Lateral Flow Immunoassay Strip Research Articles

Visual and quantitative lateral flow immunoassay based on Au@PS SERS tags for multiplex cardiac biomarkers

Rapid and sensitive detection of multiple biomarkers by lateral flow immunoassay (LFIA) remains challenging for signal amplification for commonly used nanotags. Herein, we report a novel LFIA strip for visual and highly sensitive analysis of two cardiac biomarkers based on functionalized gold nanoparticles @ polystyrene microsphere (Au@PS）microcavity as surface-enhanced Raman scattering (SERS) tags. Antibody-modified Au@PS was designed as a SERS label. The evanescent waves propagating along the surface of the PS microcavity and the localized surface plasmons of the gold nanoparticles were coupled to enhance the light-matter interaction synergistically for Raman signal enhancement. In this strategy, the proposed Au@PS SERS tags-based LFIA was carried out to quantify the content of the heart failure and infarct biomarkers synchronously within 15 min and get the limits of detection of 1 pg/mL and 10 pg/mL for cardiac troponin I (cTnI) and N-terminal natriuretic peptide precursor (NT-proBNP), respectively. The results demonstrated 10–20 folds more sensitivity than that of the standard colloidal gold strip and fluorescent strip for the same biomarkers. This novel quantitative LFIA shows promise as a high-sensitive and visual sensing method for relevant clinical and forensic analysis.

Development of lateral flow immunoassay strip for rapid detection of acute hepatopancreatic necrosis disease-causing PirAB toxins in shrimp

Acute hepatopancreatic necrosis disease (AHPND) is currently the most affected disease of shrimp farms worlwide As rapid and specific detection methods are essential to improve disease management, a lateral flow immunoassay (LFIA) was designed for the detection of PirAvp and PirBvp toxins in shrimp. In this study, polyclonal antibody based lateral flow immunoassay (LFIA) strip has been developed. The recombinant PirAvp and PirBvp toxin‐like proteins of Vibrio parahaemolyticus (VPAHPND) was used to immunize rabbits with the highest antibody titre after the third booster. This polyclonal rabbit antiserum was purified and used to develop a LFIA strip for the detection of AHPND pathogen. This test strip could detect PirAvp and PirBvp toxins up to 125 ng/mL by naked eye within 15 min. No cross-reactivity was observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. This LFIA strip could be used as a handy tool by the farmers and technicians in situ. ® 2023 Journal of Science and Technology - NTTU

Development of a sensitive lateral flow immunoassay for the rapid detection of butralin in fruits

Butralin is a dinitrobenzene pesticide, has been widely used as a herbicide in many crops. Its high levels of residues threaten the ecological balance and human health. Therefore, establishment of a sensitive monoclonal antibody (mAb) for rapid detection of butralin residues is critical. In this study, special haptens were designed with good preservation of the characteristic structure of target compound. After mice immunization, a highly sensitive monoclonal antibody was produced. The half-maximum inhibitory concentration (IC50) was 1.936 ng/mL. And we applied it to develop a method of lateral flow immunoassay (LFIA), with vLOD value of the LFIA strip to be 10 ng/mL. In watermelon and pear samples, the cLOD values of the LFIA strip were 0.432 ng/mL and 0.426 ng/mL, respectively. The recoveries of the developed method ranged from 94.4% ± 2.1%–112.1% ± 3.4% and 89% ± 5.4%–103% ± 4.1% in watermelons and pears samples, with a CV of 2.9%–3.4% and 2.2%–5.2%, respectively. The results demonstrated that the developed method showed great prospect in the detection and monitoring of butralin in real food samples.

Merging Lateral Flow Immunoassay with Electroanalysis as a Novel Sensing Platform: Prostate Specific Antigen Detection as Case of Study.

The COVID-19 pandemic highlighted lateral flow immunoassay (LFIA) strips as the most known point-of-care (POC) devices enabling rapid and easy detection of relevant biomarkers by nonspecialists. However, these diagnostic tests are usually associated with the qualitative detection of the biomarker of interest. Alternatively, electrochemical-based diagnostics, especially known for diabetes care, enable quantitative determination of biomarkers. From an analytical point perspective, the combination of the two approaches might represent a step forward for the POC world: in fact, electrochemical transduction is attractive to be integrated into LFIA strips due to its simplicity, high sensitivity, fast signal generation, and cost effectiveness. In this work, a LFIA strip has been combined with an electrochemical transduction, yielding an electrochemical LFIA (eLFIA). As a proof-of-concept method, the detection of prostate-specific antigen has been carried out by combining a printed-electrochemical strip with the traditional LFIA tests. The electrochemical detection has been based on the measurement of Au ions produced from the dissolution of the gold nanoparticles previously captured on the test line. The analytical performances obtained at LFIA and eLFIA were compared, highlighting how the use of differential pulse voltammetry allowed for a lower detection limit (2.5-fold), respectively, 0.38 and 0.15 ng/mL, but increasing the time of analysis. Although the correlation between the two architectures confirmed the satisfactory agreement of outputs, this technical note has been thought to provide the reader a fair statement with regard to the strength and drawbacks about combining the two (apparently) competitor devices in a diagnostics field, namely, LFIA and electrochemical strips.

Simultaneous rapid detection of glycyrrhizin and sennoside A in Daiokanzoto samples by lateral flow immunoassay.

Glycyrrhizin (GLY) and sennoside A (SA) are characteristic bioactive marker compounds of the Kampo medicine Daiokanzoto. Their accurate detection in blends of Rhei rhizoma and Glycyrrhizae radix of several species (4:1 or 4:2) is essential for quality control and to ensure therapeutic efficacy. A rapid, efficient assay can significantly facilitate their detection. To establish a rapid qualitative assay for GLY and SA detection, a lateral flow immunoassay (LFA) was developed using specific monoclonal antibody (mAb) nanoparticles. This assay harnesses the competitive binding of mAb nanoparticles to the immobilized analytes on test strips and free analytes in the samples. Two conjugates for detecting GLY and SA, GLY-bovine serum albumin and SA-human serum albumin, were separately immobilized on the test zones of LFA strips. The detection mechanism is reliant on the visual detection of color changes in the test zones. When GLY and SA were present in samples, they contended with the immobilized conjugates on the strip to bind with the mAb nanoparticles and produced distinct color patterns in the test zones. The limits of detection of the assay for GLY and SA were both 3.13 μg/mL. The capability of the LFA was substantiated using plant samples and Daiokanzoto, and its alignment with indirect competitive ELISA results was confirmed. The introduced LFA is a groundbreaking procedure that offers a rapid, straightforward, and sensitive method for simultaneously detecting GLY and SA in Daiokanzoto samples. It is instrumental in ensuring product quality.

A sensitive gold nanoparticle-based lateral flow immunoassay for quantitative on-site detection of Salmonella in foods

Salmonella contamination is a leading cause of foodborne infection outbreaks worldwide. Therefore, detecting this bacterium is crucial for public health. Conventional detection methods, while specific and sensitive, are costly, time-consuming, labor-intensive, and require skilled analysts, impacting various industries, particularly the food supply chain. Lateral Flow Immunoassays (LFA) present a promising solution to address the need for a fast, user-friendly, sensitive, and specific detection method. This method overcomes various challenges by providing quick results and being easy to handle, cost-effective, and portable. This study aimed at developing a gold nanoparticle-based lateral flow immunoassay for Salmonella detection in food. Gold nanoparticles coated with anti-Salmonella polyclonal antibodies were characterized using UV–Vis, Raman, and Fourier-transform infrared spectroscopy, in addition to dynamic light scattering, X-ray diffraction, and transmission electron microscopy. Sandwich-type lateral flow immunoassay strips were then constructed and the test optimized for factors like optical density, antibody concentration in the detection zone, incubation time, blocking solutions, and result reading time. The test's selectivity was confirmed by examining its efficiency in detecting Escherichia coli and Staphylococcus aureus, while specificity was assessed against different Salmonella serovars. The developed biosensor displayed superior selectivity, specificity, and sensitivity compared to existing commercial tests. It had a detection limit of 103 CFU·mL−1, providing results in just 15 min after inserting the strip into the sample. Moreover, it successfully detected the presence of Salmonella in contaminated chicken, black pepper, milk, chocolate, and egg samples. This device offers a promising alternative for screening Salmonella in food, potentially enhancing food safety.

Development of lateral flow immunoassay strip for rapid detection of acute hepatopancreatic necrosis disease-causing PirAB toxins in shrimp

Acute hepatopancreatic necrosis disease (AHPND) is currently the most affected disease of shrimp farms worlwide As rapid and specific detection methods are essential to improve disease management, a lateral flow immunoassay (LFIA) was designed for the detection of PirAvp and PirBvp toxins in shrimp. In this study, polyclonal antibody based lateral flow immunoassay (LFIA) strip has been developed. The recombinant PirAvp and PirBvp toxin‐like proteins of Vibrio parahaemolyticus (VPAHPND) was used to immunize rabbits with the highest antibody titre after the third booster. This polyclonal rabbit antiserum was purified and used to develop a LFIA strip for the detection of AHPND pathogen. This test strip could detect PirAvp and PirBvp toxins up to 125 ng/mL by naked eye within 15 min. No cross-reactivity was observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. This LFIA strip could be used as a handy tool by the farmers and technicians in situ.
 ® 2023 Journal of Science and Technology - NTTU

Pen direct writing of SERRS-based lateral flow assays for detection of penicillin G in milk.

Direct pen writing offers versatile opportunities for development of low-cost tests for point-of-care applications. In this work a lateral flow immunoassay (LFIA) test was fabricated by hand "writing" immunoprobes onto hand-cut nitrocellulose strips with a commercial fountain pen. The qualitative capabilities of the test were extended by addition of a Raman reporter and consequent design and fabrication of a Surface Enhanced Resonant Raman Scattering (SERRS)-LFIA test. As proof-of-concept, dual detection of penicillin G was achieved in milk with a visual LOD of 20 ppm and a dynamic range of 0.03-97.5 ppm. Evaluation against equivalent tests performed with conventionally prepared LFIA strips showed comparable results, thus demonstrating the validity of the test. These results demonstrate the potential for further decrease in cost and consequent broader use of LFIA tests in remote regions and resource-limited environments.

Development of a quantum dot-based lateral flow immunoassay strip for rapid and sensitive detection of SARS-CoV-2 neutralizing antibodies

To a certain extent, the development and vaccination of COVID-19 vaccine have reduced the alarming rate of transmission speed and mortality rate. At present, vaccine coverage is quite high in countries around the world. Since individual differences are unavoidable, it is necessary to assess the efficacy of the vaccine in each vaccinated person in order to reflect the protective effect of the vaccine in different populations. In this study, we developed a novel COVID-19 neutralizing antibody detection kit combining lateral flow immunochromatography and novel quantum dot technology with 85.23% sensitivity, 92.50% specificity. The novel QD-ICA could achieve an accurate detection of SARS-CoV-2 neutralizing antibodies with 10 minutes, two steps, small equipment size, and broad testing application, suggesting its capability to assess vaccine effectiveness on a large scale in areas of world that currently affected by the pandemic.

Serodiagnosis of multiple cancers using an extracellular protein kinase A autoantibody-based lateral flow platform

Extracellular protein kinase A autoantibody (ECPKA-AutoAb) has been suggested as a universal cancer biomarker due to its higher amounts in serum of several types of cancer patients than that of normal individuals. Herein, we first developed a lateral flow immunoassay (LFIA) tool, using a sandwich format, toward ECPKA-AutoAb in human serum. For this format, 3G2 as a capture antibody was identified using hybridoma technique and a series of screenings where it showed superior capacity to recognize Enzo PKA catalytic subunit alpha (Cα), compared to other PKA antibodies and antigens. Using these components, we performed sandwich ELISA toward a mimic and real sample of ECPKA-AutoAb. As per the results, limit of detection (LOD) was found to be 135 ng/mL and ECPKA-AutoAb levels were higher in various cancer patients than in normal individuals like previous studies. Based on these results, we applied this sandwich format into LFIA tool and found that the LOD of the fabricated LFIA tool showed about 3.8 ng/mL using spiked PKA-Ab, which is significantly improved compared to the LOD of sandwich ELISA. Also, the developed LFIA tool demonstrated a remarkable ability to detect significant differences in ECPKA-AutoAb levels between normal and cancer patients within 15 min, showing a potential for point-of-care (PoC) detection. One interesting point is that our LFIA strip contains an additional conjugation pad II, named because of its position behind the conjugation pad, in which PKA Cα is dried, enabling a sandwich format.

Portable SERS-based lateral flow immunoassay strips with self-calibration for detection of a prostate cancer biomarker

Serum prostate-specific antigen (PSA) is thought to be the most vital indicator for early identification, staging and monitoring of prostate cancer (PCa). Although lateral flow immunoassay (LFA) has demonstrated limitless promise for point-of-care testing (POCT) and has drawn increasing interest and attention, there are significant restrictions on the assay's sensitivity and quantitative capability. Surface-enhanced Raman spectroscopy-based LFA (SERS-LFA) technique integrates the great sensitivity of SERS with the simplicity, rapidity, and usability of the LFA technique, making it ideal for POCT. In this paper, we propose a sensitive self-calibrating SERS-LFA platform, which uses Au@MBN@Ag@PSA NPs as a SERS immunoprobe for the specific recognition of PSA and loads graphene oxide/gold nanoparticles (GO/Au NPs) on nitrocellulose (NC) membranes as an internal standard (IS) molecule. The SERS-LFA platform's detection accuracy is increased through dynamic calibration of the characteristic signals, which is accomplished by detecting the immunoprobe and IS signals simultaneously and computing their respective signal intensity ratios. The self-calibrated SERS-LFA platform demonstrated a 1.65-fold improvement in reproducibility over the conventional SERS-LFA, and PSA could only be detected to a limit of 0.08 ng/mL, meaning that it was 3.5-fold lower than the conventional SERS-LFA platform. The spike recovery experiments yielded recoveries of 96.16∼104.53% with RSD values of 9.37%, 8.66%, and 3.49%, respectively, demonstrating that our self-calibrated SERS-LFA platform for PSA quantification has an acceptable level of precision and has great prospect for early screening of PCa and other diseases.

Pt@AuNF nanozyme and horseradish peroxidase-based lateral flow immunoassay dual enzymes signal amplification strategy for sensitive detection of zearalenone

Lateral flow immunoassay (LFIA) has been employed extensively for the rapid, accurate, and portable detection of foodborne toxins. Here, the platinum gold nanoflower core-shell (Pt@AuNF) nanozyme with excellent optical properties, good catalytic ability and controllable reaction conditions were prepared to effectively improve the performance of lateral flow immunoassay (LFIA) strips. The Pt@AuNF nanozyme and horseradish peroxidase (HRP) combined with monoclonal antibody were used as signal probes based on the dual enzymes catalytic signal amplification strategy to detect Zearalenone sensitively. Dual enzymes catalyze the decomposition of hydrogen peroxide into hydroxyl radicals, and under the influence of hydroxyl radicals, colorless 3,3′,5,5′ -tetramethylbenzidine (TMB) is oxidized to blue ox-TMB, which is superimposed on the strips for signal amplification to broaden the detection range. The limit of detection (LOD) of the Pt@AuNF-HRP labeled LFIA strips after signal amplification was 0.052 ng/mL, and the detection range was 0.052–7.21 ng/mL. Compared with the Pt@AuNF labeled strips, while reducing the probes amount by half to achieve antibody conservation, the detection range was expanded by 5-fold based on achieving improved sensitivity. The study provided a meaningful reference for expanding the detection range based on immunoassay.

Lateral flow immunoassay based on gold nanoparticles for rapid and sensitive detection of zoxamide in grape, tomato and cucumber samples.

In the study, we discovered zoxamide hapten (ZOX-hapten) by introducing a carboxyl extension chain, combined it with protein to make a complete antigen to immunize mice, and generated a monoclonal antibody (mAb) against ZOX. To identify ZOX residues in grape, tomato, and cucumber samples, we used our anti-ZOX mAb to develop a lateral flow immunoassay (LFIA) strip. In grape, tomato, and cucumber samples, the calculated detection limit of the LFIA strip in grape, tomato and cucumber samples was 3.44, 4.78 and 3.53ng/g, respectively. Using the LFIA strip, the recovery rate from grape samples was 96.4-106.8%, and that from tomato samples was 98.4-107.5%, while the recovery from cucumber samples was 99.4-111.3%. These results showed that our LFIA strip could be expected to achieve rapid screening of ZOX residues in fruits and vegetables.

High extinction coefficient material combined with multi-line lateral flow immunoassay strip for ultrasensitive detection of bacteria.

Lateral flow immunoassay strips (LFIAs) are a reliable and point-of-care detection method for rapid monitoring of bacteria, but their sensitivity was limited by the low extinction coefficient of colloidal gold nanoparticles (Au NPs) and low capture efficiency of test-line. In this study, polydopamine nanoparticles (PDA NPs) were employed to replace Au NPs, due to their high extinction coefficient. And the amount of test-line was increased to 5 for further improving the efficiency of bacteria capture. Thus, under visual observation, the detection limits of PDA-based LFIAs (102 CFU/mL) were about 2 orders of magnitude lower than Au-based LFIAs (104 CFU/mL). Furthermore, the invisible signal could be collected by Image J and the detection limit can reach 10CFU/mL. The proposed test strips were successfully applied for the quantitative, accurate, and rapid screening of E. coli in food samples. This study provided a universal approach to enhance the sensitivity of bacteria LFIAs.

Ratiometric electrochemical lateral flow immunoassay for the detection of Streptococcus suis serotype 2

An electrochemical lateral flow immunoassay (eLFIA) strip with high reproducibility was developed to rapidly and accurately detect Streptococcus suis serotype 2. This proposed strip was fabricated by integrating ratiometric electrochemical detection and LFIA (R-eLFIA). The R-eLFIA exhibited excellent reproducibility, which was improved by 3.8 times compared to a single electrode. A dual-working screen-printed graphene electrode (SPGE) was designed by tuning the working electrode with electroactive species in the biosensing system. Ferrocene carboxylic acid (Fc) was used as a signal probe, and sunset yellow (SY) at one working electrode was used as an internal reference signal to provide a built-in correction for reducing the effects of inherent background current. S. suis serotype 2-specific antibodies were immobilized on a nitrocellulose membrane of LFIA, which is located on the position of Fc-SPGE. In the presence of the analyte, an immunocomplex formed on the region of Fc-SPGE, causing a decrease in Fc current while SY current remained constant. The current ratio's decrease was proportional to S. suis serotype 2's concentration. Under optimization, this biosensor showed good linearity in the range of 102–1010 CFU/mL with a limit of detection of 10 CFU/mL and achieved a rapid detection time (15 min). Moreover, the R-eLFIA biosensor exhibited excellent reproducibility and high selectivity and was applied in human serum samples. Thus, this study successfully matched the advantages of the ratiometric strategy and LFIA and has great potential to be used as an effective tool for rapidly detecting S. suis serotype 2 in clinical samples.

Modified polysulfone membrane facilitates rapid separation of plasma from whole blood for an effective anti-SARS-CoV-2-IgM diagnosis

During the outbreak of coronavirus, RT-PCR was the premier gold standard method for severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) diagnosis. However, the sophisticated procedure of RT-PCR persuades researchers to develop sustainable point-of-need immunoassay methods for tracing unwitting carriers of SARSCoV-2. Herein, by fabricating a modified polysulfone (MPSF) membrane, we developed an integrated radial flow immunoassay (IRFIA) platform as a point-of-care system, capable of multiplying the immunoassays at a short run time. The target molecule is the SARSCoV-2 IgM in separated plasma. Although the lateral flow immunoassay kits for the rapid identification of Covid-19 have already been commercially developed but, the proposed method is superior to the conventional lateral flow immunoassay. In the newly designed membrane system, we have combined the five membranes of prevalent lateral flow immunoassay (LFIA) strips in one polymeric membrane. The MPSF membrane is capable of separating plasma from whole blood sample, which will reduce the interference of red colour of hemoglobin with generated signal and enhance the immunoassay precision. The efficiency of plasma separation, reached the mean value of 97.34 v/v% in 5 s. Furthermore, the gel electrophoresis results of the separated plasma contrasted with centrifuged plasma sample, demonstrated more efficient separation by the membrane. Using the MPSF membrane, signal generation time reduced from about 20 min in conventional rapid test strip for Covid-19 to about 7 min in IRFIA platform. The sensitivity and specificity of the membrane platform were determined to be 89% and 90%, respectively and a Kappa coefficient of 0.79 showed reliable agreement between the RT-PCR and the membrane system.

Highly bright and stable CsPbBr3 perovskite nanocrystals with amphiphilic polymer binding based dual-readout lateral flow immunoassay for detection of carcinoembryonic antigen

Rapid and highly sensitive detection of tumor marker (TM) is critical for the early diagnosis and treatment of cancers. Herein, utilizing highly bright and water-stable CsPbBr3 perovskite nanocrystals (NCs) capped with amphiphilic polymer ligand of octylamine-modified polyacrylic acid (OPA) and gold nanoparticles (AuNPs) as reporters, a lateral flow immunoassay (LFIA) strip is developed for fluorescence and colorimetric dual-mode detection of carcinoembryonic antigen (CEA). The prepared CsPbBr3 NCs capped by an amphiphilic polymeric of OPA ligand showed high stability and bright fluorescence. Moreover, the AuNPs immunoprobes were captured with CEA antigen and quench the green fluorescence of CsPbBr3/OPA NCs on the T line due to the inner filter effect (IFE). Therefore, CEA could be quantitative analyzed by the dual-readout of fluorescence and colorimetric signal. The detection limits of CEA can reach as low as 0.023 ng/mL and 0.027 ng/mL for the fluorescence and colorimetric mode, respectively. Good specificity and reproducibility were also demonstrated for this method. Finally, the CsPbBr3/OPA NCs-based LFIA showed good accuracy in detection of CEA level from clinical serum samples. This work firstly enables the application of CsPbBr3 perovskite NCs in a LFIA, displaying great potential in point-of-care clinical diagnosis.

Urchin-Shaped Au-Ag@Pt Sensor Integrated Lateral Flow Immunoassay for Multimodal Detection and Specific Discrimination of Clinical Multiple Bacterial Infections.

A new lateral flow immunoassay strip (LFIA) combining sensitive detection and identification of multiple bacteria remains a huge challenge. In this study, we first developed multifunctional urchin-shaped Au-Ag@Pt nanoparticles (UAA@P NPs) with a unique combination of colorimetric-SERS-photothermal-catalytic (CM/SERS/PT/CL) properties and integrated them with LFIA for multiplexed detection and specific discrimination of pathogenic bacteria in blood samples. Unlike the conventional LFIA that relied on antibody (Ab), this novel LFIA introduced 4-mercaptophenylboronic acid (4-MPBA) as an ideal Ab replacer that was functionalized on UAA@P NPs (UAA@P/M NPs) with outstanding binding and enrichment capacities toward bacteria. Taking Staphylococcus aureus (S. aureus) as model bacteria, the limit of detection (LOD) was 3 CFU/mL for SERS-LFIA, 27 CFU/mL for PT-LFIA, and 18 CFU/mL for CL-LFIA, three of which were over 330-fold, 37-fold, and 55-fold more sensitive than ordinary visual CM-LFIA, respectively. Besides, this SERS-LFIA is capable of identifying three types of bacterial spiked blood samples (E. coli, S. aureus, and P. aeruginosa) effectively according to specific bacterial Raman "fingerprints" by partial least-squares-discriminant analysis (PLS-DA). More importantly, this LFIA was successfully applied to blood samples with satisfactory recoveries from 90.3% to 108.8% and capable of identifying the infected patients (N = 4) from healthy subjects (N = 2) with great accuracy. Overall, the multimodal LFIA incorporates bacteria discrimination and quantitative detection, offering an avenue for early warning and diagnosis of bacterial infection.

Enhanced sensitivity and accuracy via gold nanoparticles based multi-line lateral flow immunoassay strip for Salmonella typhimurium detection in milk and orange juice

Food borne pathogens threaten food safety and affect human health. The lateral flow immunoassays (LFIAs) are widely concerned because of simplicity, low cost and user friendliness, and have broad application prospects in pathogen detection. However, the sensitivity of LFIAs is limited. Herein, multi-line LFIAs are introduced into pathogen detection for the first time. Compared with traditional single-line LFIAs, the overall signal strength of multi-line LFIAs has been significantly improved. It is particularly noteworthy that multi-line LFIAs detection accuracy of 103 CFU/mL pathogen has been improved by about 55%. The proposed multi-line LFIAs reduce the possibility of judging a positive result as a false negative result. The LFIAs strip was validated in real samples of milk and orange juice. This strategy has great potential for rapid detection of pathogens in real samples, and provides new insights for improving the accuracy and sensitivity of LFIAs strips.

Development of a Lateral Flow Immunoassay Based on a Highly Specific Monoclonal Antibody To Detect 4-Methylaminoantipyrine.

To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues, especially metabolites of target compounds, the preparation of highly specific antibodies is crucial. Preserving the characteristic structure of a target compound when designing a hapten is important when preparing highly specific antibodies. Here, we designed a novel hapten, 4-(((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4yl)amino)methyl)benzoic acid, named AA-BA, to improve the specificity of antibodies for detection of 4-methylaminoantipyrine (MAA), a residual marker of dipyrone, an important antipyretic-analgesic and anti-inflammatory drug. The structural features of the hapten remained almost the same as those of MAA. After experimental validation, monoclonal antibody 6A4 (mAb 6A4) was prepared with the half maximal inhibitory concentration (IC50) value of 4.03 ng/mL and negligible cross-reactivity with dipyrone metabolites and other antibiotics. In addition, a specific lateral flow immunoassay (LFA) strip based on colloidal gold was developed for screening MAA with a cutoff value of 25 ng/mL in milk. The developed LFA is a useful tool for rapid and accurate detection of MAA.