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".

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 104 and 105 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.FigureResults achieved with multi-analyte NALFIA for E. coli virulence factors. First strip: blank; second to sixth strip: each of the STEC factors; seventh strip: all factorsElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-4334-z) contains supplementary material, which is available to authorized users.

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.FigureScheme of a NALFIA-, NAMIA, or NALMIA. Neutravidin adsorbed onto CNPs detects biotin-labelled amplicons; the discriminating tag is recognized by its respective antibody, which is immobilized onto nitrocellulose membranes or pads

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 µg 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.

The feasibility of using the Barn Owl (Tyto alba guttata) to monitor environmental quality in the Netherlands was investigated, using Cd, Cu, Pb, Mn, and Fe as indicators for environmental contamination. Throughout 1992, bird-watchers, volunteers, and officials submitted 53 birds. The age and geographical distribution of these birds, formed a representative sample of the population. The following interrelationships were investigated: cause of death, nutrient reserve, age, time of death, place of death, body measurements, sex, condition, and heavy metal concentration in kidney, liver, and tibia. Twenty-eight animals had died after collisions. Fifteen Barn Owls died of exhaustion. In total, twenty-four birds were exhausted, with coccidiosis or other parasitic gastrointestinal infections. The condition of the birds showed that as the birds' condition worsened, fat reserves were depleted before protein reserves. Significant linear relationships were found between decreasing protein reserves and decreasing dry weights of the liver, kidney, flight muscle and heart, but not of the tibia. An asymptotic, nonlinear relation was observed between dry organ weight and fat reserve. This suggested that fat reserves were only found when protein reserves exceeded 15% of the body mass at starvation. Concentrations of Cu and Fe in liver and kidney rose as protein reserves fell; the total content of Cu and Fe per organ, however, remained constant. The Mn concentration of these organs remained constant; Mn content increased with increasing organ sizes. Neither Cd nor Pb showed a clear relationship with parameters of body condition. The ratio between the organ content of Pb or Cd and the dry organ weight, however, revealed some birds from contaminated habitats. The findings suggested that concentrations of environmental contaminants should be measured on a dry weight basis. Furthermore, depending on the pharmacokinetic characteristics of a contaminant, the total content of that contaminant per organ can be more informative than the concentration. In this one year sample of Barn Owls, no indications were found of toxic levels of Cd, Cu, Pb, Mn, or Fe in the Netherlands. It is concluded that the Barn Owl is a suitable biomonitor. Furthermore, a network of volunteers can produce an informative sample of the Barn Owl population without interfering with the population.

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The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA) and the gene for E. coli 16S (hui). Optimization was performed using a Box-Behnken design, and the limit of detection for each virulence factor was established. Finally, this NAMIA using CNPs was tested with DNA from 48 field strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and specificity values higher than 0.80 and efficiency values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed fit for purpose, i.e., it is valuable for fast screening of amplified genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.