Biotin interference in susceptible CanAg Ca242 immunoassays and elimination method

Published: 20-10-2019| Version 2 | DOI: 10.17632/5b63ppnhz4.2
Contributors:
Hui Huang,
Lixian Huang,
Haibiao Lin

Description

Biotin as dietary supplement or therapy may lead to analytical interference in biotin-streptavidin immunoassay.When excess biotin is present in the specimen, the biotin molecules will saturate the streptavidin-binding sites, thus preventing the antibody-analyte sandwich from binding with the streptavidin-coated solid phase to generate assay signal after the washing phase. In order to investigate the biotin interference in CanAg Ca242, we compare the difference of experimental samples(spiked biotin solution) and control samples(spiked PBS solution). Pure biotin was dissolved in 0.01 M NaOH and stored at -20°C for reservation. Biotin stock diluted with PBS into working solutions with nine concentration gradients that were then spiked into three different baseline Ca242 samples to achieve the indicated final concentration of biotin (1000,500,250,125,62.5,31.25,15.63,7.81) ng/mL. Our data(Fig .1) shows three different concentration across the analysis of measurement ranges of Ca242 had significant interference from biotin, but the magnitude of the interference was variable at different concentration of biotin. The higher the biotin concentration, the more significant the reduction of Ca242 .The data from table 1 shows that the relative deviation between the baseline level and the biotin-spiked samples, the relative deviation greater than 10% was considered a significant difference according to the indoor quality target. The Ca242-Low level, cutoff level, and high level appeared significant difference respectively at biotin concentration of 15.63 ng/ml, 31.25 ng/ml and 62.5 ng/ml. At the biotin concentration of 250 ng/ml, the relative deviation was more than 99 %(Table 1). Now that we have confirmed that biotin interferes with Ca242 detection, the next step is to find a feasible solution. Based on the strong affinity to streptavidin, we used streptavidin microparticles to “pre-bound” biotin. The samples were absorbed with magnetic microparticles coated with streptavidin. This reagent included in the Cobas® assays kits supplied by Roche and the streptavidin concentration is 0.72 mg/mL. After pretreatment, the results had recovered firmly to the baseline levels(Table 2).Meanwhile, we performed parallel experiments on the Mindray CL2000i. The data shows that biotin interference does not appear in the Mindray CL2000i intact Ca242 assay, which does not use the streptavidin/biotin method (Fig. 2).

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Materials and Methods 1Preparation of biotin samples Pure biotin was dissolved in 0.01 M NaOH and stored at -20°C for reservation. Biotin stock diluted with PBS into working solutions with nine concentration gradients that were then spiked into three different baseline Ca242 samples to achieve the indicated final concentration of biotin (1000,500,250,125,62.5,31.25,15.63,7.81) ng/mL. For controls, sera samples were spiked with an equal volume of PBS to account for possible matrix effects. The volume of the spike was 10% of the final volume. 2 Biotin neutralization method The samples were absorbed with magnetic microparticles coated with streptavidin. This reagent included in the Cobas® assays kits supplied by Roche and the streptavidin concentration is 0.72 mg/mL. The microparticles were isolated out of Roche M reagent solution, then distribute to the polystyrene tubes and let air dry in 4℃ refrigerator that will not dilute the specimen. Samples with biotin concentration at 1000 ng/mL will add to streptavidin microparticles tubes, and the final concentration of streptavidin microparticles in all spiked sera samples were 21.6mg/mL. The streptavidin microparticles tubes were incubating with serum sample and let one hour shaking at room temperature (300 rpm), and centrifuge for 20 min at 3000 rpm/min then reanalyzed. 3 Measurement of Ca242 Serum samples measured on CanAg Ca242 ELISA kit and Mindray Ca242 platform.