Aart van Amerongen

Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey.

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”.

Amorphous carbon nanoparticles: a versatile label for rapid diagnostic (immuno)assays

AbstractCarbon 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

Molecular diagnosis of malaria in the field: development of a novel 1-step nucleic acid lateral flow immunoassay for the detection of all 4 human Plasmodium spp. and its evaluation in Mbita, Kenya

Microscopy is frequently used for malaria diagnosis, but at low parasitemia, it becomes less sensitive and time consuming. Molecular tools allow for specific/sensitive diagnosis, but current formats, such as polymerase chain reaction (PCR) combined with gel electrophoresis and real-time PCR assays, are difficult to implement in resource-poor settings. Development of a simple, fast, sensitive, and specific detection system, nucleic acid lateral flow immunoassay (NALFIA) for amplified pan-Plasmodium PCR products, is described. The NALFIA lower detection limit is 0.3 to 3 parasites/microL, 10-fold more sensitive than gel electrophoresis analysis. Evaluating 650 clinically suspected malaria cases with the pan-Plasmodium assay under field conditions (rural Kenya) revealed that NALFIA detected more positives than microscopy (agreement, 95%; kappa value = 0.85), and there was an excellent agreement between gel electrophoresis and NALFIA (98.5%; kappa value = 0.96). In conclusion, NALFIA is more sensitive than microscopy and a good alternative to detect PCR products while circumventing using electricity or expensive equipment, making NALFIA the 1st step toward molecular field diagnosis.

Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted skimmed milk. Four aminoglycosides (gentamicin, neomycine, kanamycin and a streptomycin derivative) were immobilised onto the sensor surface of a biosensor chip (CM5) in the four Fcs of the biosensor system by amine coupling. In the Biacore, milk (reconstituted from skimmed milk powder) was 10 times diluted with a mixture of the four specific antibodies and injected through the four serially connected Fcs (1 min at a flow rate of 20 μl min -1 ). The responses measured just prior to the injection (20 μl at a flow rate of 20 μl min -1 ) of the regeneration solution (0.2 M NaOH + 20% acetonitril) were indicative for the presence or absence of the aminoglycosides in reconstituted milk. The limits of detection were between 15 and 60 ng ml -1 , which was far below the maximum residue limits (MRLs) (varying from 100 to 500 ng ml -1 ) and the total run time between samples was 7 min.

Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4

Seeds of Impatiens balsamina contain a set of related antimicrobial peptides (Ib-AMPs). We have produced a synthetic variant of Ib-AMP1, oxidized to the bicyclic native conformation, which was fully active on yeast and fungal strains; and four linear 20-mer Ib-AMP variants, including two all-D forms. We show that the all-D variants are as active on yeast and fungal strains as native peptides. In addition, fungal growth inhibition nor salt-dependency of Ib-AMP4 could be improved by more than two-fold via replacement of amino acid residues by arginine or tryptophan. Native Ib-AMPs showed no hemolytic nor toxic activity up to a concentration of 100 microM. All these data demonstrate the potential of the native Ib-AMPs to combat fungal infections.

Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron

Rapid analytical methods enabling the determination of diverse targets are essential in a number of research areas, from clinical diagnostics to feed and food quality and safety. Herein, the development of a quantitative immunochromatographic assay for the detection of the synthetic phytoregulator forchlorfenuron (CPPU) is described. The competitive lateral flow immunoassay (LFIA) was based on the immobilization onto a nitrocellulose membrane of an ovalbumin-CPPU conjugate (test line) and on the use of an immunodetection ligand consisting of carbon nanoparticles labeled with an anti-CPPU monoclonal antibody through interaction with a secondary antibody. The presence of CPPU in horticultural samples was visually interpreted by the decrease in the black signal intensity of the test line, according to the competitive character of the format. The quantitative determination of the analyte was easily performed by a two-step procedure consisting of flatbed scanning of the strips followed by computer-based image analysis of the pixel gray volumes of the test lines. Under optimized conditions, the immunochromatographic test afforded a limit of quantification in buffer of 89 ng/L. The accuracy of the strip test was assessed by the analysis of fruit samples with incurred residues, and the obtained results were compared with those derived from two reference methods, ELISA and HPLC. The LOQ of the CPPU-specific LFIA in kiwifruits and grapes was established at 33.4 μg/kg. The excellent analytical performance of the developed strip test demonstrates the potential of immunochromatographic assays for the quantitative monitoring of small organic molecules in complex matrices.

Critical evaluation of the use of bioinformatics as a theoretical tool to find high-potential sources of ACE inhibitory peptides

A bioinformatics analysis to screen for high-potential sources of angiotensin converting enzyme (ACE) inhibitory peptides was conducted in the area of insect muscle proteins. Vertebrate muscle proteins are reported as good sources of ACE inhibitory peptides, while the research on invertebrate muscle proteins is limited. A phylogenetic tree constructed with actin sequences of both vertebrate and invertebrate species indicated a high homology. Furthermore, a quantitative in silico ACE inhibition analysis suggested that actin proteins of invertebrates have potentials as new sources of ACE inhibitory peptides. On one insect, Bombyx mori, a more detailed in silico analysis was done followed by a small experimental study. The in silico analysis indicated B. mori as a high-potential source of ACE inhibitory peptides and this was supported by the ACE inhibitory activity of the partially purified actin preparation. In conclusion, in food science, in silico analysis can be used as fast initial screening tool to look for high-potential sources of ACE inhibitory peptides and other peptidic bioactivities.

Attenuation of Renovascular Damage in Zucker Diabetic Fatty Rat by NWT-03, an Egg Protein Hydrolysate with ACE- and DPP4-Inhibitory Activity

Background Dipeptidyl peptidase 4 (DPP4) and angiotensin-converting enzyme (ACE) are important target enzymes in glycemic control and renovascular protection. Here, we studied the effect of NWT-03, an egg protein hydrolysate with DPP4- and ACE-inhibitory activity, on renovascular damage in Zucker diabetic fatty (ZDF) rats. Comparisons were made to rats treated with vildagliptin (VIL), included as a positive control for the effect of DPP4 inhibition. Methods ZDF rats received NWT-03 (1 g/kg/day) or VIL (3 mg/kg/day) from 10 to 25 weeks of age. Metabolic and renal functions were assessed; the kidney was removed for histological analysis of glomerulosclerosis and expression of pro-inflammatory/fibrotic markers (RT-PCR and Western blotting); and the aorta was removed for studies of endothelium-dependent relaxation (EDR). Findings Hyperinsulinemic ZDF rats typically developed signs of type-2 diabetes and renovascular damage, as evidenced by albuminuria, glomerulosclerosis, and impaired EDR. Neither NWT-03 nor VIL improved metabolic parameters; for VIL, this was despite a 5-fold increase in glucagon-like peptide (GLP)-1 levels. NWT-03 and VIL both reduced renal interleukin (Il)-1β/Il-13 mRNA expression and glomerulosclerosis. However, only NWT-03 additionally decreased renal tumor necrosis factor (TNF)-α mRNA and P22phox protein expression, reduced albuminuria, and restored aortic EDR. Indomethacin added to the organ bath instantly improved aortic EDR, indicating a role for cyclooxygenase (COX)-derived contractile prostanoids in opposing relaxation in ZDF rats. This indomethacin effect was reduced by NWT-03, but not by VIL, and coincided with decreased renal COX-1/2 protein expression. Conclusion and Interpretation Long-term supplementation with the egg protein hydrolysate NWT-03 attenuated renovascular damage in this preclinical rat model of type 2 diabetes. A comparison to the DPP4-inhibitor VIL suggests that the effects of NWT-03 were related to both ACE- and DPP4-inhibitory properties. The development of protein hydrolysates with a multiple-targeting strategy may be of benefit to functional food formulations.

The arable ecosystem as battleground for emergence of new human pathogens

Disease incidences related to Escherichia coli and Salmonella enterica infections by consumption of (fresh) vegetables, sprouts, and occasionally fruits made clear that these pathogens are not only transmitted to humans via the “classical” routes of meat, eggs, and dairy products, but also can be transmitted to humans via plants or products derived from plants. Nowadays, it is of major concern that these human pathogens, especially the ones belonging to the taxonomical family of Enterobacteriaceae, become adapted to environmental habitats without losing their virulence to humans. Adaptation to the plant environment would lead to longer persistence in plants, increasing their chances on transmission to humans via consumption of plant-derived food. One of the mechanisms of adaptation to the plant environment in human pathogens, proposed in this paper, is horizontal transfer of genes from different microbial communities present in the arable ecosystem, like the ones originating from soil, animal digestive track systems (manure), water and plants themselves. Genes that would confer better adaptation to the phytosphere might be genes involved in plant colonization, stress resistance and nutrient acquisition and utilization. Because human pathogenic enterics often were prone to genetic exchanges via phages and conjugative plasmids, it was postulated that these genetic elements may be hold key responsible for horizontal gene transfers between human pathogens and indigenous microbes in agroproduction systems. In analogy to zoonosis, we coin the term phytonosis for a human pathogen that is transmitted via plants and not exclusively via animals.

Quantitative computer image analysis of a human chorionic gonadotropin colloidal carbon dipstick assay

Colloidal particles are widely used in qualitative dipstick assays for the determination of various proteins and haptens. Recently, a new colloidal label has been introduced based on elemental carbon. With this carbon label we have prepared a human chorionic gonadotropin-specific dipstick assay with a sensitivity of 10 mIU/ml. In addition, an image- and data-processing procedure for the quantification of the dipstick assay has been developed. The sum of the pixel grey levels of a carbon line was taken as a measure for this quantitative purpose. The measurement range of the assay is almost three orders of magnitude, i.e. 10 mIU/ml to 500 mIU/ml. The deviation from the mean of two dipstick determinations was 1.22% on average. The within-run and between-run precision, expressed as coefficients of variation at 50 mIU/ml were 1.03% and 1.84%, at 150 mIU/ml 2.14% and 3.77% and at 450 mIU/ml 2.55% and 5.28%, respectively. We have correlated this quantitative sol particle immunoassay with a commercial human chorionic gonadotropin specific radioimmunoassay. In an experiment with 25 human urine samples containing the hormone in amounts from 5 to 300 mIU/ml the correlation coefficient was 0.999. The sol particle immunoassay quantified by computer image analysis has been termed Sol particle Image Processed ImmunoAssay (SIPIA).