Quantitative and equipment-free paper-based agglutination assay of bacterial cells.

Background: point-of-care (POC) tests are useful for bedside/home applications, emergencies, frequent follow-ups, and resource-limited areas. Limited quantitative and equipment-free POC assays have been reported. This study aims to develop, validate, and apply a simple, quantitative, paper-based POC assay. Methods: wax-channeled paper treated with specific anti-Brucella and anti-Salmonella antibodies was used for distance-based chromatographic elution of stained bacterial cell agglutinations. Results: a qualitative paper-based agglutination POC test was developed using color intensity, tail appearance, and "+/-" signs that clearly distinguish the positive and negative results. The optimization of the test for paper type, microfluidic channel design, antibody and bacterial cell concentrations, and elution methods was carried out. Quantitative assay transformation was successfully developed using the color intensity of the original reaction zone, intensity of elution tail, and distance-based migration that correspond to bacterial agglutination size. The migration distance of eluted bacterial agglutination bands corresponds to the target concentration with good linearity and minimal variability. Reporting of colored band migration with numbers using microfluidic patterns was used to enhance non-technical end-user applications. A distance-based POC assay prototype was then successfully used for the accurate detection of known and unknown samples in comparison with standard assays. Conclusions: the migration distance of an eluted stained bacterial agglutination correlated with anti-bacterial antibody concentrations. A simple, cheap, quantitative, and equipment-free paper-based POC assay of bacterial cell agglutination was developed. This test can be used for simple "+/-" results, thermometer-like quantification, or text reporting with numbers corresponding to target concentrations. The assay has extended applications to different human disease biomarkers.

A synthetic Protein G adsorbent based on the multi-component Ugi reaction for the purification of mammalian immunoglobulins.

Numerous efforts have been devoted to develop synthetic affinity ligands mimicking natural immunoglobulin-binding proteins, such as Proteins A and L, in order to overcome intrinsic drawbacks involving their high cost and acidic pH elution. However, few reports have focused on a Protein G mimic. This work describes the use of the solid phase multi-component Ugi reaction to generate a low cost, rationally designed, affinity ligand to mimic Protein G for the purification of mammalian immunoglobulins, including the heavy-chain only camelid IgGs, with effective elution at neutral pH. An aldehyde-functionalised Sepharose™ resin constituted one component (aldehyde) of the four-component Ugi reaction, whilst the other three components (a primary or secondary amine, a carboxylic acid and an isonitrile) were varied to generate a tri-substituted Ugi scaffold, with a wide range of functionality, suitable for mimicking peptides for immunoglobulin purification. Ligand A2C11I1 was designed to mimic Asn35 and Trp43 of Protein G (PDB: 1FCC) and in silico docking into the Fc domain showed a key binding interface closely resembling native Protein G. This candidate ligand demonstrated affinity towards IgGs derived from human, cow, goat, mouse, sheep, pig, rabbit and rat serum, chicken IgY and recombinant camelid Fc domain, out of which cow and sheep IgG demonstrated 100% binding under the conditions selected. Preparative chromatography of IgG from human serum under a standardised buffer regime eluted IgG of ∼65% purity, compared to ∼62% with Protein G. This adsorbent achieved highest elution of IgG at neutral pH (0.1M sodium phosphate pH 7.0, 30%, v/v, ethylene glycol), an advantage for purifying antibodies sensitive to extremes of pH. The ligand demonstrated a static binding capacity of 24.6 mg Ig G ml⁻¹ resin and a dissociation constant (K(d)) of 4.78 × 10⁻6 M. The solid phase Ugi scaffold provides a strategy to develop pseudo-biospecific ligands to purify immunoglobulins and other potentially high-value biotherapeutic proteins.