Design and production of recombinant antigen as biomarkers of human and animal infectious diseases

Quality

Rekom Biotech is committed to ensure the highest quality level in the design and production of recombinant antigens focused on human and animal infectious diseases for the IVD manufacturing industry.

Rekom Biotech products are designed, developed, produced and distributed according to our Quality Management System that is certified by ISO 9001 standards. Rekom recombinant antigens are always produced according to Standard Operating Procedures (SOPs) and undergo rigorous quality controls in our laboratories.

Rekom Biotech is authorised to work with genetic modified organisms (GMO), with the license number A/ES/14/I- 10, issued by National Biosafety Commission.

Rekom Biotech is registered as a Innovative SME, valid until Dec 31st 2018.

Each lot is subjected to various analyses:

  • Concentration detection by spectrophotometry

    The measurement of the protein concentration is performed with the theoretical extinction coefficient of the recombinant protein obtained from Gill and vonHippel, 1989.

    For proteins which do not contain any Trp residues, experience shows that this could result in more than 10% error in the computed extinction coefficient. Therefore, we measure the protein concentration by using the colorimetric assay based on the interaction between Coomassie brilliant blue and the arginine and aromatic residues (Bradford Method) and its maximum absorption shifts from 470 nm to 595 nm (Bradford, 1976).

  • Purity determination by SDS-PAGE

  • Immunological analyses in ELISA or Western Blot assays.

    For futher information, take a look at our technical report "Tritation experiments"

    Tritation curve by an ELISA assay

    In this plot, the optical density at 450/620 nm for positive (blue) and negative (gray) IgG sera are compared for each concentration of the recombinant antigen. An appropriate statistical test of significance for the comparison of means between both groups, the Welch's test, is employed. Eligible concentrations for the use of the antigen should present statistically significant differences between positive and negative sera. This happens when the intervals at the top do not overlap and, equivalently, when the p-value at the bottom is below 0.05. In the present figure, all p-values are below 0.05 and thus the intervals do not overlap. Therefore, any of the showed concentrations can be used to distinguish between positive and negative sera.

  • Biotin conjugation.

    We conjugate our antigens with biotin as an internal quality control for our antigens. We perform a spectroscopic estimation with HABA (4´-hydroxyazobenzene-2-carboxylic acid) of the mole-to-mole ratio of biotin to protein, also a western blot assay with streptavidin (A), and several ELISA assays (indirect ELISA assay in streptavidin coated microtiter plates, capture ELISA assay with the biotinylated recombinant antigen as detector and double-antigen-sandwich ELISA assay (B)).

    Western Blot assay with streptavidin

    (A) Western Blot assay with streptavidin

    Double antigen sandwich ELISA assay

    (B) Double antigen sandwich ELISA assay

  • Peroxidase (HRP) conjugation.

    We also conjugate our antigens with peroxidase as an internal quality control. We perform a double antigen sandwich ELISA assay (C) and a capture ELISA assay by using a commercial test (D).

    Double antigen sandwich ELISA assay

    (C) A double antigen sandwich ELISA assay (DAS) performed with positive and negative CMV IgM specimen sera pre-validated with the ELISA capture IgM VIDAS.

    Capture ELISA assay

    (D) A capture ELISA assay performed with two different dilutions of the Rekom pp150-HRP in a reference commercial test (CMV-IgM-eLA test PKS medac).