Colorimetric Enzyme-linked Immunosorbent Assay Research Articles

A paper-based microfluidic platform with shape-memory-polymer-actuated fluid valves for automated multi-step immunoassays

Smart fluid manipulation with automatically controlled paper valves will enable automated and multi-step immunoassays on paper-based microfluidic devices. In this work, we present an integrated paper-based microfluidic platform with shape-memory polymer (SMP)-actuated fluid valves capable of automated colorimetric enzyme-linked immunosorbent assays (ELISAs). A single-layer microfluidic paper-based analytical device (μPAD) was designed to store all the reagents on the chip, and sequentially transfer reagents to a paper test zone following a specific ELISA protocol through automatic fluidic flow control by the multiple SMP-actuated valves. The actuation of a paper valve was based on the thermally responsive, duel-state shape transformation of a SMP sheet attached to the root of a paper cantilever beam for driving a hydrophilic paper bridge to connect and disconnect two paper channels. A portable colorimetric reader was developed to control the on-chip valve operations, quantify the colorimetric signal output, display the assay result, and wirelessly transmit the data to a smart phone for the application of telemedicine. Reliable operations of the paper valve and the entire μPAD were demonstrated with success rates of 97% and 93%, respectively. A detection mechanism for valve malfunction was designed and confirmed effective to identify any mal-operation of individual valves, thus rendering our platform reliable in real assays. For device calibration, we conducted direct ELISAs of rabbit IgG in phosphate-buffered saline (PBS), and achieved a low limit of detection (LOD) of 27 pM (comparable to that of standard and paper-based ELISAs). In order to demonstrate the clinical application of our multi-step immunoassay platform, we also conducted sandwich ELISAs to quantify the protein level of an inflammatory cytokine, namely tumor necrosis factor (TNF)-α, in surgically injured laryngeal tissues of rats. The protein levels of TNF-α were shown similar between the conventional and μPAD ELISAs.

Association between circulating zinc/ferritin levels and parent Conner’s scores in children with attention deficit hyperactivity disorder

ADHD is one of the most common neurobehavioral disorders among children and adolescents. In this prospective study, we aimed to measure circulating zinc and ferritin levels in children with ADHD, pick up the deficient ones to give zinc and iron supplements then compare before and after treatment according to their Conner’s scores and Wecsler IQ test. Current study included fifty children diagnosed as having ADHD by DSMV criteria, their zinc and ferritin levels were measured by Colorimetric method and enzyme-linked immunosorbent assay (ELISA) respectively. They were divided into: group I (zinc only deficient),group II (zinc and ferritin deficient),group III (non-deficient), cases with mineral deficiency received zinc (55 mg/day) and/or iron (6 mg/kg/day) for 6 months then reassessed by parent Conner’s rating scale. In group 1, there was no significant difference between the Wecsler verbal and non-verbal IQ scores and oppositional and cognitive problems in Conner’s scores before and after zinc supplements, although there was significant improvement in attention, hyperactivity, emotional liability and impulsivity. In group II, there was significant improvement in verbal and total IQ but not in performance IQ, also there was significant improvement in hyperactivity, emotional liability and impulsivity with no significant difference in oppositional, cognitive problems and inattention before and after zinc/ iron supplements. In Conclusion, Zinc supplements in adjuvant to the main treatment significantly improved symptoms of ADHD children. However, a combined zinc and iron supplements was superior to zinc alone in alleviating ADHD symptoms as well as IQ improvement.

Reliable Sensing Platform for Plasmonic Enzyme-Linked Immunosorbent Assays Based on Automatic Flow-Based Methodology.

Plasmonic enzyme-linked immunosorbent assays (ELISA) using the localized surface plasmon resonance (LSPR) of metal nanoparticles has emerged as an appealing alternative to conventional ELISA counterparts for ultrasensitive naked-eye detection of biomolecules and small contaminants. However, batchwise plasmonic ELISA involving end-point detection lacks ruggedness inasmuch as the generation or etching of NP is greatly dependent on every experimental parameter of the analytical workflow. To tackle the above shortcomings, this paper reports on an automatic flow methodology as a reliable detection scheme of hydrogen peroxide related enzymatic bioassays for ultrasensitive detection of small molecules. Here, a competitive ELISA is combined with the in-line generation of plasmonic gold nanoparticles (AuNPs) followed by the real-time monitoring of the NP nucleation and growth rates and size distribution using a USB miniaturized photometer. Glucose oxidase was labeled to the secondary antibody and yielded hydrogen peroxide that acted as the measurand and the reducing agent of the Au(III)/citrate system in the flow network. High-throughput plasmonic assays were feasible by assembling a hybrid flow system composed of two microsyringe pumps, a perfluoroalkoxy alkane reaction coil, and a 26-port multiposition valve and operated under computer-controllable flow conditions. The ultratrace determination of diclofenac in high matrix samples, e.g., seawater, without any prior sample treatment was selected as a proof-of-concept application of the flow-based platform for determination of emerging contaminants via plasmonic ELISA. The detection limit (0.001 μg L-1) was 1 order of magnitude lower than that endorsed by the first EU Watch List for diclofenac as a potentially emerging contaminant in seawater and also than that of a conventional colorimetric ELISA, which in turn is inappropriate for determination of diclofenac in seawater at the levels endorsed by the EU regulation. The proposed automatic fluidic approach is characterized by the reproducible timing in AuNPs nucleation and growth along with the unsupervised LSPR absorbance detection of AuNPs with a dynamic range for diclofenac spanning from 0.01 to 10 μg L-1. Repeatability and intermediate precision (given as normalized signal readouts) in seawater were <4% and <14%, respectively, as compared to RSDs as high as 30% as obtained with the batchwise plasmonic ELISA counterpart.

Correlates of resistin and retinol-binding protein 4 in metabolic syndrome patients with and without prediabetes.

Background Resistin and retinol-binding protein 4 (RBP4) can work in an intricate in metabolic syndrome (MetS) and prediabetes (PreDM) molecular crosstalk. Materials and methods Resistin and RBP4 were evaluated using colorimetric enzyme-linked immunosorbent assays (ELISAs) in 29 normoglycemic MetS, 30 newly diagnosed drug naïve MetS-preDM patients and 29 lean and normoglycemic controls. Results In this cross-sectional design; the gradual increase in resistin levels (ng/mL), though not ascribed any statistically marked variation, was appreciable in both normoglycemic and preDM MetS groups vs. controls. RBP4 mean circulating levels (ng/mL) in both MetS groups (non-diabetic and preDM) invariably lacked discrepancy vs. controls. Except for fasting plasma glucose (FPG) and A1C; no further intergroup discrepancy could be identified between MetS arms. Adiposity indices: body mass index (BMI), body adiposity index (BAI) and lipid accumulation product (LAP) (but not conicity index) were substantially higher in both MetS (non- and preDM) groups vs. those of controls. Likewise, the atherogenicity index of plasma [but not non-high-density lipoprotein-cholesterol (nonHDL-C)/HDL-C ratio, or triglyceride (TG)/HDL-C ratio] or any of the hematological indices [red cell distribution width (RDW-CV %), monocyte to lymphocyte ratio (MLR), neutrophil to lymphocyte ratio (NLR) and platelet (PLT) to lymphocyte ratios (PLR)] had any marked variations as compared to controls. Low-density lipoprotein-cholesterol (LDL-C)/HDL-C ratio,visceral adiposity index, and waist circumference (WC)/hip circumference (HC) ratio were noticeably greater in MetS-preDM vs. normoglycemic MetS recruits. Neither biomarker could relate to each other, or any of the atherogenecity indices in 59 MetS participants (non- and preDM). Unlike RBP4; resistin associated proportionally with each of HC, BAI, MLR and NLR. Conclusions Both biomarkers can be putative indicator/surrogate prognostic tools for the prediction/prevention and pharmacotherapy of MetS anomalies.

A Colorimetric Enzyme-Linked Immunosorbent Assay with CuO Nanoparticles as Signal Labels Based on the Growth of Gold Nanoparticles In Situ

A colorimetric immunoassay has been reported for prostate-specific antigen (PSA) detection with CuO nanoparticles (CuO NPs) as signal labels. The method is based on Cu2+-catalyzed oxidation of ascorbic acid (AA) by O2 to depress the formation of colored gold nanoparticles (AuNPs). Specifically, HAuCl4 can be reduced by AA to produce AuNPs in situ. In the presence of target, CuO NPs-labeled antibodies were captured via the sandwich-type immunoreaction. After dissolving CuO nanoparticles with acid, the released Cu2+ catalyzed the oxidation of AA by O2, thus depressing the generation of AuNPs. To demonstrate the accuracy of the colorimetric assay, the released Cu2+ was further determined by a fluorescence probe. The colorimetric immunoassay shows a linear relationship for PSA detection in the range of 0.1~10 ng/mL. The detection limit of 0.05 ng/mL is comparable to that obtained by other CuO NPs-based methods. The high throughput, simplicity, and sensitivity of the proposed colorimetric immunoassay exhibited good applicability for assays of serum samples.

Anti-Inflammatory Effects of Aurantio-Obtusin from Seed of Cassia obtusifolia L. through Modulation of the NF-κB Pathway.

Aurantio-obtusin, an anthraquinone compound, isolated from dried seeds of Cassia obtusifolia L. (syn. Senna obtusifolia; Fabaceae) and Cassia tora L. (syn. Senna tora). Although the biological activities of Semen Cassiae have been reported, the anti-inflammatory mechanism of aurantio-obtusin, its main compound, on RAW264.7 cells, remained unknown. We investigated the anti-inflammatory effect of aurantio-obtusin on lipopolysaccharide- (LPS)-induced RAW264.7 cells in vitro and elucidated the possible underlying molecular mechanisms. Nitric oxide production (NO) and prostaglandin E2 (PGE2) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. Protein expression levels of cyclooxygenase 2 (COX-2) were monitored by cell-based ELISA. Interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) synthesis were analyzed using ELISA. The mRNA expression of nitric oxide synthase (iNOS), COX-2, and the critical pro-inflammatory cytokines (IL-6 and TNF-α) were detected by quantitative real-time PCR. Aurantio-obtusin significantly decreased the production of NO, PGE2, and inhibited the protein expression of COX-2, TNF-α and IL-6, which were similar to those gene expression of iNOS, COX-2, TNF-α and IL-6 (p < 0.01). Consistent with the pro-inflammatory gene expression, the Aurantio-obtusin efficiently reduced the LPS-induced activation of nuclear factor-κB in RAW264.7 cells. These results suggested that aurantio-obtusin may function as a therapeutic agent and can be considered in the further development of treatments for a variety of inflammatory diseases. Further studies may provide scientific evidence for the use of aurantio-obstusin as a new therapeutic agent for inflammation-related diseases.

Gamma-Glutamyltransferase and Risk of Acute Coronary Syndrome in Young Chinese Patients: A Case-Control Study.

Background Serum gamma-glutamyltransferase (GGT) is a biomarker of hepatic disease. Recent studies have shown that GGT may also associate with the risk of coronary artery disease. However, the underlying mechanisms of this association are still unclear. Methods This study included 216 young patients with acute coronary syndrome (aged ≤55years) and 227 age-matched controls with normal findings by coronary angiography or coronary computed tomography angiography. We use standard colorimetric techniques and sandwich enzyme-linked immunosorbent assay to measure the levels of GGT and oxidized low-density lipoprotein (ox-LDL), respectively. Traditional risk factors of coronary artery disease, including smoking, diabetes mellitus, hypertension, dyslipidemia, and obesity/overweight, were evaluated according to the current guidelines. Results The levels of GGT were significantly correlated with body mass index and levels of triglyceride, fasting plasma glucose, aspartate aminotransferase, and ox-LDL (all P < 0.05). Multivariate logistic regression analysis showed that GGT was significantly associated with the risk of acute coronary syndrome in young Chinese patients (OR = 1.53, 95% CI = 1.09–2.15) after adjusting for traditional risk factors, including sex, age, quantity of smoking, hypertension, diabetes, body mass index, dyslipidemia, and high-sensitivity C-reactive protein. However, this association was significantly attenuated (OR = 1.20, 95% CI = 0.91–1.58) after further adjusting for the levels of ox-LDL. Conclusions GGT was associated with the risk of ACS in relatively young patients. The link between GGT and the risk of ACS may be dependent on ox-LDL levels, indicating that the prooxidant action is an important pathway for GGT in the development of cardiovascular disease.

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells.

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00-1.34) and 0.97 (0.00-1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.

Gold nanoparticle-based colorimetric ELISA for quantification of ractopamine.

The work describes a gold nanoparticle-based colorimetric enzyme-linked immunosorbent assay (ELISA) for ractopamine. The ELISA is based on an indirect competitive approach. In the presence of ractopamine, gold(III) ions are oxidized by H2O2 to form red AuNPs. On the other hand, the AuNP in solution are purple-blue due to aggregation if the sample does not contain ractopamine. The absorption, best measured at 560nm, increases linearly in the 2 to 512ng·mL-1 ractopamine concentration range, and the detection limit is as low as 0.35ng·mL-1 in urine. Ractopamine can also be detected visually, even in the presence of other β-agonists and antibiotics. The results obtained by this method are consistent with those obtained by LC-MS/MS as demonstrated by analysis of sheep urine. The ELISA method described here is inexpensive, easy-to-use, and suitable for rapid screening of ractopamine in animal samples. Graphical abstract Schematic presentation of a colorimetric indirect competitive immunoassay for ractopamine. It is based on the use of catalase labeled IgG and the measurement of the absorption of red gold nanoparticles (AuNPs) that are generated by the reaction of gold ions with H2O2. In the absence of ractopamine, the solution becomes blue.

Fabrication of a Lab-on-Chip Device Using Material Extrusion (3D Printing) and Demonstration via Malaria-Ab ELISA.

Additive manufacturing, such as fused deposition modeling (FDM), has been increasingly employed to produce microfluidic platforms due to ease of use, wide distribution of affordable 3D printers and relatively inexpensive materials for printing. In this work, we discuss fabrication and testing of an FDM-printed fully automated colorimetric enzyme-linked immunosorbent assay (ELISA) designed to detect malaria. The detection platform consists of a disposable 3D-printed fluidic cartridge (with elastomeric silicone domes on top of reagent-storage reservoirs) and a nondisposable frame with servomotors and electronic controls such as an Arduino board and a rechargeable battery. The system is controlled by a novel interface where a music file (so-called “song”) is sent to the Arduino board, where the onboard program converts the set of frequencies into action of individual servomotors to rotate their arms a certain amount, thus depressing specific elastomeric domes atop reagent reservoirs and displacing the specific reagents into the detection wells, where bioassay steps are executed. Another of the distinguished characteristics of the demonstrated system is its ability to aspirate the fluid from the detection wells into the waste reservoir. Therefore, the demonstrated automated platform has the ability to execute even the most complex multi-step assays where dilution and multiple washes are required. Optimization of 3D-printer settings and ways to control leakages typical of FDM-printed fluidic systems are also discussed.

Serum levels of osteopontin predict major adverse cardiovascular events in patients with severe carotid artery stenosis

BackgroundInflammatory mediators in the blood stream and within plaques are key determinants in atherogenesis. Here, we investigated serum osteopontin (OPN) as a potential predictor of poor outcome in patients with severe carotid atherosclerosis. MethodsCarotid plaques and serum were collected from patients asymptomatic (n=185) or symptomatic (n=40) for ischemic stroke. Plaques were stained for lipids, smooth muscle cells, neutrophils, M1 and M2 macrophage subsets and matrix metallopropteinase-9 (MMP-9). Serum levels of OPN and interleukin-6 (IL-6) were determined by colorimetric enzyme-linked immunosorbent assays. ResultsSymptomatic patients showed a two-fold increase in serum OPN levels. In both symptomatic and asymptomatic patients, OPN levels positively correlated with intraplaque count of neutrophils, total macrophages, and MMP-9 content. In asymptomatic patients, OPN levels also positively correlated with lipids and M1 macrophage subsets. Receiver operating characteristic curve analysis identified serum OPN concentration of 70ng/ml as the best cut-off value to predict major adverse cardiovascular events (MACEs). Patients with high OPN levels had more vulnerable plaque phenotype and reduced levels of HDL-cholesterol and IL-6 as compared to low OPN levels. Kaplan–Meier curve confirmed that patients with OPN levels >70ng/ml had more MACEs at a 24-month follow-up. In the multivariate survival analysis, OPN levels >70ng/ml predicted MACEs, independently of age, gender, and symptomatic status. ConclusionHigh circulating OPN levels were strongly correlated with vulnerability parameters within plaques and predict MACEs in patients with severe carotid artery stenosis. Although confirmation is needed from larger trials, OPN could be a promising clinical tool to assess atherosclerotic outcomes.

Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication

Background/Aims: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). Methods: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and α-KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a “Parachute” assay and immunofluorescence. Levels of α-SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. Results: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of α-SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. Conclusion: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.

Wax-Assisted One-Step Enzyme-Linked Immunosorbent Assay on Lateral Flow Test Devices.

Lateral flow tests (LFTs) are widely used analytical tools characterized by portability, operator simplicity and short analysis times. A remaining challenge is their limited analytical sensitivity, which in classical immunoassay formats is overcome by enzyme-linked immunosorbent assay (ELISA) formats. The implementation of ELISA to an LFT format however, is hampered by the complexity of the procedure requiring the enzyme substrate addition after sample addition. In this work, a simple method for automation of this procedure without user interference is presented. Originally used sample pads of LFTs have been replaced by hydrophobic wax-modified filter paper-based sample pads to realize a delayed flow a pre-deposited colorimetric ELISA substrate without other alterations to the classical lateral-flow immunoassay format. The performance of the system has been characterized by visualizing flow behavior and final proof-of-concept is provided by a model mouse IgG assay, achieving a limit of detection of 15.8 ng mL-1 from just a single application of the sample solution.

Nominal effective immunoreaction volume of magnetic beads at single bead level.

Immunomagnetic bead (IMB)-based enzyme-linked immunosorbent assay (ELISA) has been the tool frequently used for protein detection in research and clinical laboratories. For most ELISA reactions the recommended dosage of IMBs is usually according to their weight (mg) or mass fraction (w/v) instead of the bead number. Consequently, the processes occurring in the immediate vicinity of the IMBs have always been ignored by researchers and they cannot be revealed in detail during the ELISA reaction. In this paper, we established the relationship between number of IMBs and colorimetric results, and further proposed a new concept of "nominal effective immunoreaction volume (NEIV)" to characterize a single IMB during ELISA reaction. Results showed that the NEIV of a single IMB has a constant value, which is unrelated to the amount of beads and the concentration of antigen. Optimal results of the colorimetric ELISA are achieved when the incubation volume meets each IMB's NEIV and is no longer enhanced by increasing the incubation volume. Thus, the reliable and relatively precise number of IMBs for ELISA detection during practical application could be determined. Most importantly, a study using IMB's NEIV would lay the foundation for a kinetics analysis of IMBs and antigens for future study.

Human CCDC47 sandwich immunoassay development with electrochemiluminescence technology

Coiled-Coil Domain Containing 47 (CCDC47) is an endoplasmic reticulum (ER) transmembrane protein involved in calcium signaling through utilization of its calcium binding-acidic luminal domain. CCDC47 also interacts with ERAD (endoplasmic reticulum-associated degradation) complex and is involved in ER stress relief. In this report, we developed human CCDC47 monoclonal antibodies and a sandwich immunoassay for CCDC47 measurement in biological matrices. Specificity of developed antibodies were confirmed by immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated cell lysates. To achieve high analytical sensitivity, traditional colorimetric enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence (ECL) technology were compared, and 3 logs of increased sensitivity was observed with the use of ECL. A CCDC47 sandwich ECL assay was subsequently developed and performances evaluated for calibration curves, precision and accuracy, as well as selectivity and interferences for sample measurement. Sample stability was also characterized for freeze/thaw cycles and short/long term storage conditions.

A novel Python program for implementation of quality control in the ELISA

The use of semi-quantitative assays such as the enzyme-linked immunosorbent assay (ELISA) requires stringent quality control of the data. However, such quality control is often lacking in academic settings due to unavailability of software and knowledge. Therefore, our aim was to develop methods to easily implement Levey-Jennings quality control methods. For this purpose, we created a program written in Python (a programming language with an open-source license) and tested it using a training set of ELISA standard curves quantifying the Fab fragment of an anti-cocaine monoclonal antibody in mouse blood. A colorimetric ELISA was developed using a goat anti-human anti-Fab capture method. Mouse blood samples spiked with the Fab fragment were tested against a standard curve of known concentrations of Fab fragment in buffer over a period of 133days stored at 4°C to assess stability of the Fab fragment and to generate a test dataset to assess the program. All standard curves were analyzed using our program to batch process the data and to generate Levey-Jennings control charts and statistics regarding the datasets. The program was able to identify values outside of two standard deviations, and this identification of outliers was consistent with the results of a two-way ANOVA. This program is freely available, which will help laboratories implement quality control methods, thus improving reproducibility within and between labs. We report here successful testing of the program with our training set and development of a method for quantification of the Fab fragment in mouse blood.

Quantification of enrofloxacin residues in broiler chicken tissues using competitive enzyme-linked immunosorbent assay

Irrational use of antimicrobial agents’ results in their accumulation in poultry eggs, meat and other byproducts. Less is known regarding antimicrobial residues in broiler meat, specifically for Jhang, Punjab, Pakistan. Thus, the main purpose of this research was to evaluate the prevalence of enrofloxacin residues in broiler chickens tissue. A total of 90 samples of chickens’ tissue were collected randomly from different local meat shops of Jhang city and were analyzed using competitive colorimetric enzyme-linked immunosorbent assay kit. Out of 90 samples analyzed, 53% of the breast muscle and 62% of the liver tissues samples contained residues of enrofloxacin higher than maximum residue limits. Median concentration of enrofloxacin residues in breast muscle and liver tissue was 103 ng/g and 1409 ng/g respectively. Quantification of antibiotic residues distribution in chickens’ tissues revealed that the difference between breast muscle and liver tissue was non-significant. Results of study revealed that broiler chickens being sold in Jhang city contained the enrofloxacin residues and may exposethe consumers to their deleterious effects.

An ELISA for the determination of human IgG based on the formation of a colored iron(II) complex and photometric or visual read-out

The paper describes a colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human IgG. It is based on the use of an Fe(II) coordination complex as a signal amplifier and of mesoporous silica nanoparticles modified with glucose oxidase (GOx) and secondary antibodies (Ab2). After formation of the immuno sandwich complex, the quantity of GOx is proportional to the quantity of IgG. On addition of Fe(II) and glucose, GOx catalyzes the oxidation of glucose to produce hydrogen peroxide which oxidizes Fe(II) to Fe(III). After adding a stop solution containing the complexing ligand 1,10-phenanthroline (Phen), un-reacted Fe(II) forms an orange-red complex with Phen which can be detected by plate reader and even seen with bare eyes. This sandwich ELISA has a linear response in the 1 pg.mL−1 to 100 ng.mL−1 human IgG concentration range and a 860 fg.mL−1 detection limit. This is 20 times lower than the commercial ELISA for human IgG. The assay also is selective, stable, highly sensitive and cost-effective.

Colorimetric ELISA based on glucose oxidase-regulated the color of acid–base indicator for sensitive detection of aflatoxin B1 in corn samples

This study described a sensitive direct competitive enzyme-linked immunosorbent assay (ELISA) for naked-eye detection of aflatoxin B1 (AFB1) by using glucose oxidase (GOx)-regulated bromocresol purple (BCP) color change. GOx was used as an alternative to horseradish peroxidase (HRP) for oxidization of glucose into hydrogen peroxide and gluconic acid. BCP, whose color is significantly sensitive to pH variation, was used as a signal output. Under optimal conditions, the developed method exhibited a considerably high sensitivity for AFB1 detection with a cutoff limit of 100 pg/mL by the naked eye. The reliability of the developed colorimetric ELISA using naked-eye detection showed no false negative and false positive results among 70 AFB1 spiked tests. Furthermore, the developed method showed a good linear range of 25–200 pg/mL for AFB1 quantitative detection with a half-maximal inhibitory concentration at 66.72 pg/mL, which was approximately 10-fold lower than that of conventional HRP-based ELISA (IC50 = 707 pg/mL). The recoveries from four kinds of AFB1-spiked concentrations in corn extract solutions ranged within 80.56%–108.53%, with a coefficient of variation range of 1.69%–11.86%. These results exhibited good agreement with those of LC–MS/MS method indicating an acceptable accuracy and precision for AFB1 quantitative detection in actual corn samples. In brief, this study was the first to use a GOx-mediated color change of BCP in immunoassay for naked-eye detection of AFB1. This study also provided a new method for high-throughput screening detection of other small molecular chemicals using naked eye in resource-constrained countries.

Smartphone-based colorimetric ELISA implementation for determination of women's reproductive steroid hormone profiles.

Biologists frequently collect and analyze biospecimens in naturalistic (i.e., field) conditions to ascertain information regarding the physiological status of their study participants. Generally, field-collected biospecimens need to be stored frozen in the field and then transported frozen to laboratory facilities where traditional biomarker assays, such as enzyme-linked immunosorbent assays (ELISAs), are conducted. As proper storage and transport of frozen specimens is often logistically difficult and expensive, particularly in nonurban field settings, methods that reduce the need for specimen storage and transport would benefit field-research dependent disciplines such as biology, ecology and epidemiology. One limiting factor to running assays in the field is the use of large and expensive equipment to visualize and quantify the assays, such as microplate readers. Here, we describe an implementation of colorimetric ELISA visualization and quantification using two novel and portable imaging instrumentation systems and data processing techniques for the determination of women's reproductive steroid hormone profiles. Using the light absorbance and transmittance properties of the chemical compounds that make up the hormone assay, we were able to estimate unknown hormone concentrations using a smartphone system and a webcam system. These estimates were comparable to those from a standard laboratory multiple reader (smartphone: accuracy=82.20%, R 2>0.910; webcam: accuracy=87.59%, R 2>0.942). This line of applied research, in the long run, is expected to provide necessary information for examining the extent to which reproductive function varies within and between populations and how it is influenced by psychosocial, energetic and environmental challenges. Our validation of these novel, portable visualization and quantification systems allows for the eventual development of a compact and economical closed system which can be used to quantify biomarker concentrations in remote areas.