Immunochromatographic Lateral Flow Research Articles

Ovalbumin antibody-based fluorometric immunochromatographic lateral flow assay using CdSe/ZnS quantum dot beads as label for determination ofT-2 toxin.

This work describes an anti-ovalbumin antibody-based lateral flow immunoassay (LFI) for T-2 toxin. The antibody uses acoating antigen as a bifunctional element for universality and introduces preincubation to improve the detection limits of the method. T-2 toxin and ovalbumin-modified T-2 toxin competitively binds on the anti-T-2 toxin monoclonal antibody modified on CdSe/ZnS quantum dot beads during preincubation. The modified T-2 toxin acts as a bifunctional element that forms immuno complexes during preincubation and combines with anti-ovalbumin antibody coated in the test line through the ovalbumin terminal. Fluorescence is detected at 610nm on the test zone following photoexcitation at 365nm. It has a reverse dose-effect relationship with the amount of T-2 toxin. The calibration plot is linear inthe 20-110fgmL-1T-2 toxin concentration range, and the limit of detection (LOD) is 10fgmL-1, which is lower by 8-fold than that of the traditional LFI system (LOD 80fgmL-1) and one order of magnitude than those of LFIs with labels of colloidal gold nanoparticles (LOD 150fgmL-1) or fluorophores (LOD 190ngmL-1). Universality was verified through aflatoxin B1 detection using the established ovalbumin antibody-based LFI system (LOD 10fgmL-1). The performance of the method was compared with that of established systems and a commercial ELISA kit (LOD 360fgmL-1). Graphical abstractSchematic representation of ovalbumin antibody-based immunochromatographic lateral flow assay for T-2 toxin. Preincubation is introduced for high sensitivity. T-2- anti-ovalbumin acts as a bi-functional element for universality. CdSe/ZnS quantum dot beads act as label. Fluorometric signal is detected at 610nm.

Serological and molecular detection of infection with Mycobacterium leprae in Brazilian six banded armadillos (Euphractus sexcinctus)

Leprosy was recognized as a zoonotic disease, associated with nine-banded armadillos (Dasypus novemcinctus) in the Southern United States of America in 2011. In addition, there is growing evidence to support a role for armadillos in zoonotic leprosy in South America. The current study evaluated twenty specimens of the six-banded armadillo (Euphractus sexcinctus), collected from rural locations in the state of Rio Grande do Norte (RN), Brazil for evidence of infection with Mycobacterium leprae. Serum was examined using two "in-house" enzyme-linked immunosorbent assays (ELISAs) and via two commercially available (ML flow and NDO-LID®) immunochromatographic lateral flow (LF) tests, for detection of the PGL-I and/or LID-1 antigens of the bacterium. The presence of M. leprae DNA in liver tissue was examined using the multi-copy, M. leprae-specific repetitive element (RLEP), as target in conventional and nested PCR assays. Molecular and anti-PGL-I-ELISA data indicated that 20/20 (100 %) of the armadillos were infected with M. leprae. The corresponding detection levels recorded with the LF tests were 17/20 (85 %) and 16/20 (85 %), for the NDO-LID® and ML flow tests, respectively. Our results indicate that, in common with D. novemcinctus, six banded armadillos (a species hunted and reared as a food-source in some regions of Brazil, including RN), represent a potential reservoir of M. leprae and as such, their role in a possible zoonotic cycle of leprosy within Brazil warrants further investigation.

Screening for human immunodeficiency virus using a newly developed fourth generation lateral flow immunochromatography assay

BackgroundHigh sensitivity for detection of HIV-1 p24 antigen allows for early detection of primary HIV-1 infections. ObjectivesTo evaluate the detection sensitivity and specificity of the Daina Screen® HIV Combo assay using clinical specimens in Japan where the pretest probability (prevalence) is low. Study designWe screened 17,373 preoperative outpatient blood samples using 4th generation lateral flow immunochromatography Daina Screen® HIV Combo assay for simultaneously detecting anti-HIV-1/2 and HIV-1 p24 antigen. ResultsOf the samples tested, 24 were positive for HIV-1 p24 antigen and 49 for HIV-1/2 antibody. Of the 49 samples, 36 were WB and HIV-1 RNA negative, 10 were WB and HIV-1 RNA positive, and 3 were WB positive, HIV-1 RNA negative, and in-house HIV-1 proviral DNA positive. RT-PCR revealed that of the 24 samples that were p24 antigen positive, one sample was HIV-1 RNA positive, which was reconfirmed using an in-house HIV-1 provirus DNA assay. From the 17,300 HIV-1 p24 antigen and anti-HIV-1/2 negative samples, pools containing 10 negative samples each were tested for HIV-1 by RT-PCR; all results were negative. ConclusionThe Daina Screen® HIV Combo assay had a sensitivity and specificity of 100% and 99.7%, respectively, which sufficiently detected HIV infection in the cohort.

Cryptosporidium Diagnostic Assays: Microscopy.

Stained microscopy of fecal smears was the cornerstone of Cryptosporidium diagnosis for many years, and still provides a low-cost method for detecting oocysts. The development and commercialization of improved enzyme immunosorbent assays (EIA) for coproantigen detection provided an automatable method for mass testing, and rapid diagnostics when incorporated onto a cartridge format. Similarly, immunochromatographic lateral flow assays (ICLF) enable rapid diagnostics. Nevertheless, it is important that positive reactions by EIA or ICLF are confirmed. Here we describe microscopical methods using tinctorial stains for the diagnosis of acute cryptosporidiosis, and using immunofluorescent reagents for diagnosis or for confirmation of EIA or ICLF positive reactions.

Aflatoxin Contamination of Non-cultivated Fruits in Zambia.

Wild fruits are an important food and income source for many households in Zambia. Non-cultivated plants may be as susceptible as crops to aflatoxin contamination. Concentrations of aflatoxins in commonly consumed wild fruits from markets and characteristics of associated aflatoxin-producers need to be determined to assess the aflatoxin risk posed by handling, processing, storage, and consumption. Samples of Schinziophyton rautanenii (n = 22), Vangueriopsis lanciflora (n = 7), Thespesia garckeana (n = 17), Parinari curatellifolia (n = 17), Ziziphus spp. (n = 10), Adansonia digitata (n = 9), and Tamarindus indica (n = 23) were assayed for aflatoxin using lateral-flow immunochromatography from 2016 to 2017. Aflatoxins were above Zambia’s regulatory limit (10 μg/kg) in S. rautanenii (average = 57 μg/kg), V. lanciflora (average = 12 μg/kg), and T. garckeana (average = 11 μg/kg). The L strain morphotype of Aspergillus flavus was the most frequent member of Aspergillus section Flavi in market samples, although Aspergillus parasiticus and fungi with S morphology were also found. All fruits except T. indica supported both growth (mean = 3.1 × 108 CFU/g) and aflatoxin production (mean = 35,375 μg/kg) by aflatoxigenic Aspergillus section Flavi. Innate resistance to aflatoxin producers was displayed by T. indica. For the other fruits, environment and infecting fungi appeared to have the greatest potential to influence aflatoxin concentrations in markets. This is the first report of aflatoxins and aflatoxin-producers on native fruits in Zambia and suggests mitigation is required.

An Immunochromatographic Lateral Flow Strip Test for the Rapid Detection of Danofloxacin in Milk

An immunochromatographic lateral flow strip test (ILFST) for the rapid detection of danofloxacin (DAN) in milk samples was developed based on colloidal gold-labeled anti-DAN monoclonal antibody 3E9 and a competitive format. Quantitative detections of DAN-spiked samples using the ILFST gave a half inhibitory concentration (IC50) of 0.513 ng/mL and a limit of detection (LOD) of 0.092 ng/mL. Recoveries of DAN from spiked milk samples were from 80.0 to 95.1% within an assay (intra-assay) and from 87.0 to 89.8% between assays (inter-assay). The coefficients of variation (CV) for intra-assay and inter-assay were 2.09–9.93% and 2.42–12.07%, respectively. These data indicated that the ILFST was highly sensitive and reproducible. Detections of authentic milk samples using ILFST and high-performance liquid chromatography (HPLC) showed no significant differences between the two methods. Hence, the ILFST can be widely used for both qualitative and quantitative detection of DAN residues in milk samples.

Detection of Tadalafil and Its Analogues in Health Wine and Oral Liquid by Microwell Lateral Flow Immunochromatography Assay

To monitor illegal addition of tadalafil and its analogues in health food, a new polyclonal antibody was prepared by immunizing New Zealand white rabbits with a common skeleton structure hapten combined with an unsaturated linear alkyl arm hapten strategy, which could simultaneously recognize tadalafil, amino tadalafil and N -Desmethyl tadalafil. A microwell lateral flow immune- chromatographic assay ( mw LFIA) method combined with simple sample pretreatment was developed. The mw LFIA showed that the LOD for three tadalafil drugs was between 0.05 ng mL −1 and 0.30 ng mL −1 , the cut-off values were between 50 ng mL −1 and 100 ng mL −1 . Cross-reactivity (CR) results showed no interference from substances with similar structures, indicating a good specificity against tadalafil, amino tadalafil and N -Desmethyl tadalafil. The average recoveries of three drugs in spiked health wine and oral liquid ranged from 78.7% to 117.8% with an RSD of less than 15%, showing a good aggreement with the results of HPLC-MS/MS method. The developed mw LFIA strip was suitable for the rapid detection of tadalafil and its analogues in health wine and oral liquid samples. A microwell lateral flow immunochromatographic assay ( mw LFIA) for simultaneous determination of tadalafil, amino Tadalafil and N -desmethyl Tadalafil was developed with high accuracy and sensitivity. The mw LFIA method was successfully applied to the detection of Tadalafil and its analogues residue in analogues health wine and oral liquid samples.

Evaluation of the RESIST-4 O.K.N.V immunochromatographic lateral flow assay for the rapid detection of OXA-48, KPC, NDM and VIM carbapenemases from cultured isolates.

Purpose This study aimed to evaluate the performance of the RESIST-4 O.K.N.V immunochromatographic lateral flow assay for the detection of OXA-48, KPC, NDM and VIM carbapenemases in 100 clinical Enterobacteriaceae isolates using solid culture media.Methodology In total, 100 clinical Enterobacteriaceae isolates with characterized β-lactamase enzymes (OXA-48 n=46, KPC n =4, NDM n =43 and VIM n =10) were evaluated using the RESIST-4 O.K.N.V assay. The assay was also evaluated using carbapenem-sensitive control strains and confirmed non-carbapenemase-producing Enterobacteriaceae clinical isolates resistant to carbapenems. Inter-rater agreement of the test was evaluated by four different users who tested 11 randomly selected isolates daily over 3 days.Results Overall accuracy of the assay was 99.5 %. For the detection of KPC, OXA-48 and its variants and VIM the assay correctly identified 100 % of the isolates when compared to PCR. Initial performance for NDM detection was sensitivity=95.3 %, specificity=100 %. Two PCR positive Providencia rettgeri isolates rendered false negative results on the assay. Retesting from a carbapenem zone of inhibition rendered a positive result for both isolates increasing the sensitivity to 100 %. No false positive results or cross reactions were detected.Conclusion The RESIST-4 O.K.N.V is reliable, sensitive and specific for the detection of OXA-48, KPC, NDM and VIM carbapenemases. Further evaluation on improving NDM detection in organisms from the Proteeae tribe is warranted to determine optimal test conditions.

Generation and selection of antibodies for a novel immunochromatographic lateral flow test to rapidly identify OXA-23-like-mediated carbapenem resistance in Acinetobacter baumannii.

The spread of carbapenem-resistant Acinetobacter baumannii has led to a worldwide healthcare problem. Carbapenem resistance in A. baumannii is mainly mediated by the acquisition of the carbapenem-hydrolyzing oxacillinase OXA-23. The phenotypic detection of carbapenem-producing A. baumannii is challenging and time-consuming. Hence, there is an unmet medical need for reliable and rapid diagnostic tools to detect OXA-23-producing Acinetobacter isolates to enable successful patient management. Development of an immunochromatographic lateral flow test (ICT) for the rapid and reliable detection of OXA-23-producing carbapenem-resistant Acinetobacter isolates. For the development of an antibody-based ICT, we generated anti-OXA-23 monoclonal antibodies (MoAbs) and screened them sequentially for their ability to bind native OXA-23. Selected OXA-23-specific MoAbs were tested in different combinations for their capacity to capture and detect OXA-23His6 by sandwich enzyme-linked immunosorbent assay (ELISA) and ICT. A well-characterized collection of carbapenem-resistant Acinetobacter isolates with defined carbapenem resistance mechanisms were used to evaluate the specificity of the final OXA-23 ICT prototype. The antibody pairs best suited for the sandwich ELISA format did not match the best pairs in the ICT format selected during the development process of the final prototype OXA-23 ICT. This prototype was able to differentiate between OXA-23 subfamily-mediated carbapenem resistance and carbapenem-resistant Acinetobacter isolates overexpressing other OXAs with 100 % specificity and a turnaround time of 20 min from culture plate to result. With this rapid detection assay one can save 12-48 h of diagnostic time, which could help avoid inappropriate use of carbapenems and enable earlier intervention to control the transmission of OXA-23-producing carbapenem-resistant Acinetobacter isolates to other patients and healthcare workers.

Up-Converting Nanoparticle-Based Immunochromatographic Strip for Multi-Residue Detection of Three Organophosphorus Pesticides in Food.

Organophosphorus (OP) pesticides are widely used to control pests because of their high activity. This study described a rapid and sensitive lateral flow immunochromatographic (LFIC) assay based on up-converting nanoparticles (UCNPs) for multi-residue detection of three OP pesticides. The developed assay integrated novel fluorescent material UCNPs labeled with a broad-specific monoclonal antibody. Based on the competitive platform by immobilized antigen in the test zone, the optimized UCNPs-LFIC assay enabled sensitive detection for parathion, parathion-methyl, and fenitrothion with IC50 of 3.44, 3.98, and 12.49 ng/mL (R2 ≥ 0.9776) within 40 min. The detectable ability ranged from 0.98 to 250 ng/mL. There was no cross-reactivity with fenthion, phoxim, isocarbophos, chlorpyrifos, or triazophos, even at a high concentration of 500 ng/mL. Matrix interference from various agricultural products was also studied in food sample detection. In the spiked test, recoveries of the three OP pesticides ranged from 67 to 120% and relative standard deviations were below 19.54%. These results indicated that the proposed strip assay can be an alternative screening tool for rapid detection of the three OP pesticides in food samples.

Rapid detection of adulteration of dehydroepiandrosterone in slimming products by competitive indirect enzyme-linked immunosorbent assay and lateral flow immunochromatography

ABSTRACTIn this study, dehydroepiandrosterone (DHEA) hapten and high sensitivity polyclonal antibody against DHEA were prepared, a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and lateral flow immunochromatography (LFA) for the determination of DHEA in slimming products were developed for the first time. The sample pretreatment was very simple and fast, the limit of detection (LOD) of the ciELISA for DHEA in slimming products was 9.6 μg/kg. The average recoveries of slimming tea, slimming tablets and slimming capsules ranged from 93.2% to 99.4%, 86.8% to 100.0%, and 80.5% to 115.4%, respectively, with the coefficient of variations (CVs) between 7.7% and 14.1%, 2.7% and 11.0%, and 2.2% and 8.6%, respectively. The pAb had negligible cross-reactivity to the structural and functional analogs of DHEA. Simultaneously, the LFA showed a cut-off value of 500 μg/kg in slimming products, and the results could be attained within 3 min. A parallel HPLC analysis in 30 commercial slimming products validated a good agreement with the proposed ciELISA and LFA.

Antibodies produced by dogs successfully challenged with live Leptospira spp. did not cross-react to Brucella antigen in a commercial rapid slide agglutination test that detects antibodies to Brucella canis.

Brucella canis is a cause of canine infertility and abortion. Veterinarians and veterinary laboratorians screen for antibodies to B. canis with serologic tests including a rapid slide agglutination test (RSAT; D-Tec CB, Zoetis, San Diego, CA). False-positive results are possible because of cross-reactivity to antibodies to some gram-negative bacteria. Cross-reactivity has been reported between antibodies of Brucella abortus and Leptospira spp. with serologic tests for bovine brucellosis; however, this has not been documented with serologic tests for canine brucellosis, to the author's knowledge. The RSAT was evaluated with the sera from dogs experimentally challenged with 1 of 4 serovars of Leptospira spp.: L. kirschneri serovar Grippotyphosa, or L. interrogans serovars Canicola, Icterohaemorrhagiae, or Pomona. Experimental infections were confirmed through results of microscopic agglutination testing and/or lateral flow immunochromatography testing. The sera of 32 dogs collected at day 0 and days 7, 10, and 14 yielded negative results with the RSAT. Antibodies produced through experimental infections to these 4 serovars of Leptospira spp. did not cross-react with Brucella antigen with the RSAT; therefore, cross-reactivity of anti-leptospiral antibodies may not be of concern for B. canis rapid slide agglutination testing of dogs.

Generation of monoclonal antibodies against foot-and-mouth disease virus SAT 2 and the development of a lateral flow strip test for virus detection.

Foot-and-mouth disease (FMD) remains a major economic concern for the livestock productivity in many developing countries and a continued threat to countries that are disease free because of its potential devastating impact on agricultural, food chain and tourism sectors. FMD virus (FMDV) is recognized as having seven serotypes: O, A, C, Asia 1, South African Territories (SAT) 1, 2, 3 and multiple subtypes within each serotype. FMD outbreaks due to SAT 2 have been reported in many African countries. The development of a rapid and easily performed test for FMD detection is critical for controlling FMD outbreaks and containing its spread. The present project developed a lateral flow immunochromatographic (LFI) strip test for the rapid detection of FMDV SAT 2. A panel of monoclonal antibodies (mAbs) against FMDV serotype SAT 2 was produced and characterized. One mAb (#10) was selected as the capture mAb because it reacted to all 23 SAT 2 isolates archived at the National Center for Foreign Animal Disease. The LFI strip test was developed using biotin-conjugated mAb #10, and the colloid gold-conjugated FMDV serotype-independent mAb as the detection mAb. A generic Rapid Assay Device (gRAD) with one test line and a control line was used for the test. The LFI strip test detected all 23 tested SAT 2 isolates and recent outbreak strains. The results indicated that the diagnostic specificity and sensitivity of the LFI strip test were greater than the double antibody sandwich (DAS) DAS ELISA. The ability of the LFI strip test to produce rapid diagnostic results will be useful for early on-site diagnosis during FMD outbreaks.

Rapid and easy determination of morphine in chafing dish condiments with colloidal gold labeling based lateral flow strips

In order to enhance the flavor of chafing dish and increase the attraction of consumers, the poppy shell is reported to be illegally added to the condiments of chafing dish. In this research, a rapid, simple, and convenient method based on the classic immunochromatographic lateral flow strip (LFS) with gold nanoparticles (GNPs) labeling was developed for easy monitoring of morphine (MOP), an effective component of poppy shell. Under optimized conditions, this developed LFS can well realize the detection of target MOP in the condiments of chafing dish in less than 10 min without any complicated pre-treatments. The limit of detection (LOD) can be achieved as low as 0.1 ppb for standard MOP or the MOP spiked condiments of chafing dish. All these results of the research strongly demonstrate that this established LFS method can be successfully applied in practical rapid and accurate on-site screening of poppy shell in condiments of chafing dish.

2083. Rapid Diagnosis and Differentiation of Dengue During Peri-monsoon Season in Tropical Resource Limited Facilities

BackgroundDengue is a re-emerging public health problem threatening the tropical developing world, mandating rapid diagnosis and supportive management in the absence of licensed vaccines or anti-dengue therapy. Regions endemic for dengue and related viruses are overwhelmed by the sudden surge of cases during outbreaks. It is difficult to justify confirmatory diagnosis of every case using WHO criteria or differentiate it from other concurrent viral illnesses. The study evaluated a rapid, sensitive and specific diagnostic methodology suitable for dengue outbreaks in resource limited facilities.MethodsOne hundred dengue patients as per WHO Criteria as well as 100 healthy controls from New Delhi, India were included. Samples collected on fifth day on onset of fever were tested by lateral flow immunochromatography (LF-ICT), IgM ELISA and reverse transcriptase polymerase chain reaction (RT-PCR), and results were compared. Diagnostic accuracy indices and Kappa analysis were calculated.ResultsThe sensitivity, specificity, positive and negative predictive values (PPV and NPV) of NS1 against RT-PCR was 98.31, 100, 100, and 99.3% and strength of agreement was perfect.ConclusionAntigen-based and molecular tests are a better tool for early diagnosis of dengue. The combined LF-ICT kits are highly sensitive, specific, user-friendly, compact, frugal and thus recommended for use in dengue outbreaks, field conditions and as bed side diagnostic tests, for confirmatory dengue diagnosis. Further studies are required to assess their utility in prognosis, surveillance and establishment of guidelines for dengue outbreaks.Disclosures All authors: No reported disclosures.

Rapid Detection of Severe Fever with Thrombocytopenia Syndrome Virus via Colloidal Gold Immunochromatography Assay.

To develop the point-of-care testing method to facilitate the clinical detection of severe fever with thrombocytopenia syndrome virus (SFTSV), colloidal gold paper-based lateral flow immunochromatography test strips (LFITSs) have been fabricated for the rapid detection for the first time. The pH value and the amount of monoclonal antibody to prepare colloidal gold nanoparticle-labeled monoclonal antibody bioconjugates were optimized. In addition, 0.4% bovine serum albumin was considered to be the best concentration for blocking nitrocellulose membranes. Under optimal conditions, the limit of detection for SFTSV was as low as 1 ng/mL depending on a visual line. Meanwhile, the entire detection process required no more than 10 min with a volume of only 50 μL of the analyte solution. Moreover, paper-based LFITSs were evaluated in real samples of human serum of patients with satisfactory results. In addition, all strips were of high stability and specificity. In the light of advantages such as simple, portable, rapid, and low cost, the developed LFITSs will extensively come into service, especially in remote areas.

Aflatoxin Contamination of Dried Insects and Fish in Zambia

Dried insects and fish are important sources of income and dietary protein in Zambia. Some aflatoxin-producing fungi are entomopathogenic and also colonize insects and fish after harvest and processing. Aflatoxins are carcinogenic, immune-suppressing mycotoxins that are frequent food contaminants worldwide. Several species within Aspergillus section Flavi have been implicated as causal agents of aflatoxin contamination of crops in Africa. However, aflatoxin producers associated with dried fish and edible insects in Zambia remain unknown, and aflatoxin concentrations in these foods have been inadequately evaluated. The current study sought to address these data gaps to assess potential human vulnerability through the dried fish and edible insect routes of aflatoxin exposure. Caterpillars ( n = 97), termites ( n = 4), and dried fish ( n = 66) sampled in 2016 and 2017 were assayed for aflatoxin by using lateral flow immunochromatography. Average aflatoxin concentrations exceeded regulatory limits for Zambia (10 μg/kg) in the moth Gynanisa maja (11 μg/kg), the moth Gonimbrasia zambesina (Walker) (12 μg/kg), and the termite Macrotermes falciger (Gerstacker) (24 μg/kg). When samples were subjected to simulated poor storage, aflatoxins increased ( P < 0.001) to unsafe levels in caterpillars (mean, 4,800 μg/kg) and fish ( Oreochromis) (mean, 23 μg/kg). The L strain morphotype of A. flavus was the most common aflatoxin producer on dried fish (88% of Aspergillus section Flavi), termites (68%), and caterpillars (61%), with the exception of Gynanisa maja, for which A. parasiticus was the most common (44%). Dried fish and insects supported growth (mean, 1.3 × 109 CFU/g) and aflatoxin production (mean, 63,620 μg/kg) by previously characterized toxigenic Aspergillus section Flavi species, although the extent of growth and aflatoxigenicity depended on specific fungus-host combinations. The current study shows the need for proper storage and testing of dried insects and fish before consumption as measures to mitigate human exposure to aflatoxins through consumption in Zambia.

Romer Labs RapidChek®Listeria monocytogenes Test System for the Detection of L. monocytogenes on Selected Foods and Environmental Surfaces.

The Romer Labs RapidChek® Listeria monocytogenes test system (Performance Tested Method 011805) was validated against the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (USDA-FSIS/MLG), U.S. Food and Drug Association Bacteriological Analytical Manual (FDA/BAM), and AOAC Official Methods of Analysis (AOAC/OMA) cultural reference methods for the detection of L. monocytogenes on selected foods including hot dogs, frozen cooked breaded chicken, frozen cooked shrimp, cured ham, and ice cream, and environmental surfaces including stainless steel and plastic in an unpaired study design. The RapidChek method uses a proprietary enrichment media system, a 44-48h enrichment at 30 ± 1°C, and detects L. monocytogenes on an immunochromatographic lateral flow device within 10min. Different L. monocytogenes strains were used to spike each of the matrixes. Samples were confirmed based on the reference method confirmations and an alternate confirmation method. A total of 140 low-level spiked samples were tested by the RapidChek method after enrichment for 44-48h in parallel with the cultural reference method. There were 88 RapidChek presumptive positives. One of the presumptive positives was not confirmed culturally. Additionally, one of the culturally confirmed samples did not exhibit a presumptive positive. No difference between the alternate confirmation method and reference confirmation method was observed. The respective cultural reference methods (USDA-FSIS/MLG, FDA/BAM, and AOAC/OMA) produced a total of 63 confirmed positive results. Nonspiked samples from all foods were reported as negative for L. monocytogenes by all methods. Probability of detection analysis demonstrated no significant differences in the number of positive samples detected by the RapidChek method and the respective cultural reference method.

Validation of a rapid lateral flow method for the detection of cows’ milk in water buffalo, sheep or goat milk

ABSTRACTFor many years, the adulteration of milk from sheep, goats or water buffalos with cows’ milk has been a widespread practice due to the higher cost of milk from those other species. Because of this, great concern has been shown by many Protected Designation of Origin councils that have to assure the quality and genuineness of the cheese produced by their associates. Therefore, the whole production chain needs analytical tools that allow the control of potential adulteration. Rapid methods to be used in the field are scarce and have not been validated according to international guidelines. The aim of this work has been to validate a rapid test based on lateral flow immunochromatography to detect cows’ milk in milk from other species, including buffalo’s milk, according to AOAC guidelines. No false-positive result was found after analysing 146 known negative samples from individual animals. The lowest level of adulteration with a Probability of Detection (POD) of 1.00 (confidence interval between 0.94 and 1.00) was found at 0.5% of cows’ milk. This level is below the current EU allowed level of cows’ milk, set at 1%. Variations in the time of assay, volume of the analysis buffer and different batches of the test were evaluated to detect any effect on the false-positive rate or on the limit of detection of the test. The effects of compositional factors (such as high level of fat, protein and somatic cell counts) were also evaluated. The new rapid test to detect cows’ milk in milk from other species is shown to be an adequate tool to control milk quality in routine analysis. This kind of test is very easy to use and it can be performed by untrained staff during milk collection at the farm or upon arrival at dairies.

Development of an immunochromatographic lateral flow strip for the simultaneous detection of aminoglycoside residues in milk.

A colloidal gold-based immunochromatographic strip with a competitive format has been developed for the rapid, simultaneous, semi-quantitative and quantitative detection of several aminoglycoside residues in milk, including gentamicin sulfate (GM), neomycin sulfate (NEO) and kanamycin sulfate (KN). Three monoclonal antibodies against the three corresponding aminoglycosides were conjugated to colloidal gold particles and applied to the conjugate pads of the strip. The competitors [GM-bovine serum albumin (GM-BSA), NEO-BSA and KN-BSA conjugates] of GM, NEO and KN were immobilized onto a nitrocellulose (NC) membrane at three detection zones, T1, T2, and T3, respectively. The minimal cut-off values of the strip were 10 ng mL−1 for GM, and 100 ng mL−1 for NEO and KN, which are lower than the maximum residue levels (MRLs) established for aminoglycosides. The IC50 values of the strip were 0.737 ng mL−1, 8.971 ng mL−1 and 11.110 ng mL−1 for GM, NEO and KN respectively. In conclusion, the immunochromatographic lateral flow strip could be used for rapid, simultaneous, semi-quantitative and quantitative detection of GM, NEO and KN residues in milk.