Lateral Flow Immunoassay Test Strips Research Articles

A novel integrated lateral flow immunoassay platform for the detection of cardiac troponin I using hierarchical dendritic copper-nickel nanostructures

Cardiac troponin I (cTnI) is a critical biomarker for the diagnosis of acute myocardial infarction (AMI). Herein, we report a novel integrated lateral flow immunoassay (LFIA) platform for highly sensitive point-of-care testing (POCT) of cTnI using hierarchical dendritic copper-nickel (HD-nanoCu-Ni) nanostructures. The electrodeposited HD-nanoCu-Ni film (∼22 μm thick) on an ITO-coated glass substrate exhibits superior capillary action and structural integrity. These properties enable efficient sample transport and antibody immobilization, making it a compelling alternative to conventional multi-component paper-based LFIA test strips, which are often plagued by structural fragility and susceptibility to moisture damage. The biofunctionalized HD-nanoCu-Ni substrates were laser-etched with lateral flow channels, including a sample loading/conjugate release zone, a test zone, and a control zone. Numerical simulations were used to further optimize the design of these channels to achieve optimal fluid flow and target capture. The HD-nanoCu-Ni LFIA device utilizes a fluorescence quenching based sandwich immunoassay format using antibody-labeled gold nanoparticles (AuNPs) as quenchers. Two different fluorescent materials, fluorescein isothiocyanate (FITC) and CdSe@ZnS quantum dots (QDs), were used as background fluorophores in the device. Upon the formation of a sandwich immunocomplex with cTnI on the HD-nanoCu-Ni device, introduced AuNPs led to the fluorescence quenching of the background fluorophores. The total assay time was approximately 15 min, demonstrating the rapid and efficient nature of the HD-nanoCu-Ni LFIA platform. For FITC, both inner filter effect (IFE) and fluorescence resonance energy transfer (FRET) contributed to the AuNP-mediated quenching. In the case of CdSe@ZnS QDs, IFE dominated the AuNP-induced quenching. Calibration curves were established based on the relationship between the fluorescence quenching intensity and cTnI concentration in human serum samples, ranging from 0.5 to 128 ng/mL. The limits of detection (LODs) were determined to be 0.27 ng/mL and 0.40 ng/mL for FITC and CdSe@ZnS QDs, respectively. A method comparison study using Passing-Bablok regression analysis on varying cTnI concentrations in human serum samples confirmed the equivalence of the HD-nanoCu-Ni LFIA platform to a commercial fluorescence cTnI LFIA assay kit, with no significant systematic or proportional bias observed.

Conjugation of tetracycline with carrier proteins and production of its polyclonal antibody for the development of rapid test

Tetracycline (TC) is a broad-spectrum antibiotic used in the treatment of several infectious diseases. The excessive use of tetracycline antibiotics, especially tetracyclines in livestock, has raised concerns about residues of tetracycline antibiotics in food that may affect consumers' health. It is necessary to develop a rapid test strip for detecting tetracycline antibiotic residues in food and animal feed. This paper presents the suitable conditions for the conjugation of tetracycline to BSA or KLH: molar ratio of TC:BSA/KLH was 25:1, formaldehyde concentration of 5 %, temperature of 4oC and reaction time of 4 hours. The KLH-TC conjugate was used to successfully raise polyclonal antibodies against TC in rabbits. The anti-TC polyclonal antibodies were purified and could be used for the development of the lateral flow immunoassay test strip for the detection of tetracycline.

A Magnetic-Optical Triple-Mode Lateral Flow Immunoassay for Sensitive and Rapid Detection of Respiratory Adenovirus.

Human respiratory adenovirus (ADV) is a highly infectious respiratory virus with potential for pandemics. There are currently no specific drugs to treat ADV worldwide, so early rapid detection of ADV infection is essential. In this study, we developed an innovative magnetic-optical triple-mode lateral flow immunoassay (LFIA) using magnetic quantum dots as immunomarkers. This novel approach addresses the need for rapid and accurate ADV detection, allowing for multimodal quantitative/semiquantitative analysis of magnetic, fluorescent, and visible signals within a mere 15 min. The lower limit of detection (LOD) for magnetic, fluorescent, and visual signals was determined to be 5.6 × 103, 1.2 × 103, and 1.95 × 104 copies/mL, respectively. The detection range for ADV using this approach was 1.2 × 103-5 × 107 copies/mL. Additionally, semiquantitative analysis, which is user-friendly and does not necessitate specialized equipment, was successfully implemented. Notably, seven respiratory viruses showed no cross-reactivity with the generated LFIA test strips. The intrabatch repeatability exhibited a coefficient of variation (CV) of less than 5%, while the interbatch repeatability had a CV of less than 15%. Furthermore, recovery values ranged from 95% to 106.8% for samples analyzed concurrently with dual signals at the same spiking concentration. The assay developed in this study boasts a wide detection range and exceptional sensitivity and specificity. This technique is exceptionally well-suited for on-site rapid detection, with the potential for personal self-testing and early ADV infection diagnosis. Its versatility extends to a broad array of application scenarios.

Understanding sensitivity and cross-reactivity of xylazine lateral flow immunoassay test strips for drug checking applications.

The continued prevalence of xylazine in the illicit drug market has necessitated development of quick and simple methods for identification, including lateral flow immunoassays (also known as "test strips"), like those frequently used to detect fentanyl. This study explored the drug checking applicability of the first publicly available xylazine test strips (XTS) using four sub-studies: reproducibility (i.e., consistency of positive results in a highly-concentrated xylazine solution); limit of detection on a calibration curve of xylazine concentrations; cross-reactivity against 77 commonly encountered drugs, cutting agents, and other structurally similar compounds; and applicability for analyzing community-acquired samples-where 100 drug residue samples were analyzed using XTS, direct analysis in real time mass spectrometry (DART-MS), and gas chromatography tandem mass spectrometry (GC-MS/MS). XTS consistently detected xylazine at concentrations ≥2.5μg/ml, and XTS results were reproducible. Sensitivity and specificity of XTS were calculated by comparing expected versus obtained results based on xylazine concentration of community-acquired samples measured by GC-MS/MS. XTS consistently detected xylazine in samples with concentration >2μg/ml and yielded a sensitivity of 0.974, specificity of 1.00, and overall accuracy of 0.986. Cross-reactivity with lidocaine, a common cutting agent, and lack of XTS reactivity with other α2 -agonists found in the illicit drug supply highlight the need to offer consumers comprehensive drug checking services that identify a range of substances and better inform them about drug contents.

A flexible gradient lateral flow immunochromatographic assay for qualitative, semi-quantitative, and quantitative determination of serum amyloid A

Serum amyloid A (SAA) is an acute-phase protein produced in response to inflammatory proteins during infections, inflammation, trauma, surgery, cancer, and other conditions. Early and accurate detection of SAA is necessary for diagnosis and monitoring of disease progression. To meet this need, we developed a gradient lateral flow immunoassay test strip using Au nanoparticles as signal reporters. The test strip has three test (T1, T2, and T3) lines with progressively decreasing concentrations of SAA antibody, enabling the determination of high, medium, and low concentrations of SAA in serum. The test strip results were analyzed using three distinct readout methods, each with different sensitivity, accuracy, and precision for SAA concentration measurements. Qualitative judgment is based on the color of the T1 line. Semi-quantitative assessment of SAA concentration is determined by the number of colored T-lines. Specifically, color development in T1 line alone indicates a concentration range of 10–50 μg/mL, while T1 and T2 lines together indicate a range of 50–100 μg/mL, and development in all three lines (T1, T2, and T3) indicates a concentration of >100 μg/mL. Quantitative analysis was performed using either smartphone imaging or image scanning with ImageJ software. By using a five-parameter logistic function, we found a strong correlation (R2 = 0.998) between the ratio of signal intensities of (T1 + T2 + T3) to the control (C) line and SAA concentrations ranging from 5 to 1000 μg/mL. At lower concentrations (0–100 μg/mL), we observed a proportional relationship between the value of (T1 + T2 + T3)/C and SAA concentration. The limit of detection for SAA was 9.33 ng/mL (or 6.53 μg/mL of SAA in undiluted serum samples) for the smartphone method and 3.06 ng/mL (or 2.14 μg/mL of SAA in undiluted serum samples) for the scanner method. The gradient test strip was highly consistent with a commercially available SAA immunochromatographic test strip when tested with real human serum samples. Passing-Bablok regression indicated that results obtained using the smartphone app and scanner methods of the gradient test strip were comparable to those obtained using the commercial test strip. The gradient test strip is flexible and adaptable, providing solutions for qualitative, semi-quantitative, and quantitative SAA measurements.

Rapid and portable bunyavirus SFTSV RNA testing utilizing catalytic hairpin assembly coupled with lateral flow immunoassay.

Severe fever with thrombocytopenia syndrome (SFTS) is a prevalent, life-threatening, emergent infectious disease. Currently, reverse transcription-polymerase chain reaction is the gold standard for diagnosing SFTS virus (SFTSV) infection, which requires sophisticated equipment and professional personnel that are frequently unavailable in most SFTS endemic rural areas. Here, we reported a simple, rapid nucleic acid amplification system that combined the catalytic hairpin assembly (CHA) with a lateral flow immunoassay (LFIA) strip-based detection method for SFTSV detection. The detection of SFTSV RNA could be realized by generation of H1-H2 hybrid duplexes labeled with biotin and digoxin, which subsequently added to the LFIA test strips containing streptavidin conjugated with Alexa Fluor 647 as well as anti-digoxin antibodies. Our CHA-based LFIA assay offered high amplification efficiency and specificity with a detection limit of 1 aM. Crucially, this method enabled stable detection of 500 copies/mL of SFTSV within 30 min using clinical serum samples. Therefore, our CHA-based LFIA approach provided a potential useful tool to facilitate early and precise diagnosis of SFTS patients in poorly resourced SFTS endemic areas.IMPORTANCESevere fever with thrombocytopenia syndrome (SFTS) is an emerging and potentially fatal infectious disease prevalent in China. Here we report a simple, rapid nucleic acid amplification system, the catalytic hairpin assembly (CHA) in conjunction with a lateral flow immunoassay (LFIA) strip-based detection method for SFTS virus detection, which demonstrated high amplification efficiency and specificity with limit of detection of 1 aM. Most importantly, we also validate our CHA-based LFIA assay using the clinical serum samples, which was fully compatible with reverse transcription-PCR results. Therefore, our strategy provides a potential useful tool to facilitate early and precise diagnosis of SFTS patients especially in poorly resourced SFTS endemic areas.

P-675 Analysis of a digitally integrated ovulation date kit to determine ovulation precisely

Abstract Study question Do digitally integrated ovulation prediction kits (OPK) predict the fertile window and the ovulation date accurately? And can this prediction be proven by sonography? Summary answer Particularly for subfertile women with irregular cycles, OPKs can help predict their fertile window and ovulation day, in accordance with vaginal sonography. What is known already The fertile window is defined as the period of five days before ovulation including the day of ovulation itself. Prediction based only on cohort observations or historical datasets may be inaccurate for women with irregular cycles, particularly when unforeseen changes of ovarian function occur during reproductive life. Urinary luteinizing hormone (LH) assays and smartphone applications are already widely used to detect the optimal time for conception, but can only predict a likelihood of ovulation without proof. Study design, size, duration 100 healthy women between the ages of 21 and 45, cycle length up to 42 days, trying to conceive and without hormone intake were to be recruited in the gynecological outpatient clinics of the Technical University of Munich (TUM) and of the University of Ulm. Each participant entered the study for up to three consecutive cycles. Two vaginal ultrasound scans were performed per cycle – before and after the ovulation date prognosis by the app. Participants/materials, setting, methods After informed consent, participants downloaded the smartphone application “Pearl Fertility” and received OPKs consisting of four different sets of lateral flow immunoassay test strips (LFIA): for luteinizing hormone, follicle-stimulating hormone, progesterone and human chorionic gonadotropin. Home measurements with LFIAs were performed with morning urine. The phone camera feed analyzed the color information of the LFIAs. Three differently prioritized algorithms calculated the ovulation date. Two questionnaires evaluated patient baseline characteristics and their perception of the app. Main results and the role of chance 89 women were recruited. Mean age was 34.6 yrs. (SD ± 4.2). On average, the included women had tried to conceive for 7.5 (±5) months before joining the study. 8% (n = 7) became pregnant before the first cycle started.140 completed cycles in 71 remaining women were analyzed so far. 46% of the recruited women completed the study after 3 cycles, the drop-out-rate was 32.4%. The app predicted ovulation for cycle day 14.3 (mean, SD ± 3.5). In 25 cycles the date of ovulation was before day 12 and in 33 cycles on day 16 or later. The first ultrasound was done on cycle day 13.5 (mean, SD ± 3), the second ultrasound on cycle day 17.6 (mean, SD ± 3.6). In several cycles at least one (up to three) corrections of the prognosis occurred by the app, resulting in the day of predicted ovulation being moved either backward (retrospectively) or forward by 2.5 (mean, SD ± 1.9) days. Urinary progesterone measured with the OPK and post-ovulatory ultrasound were in accordance in the majority of cycles. 17% (n = 12) of the women became pregnant in the current analysis (mean duration to pregnancy: 1.3 cycles). In the ITT population (after informed consent) the rate was 23%. Limitations, reasons for caution This study was performed without a control group. Therefore, as an intraindividual control, a follow-up is planned to calculate the pregnancy rates and outcomes with and after the use of the app. Wider implications of the findings Our study shows that digitally integrated OPK are able to predict the fertile window and the ovulation date in a slightly subfertile cohort unselected for mild cycle irregularities. An early and accurate prediction may help couples to conceive, particularly if the date of ovulation is divergent from standard information. Trial registration number Not applicable

Utilization of a lateral flow colloidal gold immunoassay strip based on surface-enhanced Raman spectroscopy for rapid detection of glycinin

Glycinin is an important storage protein in soybean, but it can also lead to allergic reactions in humans. In this study, based on a low-cost, simple, rapid and portable lateral flow immunoassay test strip, combined with high sensitivity surface enhanced Raman spectroscopy (SERS) technology, a sandwich lateral flow immunochromatographic test strip for rapid detection of soybean allergen glycinin is established. In the experiment, colloidal gold was covalently conjugated with rabbit-derived polyclonal antibodies of glycinin and Raman probe molecule 4-aminothiophenol(4-PATP) to prepare the immunoprobe. The respective optimal PATP and optimal antibody labeling amounts of colloidal gold solution were 1.05 × 10-2mol/L and 4.6 × 10-8mol/L. The detection limit of the test strip for glycinin was 4.87 ng/mL. The recovery rate ranged from 91 to 107 % and the CV was between 3 and 10 %. The test strip underwent no cross-reaction with β-conglycinin, sesame protein, peanut protein, wheat protein or whey protein. The results of the experiment showed that this method exhibits high sensitivity and specificity.

Rapid detection of SARS-CoV-2: The gradual boom of lateral flow immunoassay.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is still in an epidemic situation, which poses a serious threat to the safety of people and property. Rapid diagnosis and isolation of infected individuals are one of the important methods to control virus transmission. Existing lateral flow immunoassay techniques have the advantages of rapid, sensitive, and easy operation, and some new options have emerged with the continuous development of nanotechnology. Such as lateral flow immunoassay test strips based on colorimetric-fluorescent dual-mode and gold nanoparticles, Surface Enhanced Raman Scattering, etc., these technologies have played an important role in the rapid diagnosis of COVID-19. In this paper, we summarize the current research progress of lateral flow immunoassay in the field of Severe Acute Respiratory Syndrome Coronavirus 2 infection diagnosis, analyze the performance of Severe Acute Respiratory Syndrome Coronavirus 2 lateral flow immunoassay products, review the advantages and limitations of different detection methods and markers, and then explore the competitive CRISPR-based nucleic acid chromatography detection method. This method combines the advantages of gene editing and lateral flow immunoassay and can achieve rapid and highly sensitive lateral flow immunoassay detection of target nucleic acids, which is expected to be the most representative method for community and clinical point-of-care testing. We hope that researchers will be inspired by this review and strive to solve the problems in the design of highly sensitive targets, the selection of detection methods, and the enhancement of CRISPR technology, to truly achieve rapid, sensitive, convenient, and specific detection of novel coronaviruses, thus promoting the development of novel coronavirus diagnosis and contributing our modest contribution to the world's fight against epidemics.

Using an ultra-compact optical system to improve lateral flow immunoassay results quantitatively

The lateral flow immunoassay (LFIA) is a paper-based platform with extensive application in point-of-care (POC) testing and many fields. However, its clinical application is severely limited due to the lack of quantitative ability of standard LFIA tests; this augmentation provides the system with quantifying the signal from magenta-colored AuNPs. To address this issue, we proposed an ultra-compact optical system that allowed LFIAs to be performed more accurately and objectively. The experimental setup consisted of multiple optical accessories manufactured by 3D printing (STEP files were included). A high-resolution printer was used to print out a magenta card model for the LFIA, whose color code, ranging from 255, 255, 255 to 255, 0, 255 in the RGB (red, green, blue) format, represents different levels of magenta color intensity (from 0% to 100%) and thus the results of LFIA test strips. A mathematical model was built using a calibration curve to describe the relationship between magenta color value and reflectance spectrum. In addition, a spectrum module was integrated into the proposed system to identify and quantify LFIA results. This integration represents a pioneering step in developing portable detection techniques that facilitate quantifying LFIA results. Finally, we expect this ultra-compact optical spectroscopy system to have great potential for novel clinical applications.

P-637 Evaluation of a digitally integrated ovulation prediction kit with proof of ovulation – a pilot study

Abstract Study question Is a digital ovulation prediction kit (OPK) with integrated progesterone measurements a useful tool in predicting and proving ovulation? Summary answer The digitally integrated OPK with progesterone integration may be a helpful tool for couples to identify the woman’s fertile window and detect anovulation. What is known already Apart from detecting the coming fertile window (i.e. the period of the highest probability of conception during the menstrual cycle, including the 5 days previous to and the day of ovulation itself), knowledge whether the likely ovulation indeed occurs, is crucial for achieving pregnancy. As women’s hormone profiles show intra- and interindividual variations, an individual prediction of the fertile window is a promising approach for couples, particularly in early premature ovarian insufficiency and perimenopause. Information whether the presumed ovulation has actually happened is not provided by most cycle apps. Study design, size, duration Healthy women, aged 21 to 44 years, who had not been taking hormonal contraceptives for at least three months were recruited via social media, paper postings and hand-outs. Women with infertility for more than 24 months were excluded. Up to three cycles were measured in every woman in case of a lacking pregnancy. Participants/materials, setting, methods Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH) in first morning urine were gaged via lateral flow immunoassay test strips (LFIA) (RunBio Biotechnology Ltd.). Three algorithms of the smartphone application Pearl Fertility (iOS 1.0.3-1.0.5, Colorimetrix GmbH) were applied to predict the day of ovulation. Photographic visual control of the LH test strips, vaginal ultrasound close to the predicted ovulation and progesterone (PdG LFIA) were used to confirm ovulation, human chorionic gonadotropin (HCG LIFA) to confirm pregnancy. Main results and the role of chance 36 cycles of 19 women recruited for this pilot study were started. 6 participants dropped out or were excluded, leaving 25 evaluable cycles in 13 healthy women, average age 37 years (range 30 to 44), who had not been taking hormonal contraceptives for at least three months. 76 % of the analysable cycles had corresponding prediction of ovulation by algorithm and by visual control of the measured maximum value of LH (OVV). OVV corresponded with the day of ovulation determined retrospectively by length of luteal phase in 75 % (Bland-Altman plot (acceptable deviation Δ≤|2| days)). Prediction of ovulation differed by 2.12±3.13 days (mean±SD) between OVV and prospective cycle-length-based data (Wilcoxon signed-rank test (p < 0,05)). The PdG LIFA suggested ovulation in 84 % of the cycles with presumed ovulation by postovulatory PdG measurement. 6 of the participating women got pregnant during the study. Analysis of cumulative rate of pregnancy with 95 % confidence interval resulted in 25±21 %, 40±19 % and 40±17 % within the first, second and third cycle. Limitations, reasons for caution In order to make valid statements about the benefit of digitally integrated OPKs, further research with a larger population is needed. Perimenopausal women and patients with irregular cycles may specifically benefit from individual self-monitoring. Wider implications of the findings The knowledge of the fertile window is expected to help fertile couples to achieve pregnancy and proof of ovulation or anovulation may detect infertility due to anovulation sooner, thereby reducing the interval until diagnosis and treatment. Trial registration number 'not applicable'

Sensitive detection of hepatitis C virus using a catalytic hairpin assembly coupled with a lateral flow immunoassay test strip

Currently, PCR is the gold standard for the detection of hepatitis C virus (HCV). However, the PCR technique is complicated and time-consuming, which prevents its application and, clinical point-of-care testing (POCT). Herein, we report a POCT method with simplicity, high sensitivity and specificity, which consists of a catalytic hairpin assembly (CHA) signal amplification system coupled with a lateral flow immunochromatographic (LFIA) test strip for the detection of HCV. Two ingeniously designed hairpin probes were hybridized to form the H1–H2 duplex in the presence of the target DNA. The catalytic hairpin assembly which was characterized of isothermal and enzyme-free, was accomplished within 40 min and the reaction was then applied to a LFIA test strip. Only the H1–H2 duplex labeled with both digoxin and biotin could be captured by the test strip, and the fluorescence value was determined. In addition, we evaluated the application potential for the detection of clinical samples. The reported method demonstrated high sensitivity with a detectable minimum concentration at 1 fM and showed a good linear range from 10 nM to 10pM, and high specificity for various mismatched sequences. The results demonstrated that clinically positive samples could be successfully detected. In conclusion, the reported method is simple, rapid, and free of large-scale equipment. POCT is expected to be useful for HCV detection in clinic.

IQVision: An Image-Based Evaluation Tool for Quantitative Lateral Flow Immunoassay Kits.

The development of quantitative lateral flow immunoassay test strips involves a lot of research from kit manufacturers’ standpoint. Kit providers need to evaluate multiple parameters, including the location of test regions, sample flow speed, required sample volumes, reaction stability time, etc. A practical visualization tool assisting manufacturers in this process is very much required for the design of more sensitive and reliable quantitative LFIA test strips. In this paper, we present an image-based quantitative evaluation tool determining the practical functionality of fluorescence-labelled LFIA test cartridges. Image processing-based algorithms developed and presented in this paper provide a practical analysis of sample flow rates, reaction stability times of samples under test, and detect any abnormalities in test strips. Evaluation of the algorithm is done with Glycated Hemoglobin (HbA1C) and Vitamin D test cartridges. Practical sample flow progress for HbA1C test cartridges is demonstrated. The reaction stability time of HbA1C test samples is measured to be 12 min, while that of Vitamin D test samples is 24 min. Experimental evaluation of the abnormality detection algorithm is carried out, and sample flow abnormalities are detected with 100% accuracy while membrane irregularities are detected with 96% accuracy.

Replacement of antibodies with bacteriophages in lateral flow assay of Salmonella Enteritidis

In this study, the analytical performance of bacteriophages for Salmonella Enteritidis was investigated using lateral flow assay (LFA) technique. The analytical performance characteristics of bacteriophages were compared with antibodies which are regularly used as analyte-specific agents in the lateral flow immunoassay test strip. Bacteriophages could be an alternative analyte-specific agents to antibodies in lateral flow assay testing of bacteria since they offer comparable sensitivity, specificity, and accuracy. In the present study, Surface Enhanced Raman Spectroscopy (SERS) and colorimetric measurements were combined in one platform and sensitive quantitation of target bacteria was accomplished with a total quantitative analysis time of less than 30 min. The developed Salmonella Enteritidis F5-4 phage-based LFA specifically responds to Salmonella Enteritidis, while lower SERS responses to different bacteria types including Bacillus subtilis, Micrococcus luteus, Escherichia coli, Salmonella Typhimurium were observed. The developed test strips were also applied for the determination of Salmonella Enteritidis in spiked chicken and egg samples.

Point-of-care test system for detection of immunoglobulin-G and -M against nucleocapsid protein and spike glycoprotein of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) epidemic continues to ravage the world. In epidemic control, dealing with a large number of samples is a huge challenge. In this study, a point-of-care test (POCT) system was successfully developed and applied for rapid and accurate detection of immunoglobulin-G and -M against nucleocapsid protein (anti-N IgG/IgM) and receptor-binding domain in spike glycoprotein (anti-S-RBD IgG/IgM) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Any one of the IgG/IgM found in a sample was identified as positive. The POCT system contains colloidal gold-based lateral flow immunoassay test strips, homemade portable reader, and certified reference materials, which detected anti-N and anti-S-RBD IgG/IgM objectively in serum within 15 min. Receiver operating characteristic curve analysis was used to determine the optimal cutoff values, sensitivity, and specificity. It exhibited equal to or better performances than four approved commercial kits. Results of the system and chemiluminescence immunoassay kit detecting 108 suspicious samples had high consistency with kappa coefficient at 0.804 (P < 0.001). Besides, the levels and alterations of the IgG/IgM in an inpatient were primarily investigated by the POCT system. Those results suggested the POCT system possess the potential to contribute to rapid and accurate serological diagnosis and epidemiological survey of COVID-19.

A lateral flow-based portable platform for determination of reproductive status of cattle

Our objective was to develop and validate a tool integrating a disposable fluorescence-based lateral flow immunoassay (LFIA) coupled with a portable imaging device for estimating circulating plasma concentrations of progesterone (P4). First, we developed and optimized a competitive LFIA test strip to measure P4 in bovine plasma. The LFIA design included a sample pad, a conjugate pad that stores R-phycoerythrin-anti-P4 conjugates, a glass-fiber spacer pad, a nitrocellulose membrane with printed test and control lines, and a cellulose-fiber absorbent pad. To perform a test, 20 µL of plasma and 50 µL of running buffer were added on the sample pad. After 3 min, 45 µL of running buffer was added to initiate sample flow. After allowing 15 min to stabilize the colorimetric signal, strips were introduced in an LFIA portable reader wirelessly linked to a laptop to determine P4 concentration based on test-to-control-line signal (T/C ratio). In a series of experiments (n = 6), the ability of the LFIA to differentiate plasma samples with ≥1 or <1 ng/mL of P4 was evaluated. For each experiment, a calibration curve was constructed using plasma with known concentrations of P4 (0.1 to 3.7 ng/mL; n = 5). The resulting linear equation was then used to determine a T/C ratio cutoff to differentiate samples with ≥1 or <1 ng/mL of P4. In addition, to evaluate the ability of the platform to assign samples to P4 concentration groups without a calibration curve for individual batches, we performed a receiver operating characteristic analysis to identify a single cutoff value for T/C ratio that could potentially be used for all batches. Overall, calibration curves showed a linear relationship between T/C ratio and P4 levels (mean coefficient of determination = 0.74; range 0.42 to 0.99). Next, plasma samples from lactating dairy cows (n = 58) were tested in triplicate to determine the ability of the LFIA system to differentiate plasma samples with ≥1 or <1 ng/mL of P4 using a RIA for P4 as reference test. Overall, the LFIA assay correctly classified 90% of the samples, with 97% sensitivity, 83% specificity, 85% positive predictive value, and 96% negative predictive value. Agreement between the tests was substantial (kappa = 0.79; 95% confidence interval 0.64 to 0.95). When using a single cutoff value for T/C ratio selected by receiver operating characteristic analysis, sensitivity and specificity to determine CL presence were 97 (95% confidence interval 82 to 99) and 79% (95% confidence interval 60 to 92), respectively. These data suggest that the developed portable LFIA system can accurately differentiate plasma samples with ≥1 or <1 ng/mL of P4.

Development of a Lateral Flow Immunoassay using recombinant GRA7 antigen to detect Toxoplasma gondii IgG antibodies

Toxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis and is of medical importance in pregnant women and immunosuppressed patients. In recent years, many methods have been developed for the detection of infection caused by this parasite; however, most of the developed methods are not adequately sensitive. The dense granular antigen (GRA) 7 is a highly immunogenic protein that is used as a specific antigen for the diagnosis of toxoplasmosis. This study was designed to produce recombinant GRA7 (rGRA7) antigen in bacterial system in order to be applied as an antigen for developing a simple and rapid lateral flow immunoassay test strip using a gold nanoparticle-pAb conjugate probe to detect Toxoplasma IgG-specific antibodies in human sera. After the extraction of genomic DNA from RH strain tachyzoites, polymerase chain reaction (PCR) was performed using specific primers considering restriction sites and BglII and XhoI enzymes. Subsequently, the GRA7 gene was cloned in pET-32a (+) expression vector, and then pET-32a(+)-GRA7 was transformed into E. coli Rosetta (DE3). The induction of protein production was accomplished by IPTG, and the product was finally purified by Ni-NTA affinity chromatography. In order to make the strip test, the anti-human gold nanoparticle conjugate was applied on conjugate pad, rGRA7 antigen was immobilized to a nitrocellulose membrane as the capture agent, and sample and absorbance pads were assembled on a backing card. For the analysis of the sensitivity and specificity of the assay, the selected patients’ sera samples were tested by standard chemiluminescence immunoassay (CLIA) method, and then compared with TOXO-IgG strip results. The findings showed that the use of rGRA7 is an accurate, sensitive, and inexpensive technique for the rapid detection of anti-Toxo­plasma IgG in human sera. Therefore, rGRA7 can be applied as a diagnostic agent in laboratories.

Tobacco Use Classification by Inexpensive Urinary Cotinine Immunoassay Test Strips.

Urinary cotinine is one of the most commonly measured biomarkers reflecting recent exposure to nicotine. In some cases a simple qualitative dichotomization of smokers and non-smokers is all that is required. NicAlert® test strips have been evaluated for this purpose, but other recently introduced, inexpensive single-line test strips have not. In this study we evaluated two such strips with nominal cutoffs of 200 and 10 ng/mL. A total of 800 urine samples with known cotinine concentrations determined by an LC-MS-MS method were examined, including 400 urine samples ranging from 0.23 to more than 24,000 ng/mL by the 200 ng/mL strip, and 400 samples with concentrations <200 ng/mL by the 10 ng/mL cutoff strip. Both test strips performed well in these evaluations. Classification relative to LC-MS-MS by the 200 ng/mL strips had a sensitivity of 99.5% and specificity of 92%, with 95.8% accuracy. The 10 ng/mL strips had a sensitivity of 98.7% and specificity of 90.1%, with 93.3% accuracy. The positive predictive value for the 200 ng/mL strips was 92.6% and the negative predictive value was 99.5%. For the 10 ng/mL strips, the corresponding values were 85.4 and 99.2%, respectively. The prevalence of positive samples was 50% in the 200 ng/mL group, and 37% in the 10 ng/mL set. Each strip was read by two readers with an overall agreement of >98%. Our results suggest that these simple and inexpensive lateral flow immunoassay test strips can provide useful qualitative estimates of nicotine exposures for appropriate applications within the inherent limitations of sensitivity and precision of the immunoassay test strip format.

EFFICIENT CONJUGATION OF AFLATOXIN B1 WITH BOVINE SERUM ALBUMIN APPLYING FOR DEVELOPMENT OF AFLATOXIN B1 QUICK TEST

Aflatoxins are secondary metabolites mostly produced by Aspergillus flavus and Aspergillus parasiticus and found in agricultural foodstuff such as maize grains, peanuts, animal feeds,... These are toxic and cancerous agents for humans and animals. Among them, aflatoxin B1 is the most consideration due to its highest toxicity and presence in samples. Several methods were developed to detect aflatoxin in food, feed and other foodstuffs such as chromatographic methods (TLC, HPTLC, HPLC), LC-MS/MS, FTIR, RIA, ELISA, SPR, electrochemical, and immunodipstick. Among them only immunodipstick method is compatible for field usage. Therefore, aim of the present study is to determine optimal condition for conjugation of aflatoxin B1 (AFB1) with bovine serum albumin (BSA) to develop a lateral flow immunoassay test strip for detection of aflatoxin B1. Optimal conditions for generating mediate compound of aflatoxin B1 – CMO was investigated: AFB1:CMO ratio of 1:2, reflux temperature of 100oC for 2 hours. Concentration of aflatoxin B1 in reaction was 0.27 µg/µl. Conjugation of AFB1 with CMO was confirmed by thin layer chromatography, HPLC and FT-IR methods. Furthermore, optimal conditions for conjugation of mediate AFB1-CMO derivative with BSA were also investigated. Ratio of AFB1-CMO with BSA was determined as 40:1 and equal to predicted theoretical ratio. Efficient conjugation conditions were 25oC for 2 hours in bicarbonate buffer pH 9.5. Generated conjugate of AFB1-BSA was successfully applied to construct a lateral flow immunoassay test strip for detection of aflatoxin B1

IronPhone: Mobile device-coupled point-of-care diagnostics for assessment of iron status by quantification of serum ferritin

Iron deficiency (ID) is an urgent public health problem that has devastating effects on maternal and child health. However, due to poor access and affordability, screening and diagnosis for ID is often limited to proxy hemoglobin measurements alone. Here, we report the development and validation of ironPhone, a mobile-device coupled portable diagnostics for quantification of serum ferritin concentrations, an iron status biomarker, within a few minutes, from a drop of fingerprick blood. The ironPhone diagnostic platform comprises of a smartphone accessory, an app, and a disposable lateral flow immunoassay test strip to quantify serum ferritin. For initial validation in the lab, we optimized and evaluated the performance of ironPhone with known ferritin concentrations in spiked buffer and serum samples. Following lab validation, we performed a human validation by collecting fingerprick whole blood samples from 20 participants to assess iron status using ironPhone and compared the results with the laboratory standard IMMULITE 2000 analyzer. Findings from the ironPhone for the buffer and spiked serum samples provided a calibration curve with R2 values of 0.97 (n=27) and 0.93 (n=12), respectively. On comparison with the laboratory standard IMMULITE analyzer in whole blood samples, a correlation of 0.92 (P<0.0001) was observed with a sensitivity of over 90% for predicting ID (ferritin<15.0µg/L) via the ironPhone, demonstrating its promise for iron status assessment at the point-of-care.