Geertruida A. Posthuma‐Trumpie

Lateral flow (immuno)assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food and environmental settings. We describe principles of current formats, applications, limitations and perspectives for quantitative monitoring. We illustrate the potentials and limitations of analysis with lateral flow (immuno)assays using a literature survey and a SWOT analysis (acronym for "strengths, weaknesses, opportunities, threats"). Articles referred to in this survey were searched for on MEDLINE, Scopus and in references of reviewed papers. Search terms included "immunochromatography", "sol particle immunoassay", "lateral flow immunoassay" and "dipstick assay".

Carbon nanoparticles (CNPs) labeled with reporter molecules can serve as signaling labels in rapid diagnostic assays as an alternative to gold, colored latex, silica, quantum dots, or up-converting phosphor nanoparticles. Detailed here is the preparation of biomolecule-labeled CNPs and examples of their use as a versatile label. CNPs can be loaded with a range of biomolecules, such as DNA, antibodies, and proteins (e.g., neutravidin or a fusion protein of neutravidin with an enzyme), and the resulting conjugates can be used to detect analytes of high or low molecular mass.

The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 10(4) and 10(5) colony forming units/mL or 0.1-0.9 ng/μL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification.

Several aspects of the development of competitive lateral flow immunoassays (LFIAs) are described. The quantitation of progesterone is taken as an example. The LFIA format consisted of a nitrocellulose membrane spotted with various progesterone conjugates as the test line. A mixture of primary antibody and secondary antibody adsorbed to colloidal carbon was used for signal generation. A digital scanner and dedicated software were used to quantitate the response. A reappraisal of the checkerboard titration, often used in the optimisation of immunoassays, is discussed. Surprisingly, the highest sensitivity of the LFIA format (IC(50) of 0.6 microg L(-1) progesterone in buffer) was achieved by using a high coating concentration of the analyte-protein conjugate and a high dilution of the antibody solution. Immediate addition of all reagents in LFIA was superior to premixing the components and allowing prereaction. Of several blocking agents tested bovine serum albumin was superior in performance, whereas the combination of ovalbumin and progesterone substantially influenced test results.