effect of various procedural conditions on the variability of an in-house IgG multiplex assay designed to detect different types and classes of antibodies against multiple P. falciparum antigens.

The assay comprised different parasite antigens coupled to microspheres. These beads were tested to determine if they were recognized by plasma samples from individuals who may have been exposed to malaria. The assay was designed to detect different types and classes of antibodies against multiple P. falciparum antigens, in a sensitive and specific manner. The simple multiplex assay was found to be highly reproducible not only between experiments but also between operators and laboratories.

The investigators identified the key factors requiring optimization to reduce variability of the assay. Then, they used the assay to better characterize the antibody profile of the anti-malaria human plasma pool provided by the WHO as reference reagent.

IgG levels against seven P. falciparum antigens with heterogeneous immunogenicities were measured in test samples, in a positive control, and in blanks. Variability associated with each combination of conditions and their interactions was assessed, and the minimum number of samples and blank replicates required to achieve good replicability was determined. Results showed that antigen immunogenicity and sample seroreactivity defined the optimal dilution for assessing the effect of assay conditions on variability. Furthermore, unique antigen-bead coupling, pre-dilution of samples, incubation at 22 degrees Celsius, and an automated washing procedure reduced variability.

"These simple and reproducible multiplex protocols will allow us to analyze in detail natural and vaccine-induced antibody responses to large panels of P. falciparum antigens, and elucidate correlates of malaria protection," said senior author Dr. Carlota Dobaño, an associate research professor at the Barcelona Institute for Global Health. "Furthermore, the assay is highly versatile and can be adapted to study responses to antigens from other microbes or vaccines. We are currently collaborating with several groups outside the malaria field."

The study was published in the July 2, 2018, online edition of the journal PLOS One.

Related Links:
Barcelona Institute for Global Health