Optimal Assay Conditions Research Articles

Immunoassays for riboflavin and flavin mononucleotide using antibodies specific to d-ribitol and d-ribitol-5-phosphate

Riboflavin (vitamin B2), a water-soluble vitamin, plays a key role in maintaining human health. Though, numerous methods have been reported for the determination of total riboflavin (TRF) content in foods and biological samples, very few methods are reported for quantifying riboflavin and its coenzymes [flavin mononucleotide (FMN); flavin adenine dinucleotide (FAD)] individually. Recently, we have demonstrated that antibodies specific to d-ribitol and d-ribitol-5-phosphate also recognize riboflavin and FMN, respectively, and not vice-versa. In this study, we have evaluated these two antibodies for the analysis of riboflavin and FMN by indirect competitive ELISA (icELISA) in selected foods and pharmaceuticals. Under the optimal assay conditions, 50% inhibition concentration (IC50) and limit of detection (LOD, IC10) were 3.41ng/mL and 0.02ng/mL for riboflavin, and 7.84ng/mL and 0.24ng/mL for FMN, respectively, with detectable concentration range between 0.1 and 100ng of analytes and <0.1% cross-reactivity with other water-soluble vitamins. The amounts of TRF in food samples, as analyzed by icELISA using ribitol antibody, were 90–95% of the reported values in the literature or label values. Quantification of individual flavins (riboflavin and FMN) from the same food samples showed variation in their values compared to TRF, and were in good agreement with values obtained from HPLC and AOAC methods. Further, spiking and recovery analysis of food samples and pharmaceuticals showed no significant matrix effects. The immunoassays were validated in terms of accuracy and precision using inter- and intra-assays. The immunoassays developed in this study are sensitive and appears feasible for screening a large number of samples in the quantification of riboflavin and FMN in various biological samples, pharmaceuticals and natural/processed foods.

Rapid and ultrasensitive detection of 3-amino-2-oxazolidinone in catfish muscle with indirect competitive enzyme-linked immunosorbent and immunochromatographic assays

ABSTRACTIn this study, we developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic assay to detect the furazolidone metabolite, 3-amino-2-oxazolidinone (AOZ). The detection of AOZ was based on the AOZ derivative 3-([2-nitrophenyl]methyleneamino]-2-oxazolidinone (2-NPAOZ). 3-([(3-Carboxyphenyl)methylene]-amino)-2-oxazolidinone (2-CPAOZ) was used as the immunizing hapten and a conjugate of AOZ and glyoxylic acid (AOZ-A) was used as the coating hapten. The monoclonal anti-2-NPAOZ antibody, 5G12, was generated with a half-inhibitory concentration (IC50) of 0.2 ng/mL and a line of 0.06–0.66 ng/mL. Its cross reactivity with other analogues was less than 8%. Spiked catfish samples (0.1, 0.2, and 0.5 μg/kg) were analyzed with the proposed system. The ic-ELISA showed a recovery range of 86.2–118.5% and the intra-assay and inter-assay coefficients of variation ranged from 4.3% to 9.4%. Under the optimum assay conditions, the immunochromatographic assay had a visual cut-off value of 0.5 μg/kg in catfish samples. Both methods can be used to detect AOZ in catfish samples.

In Situ Growth of Plasmonic Gold Nanoparticles for the Direct and Sensitive Colorimetric Assay of Glucose

Gold nanoparticles (Au NPs)‐based colorimetric assay for the highly sensitive and selective detection of glucose has been conducted. The sensing, which was based on the in situ growth of Au NPs in the presence of glucose, was developed with the solution changing from colorless to red. This change could be observed with the naked eye or a ultraviolet–visible (UV–vis) spectrophotometer. Moreover, adding trace amounts of Tween‐20 kept Au NPs well spaced. Under the optimal assay conditions, the absorbance of Au NPs at 550 nm was proportional to glucose concentration within 400–1800 μM with a linear regression equation, and the detection limit was 6.28 μM. Some common interfering compounds in beverages, including other sugars, food additives, and amino acids, did not affect glucose sensing. Therefore, the proposed Au NP‐based colorimetric assay could be successfully used to determine glucose in beverages.

Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237

Description of the subject. Alkaline phosphatases (APases) are commonly used as nonradioactive markers for detecting specific proteins or DNA targets in clinical medicine and molecular biology. However, their applications in the biotechnology industry require thermostability and storage stability. Our preliminary study revealed that APase from Thermus sp. NTU-237 (TsAPase) is thermostable and exhibits high activity. Therefore, it is desirable to establish the optimal conditions for its application. Objectives. To characterize APase from thermophile Thermus sp. NTU-237 and to evaluate its potential applications. Method. The APase gene of Thermus sp. NTU-237 was cloned and expressed in Escherichia coli. Subsequently, the effects of buffer, glycerol, sodium dodecyl sulfate (SDS), NaCl, and temperature on enzyme activity were studied to establish the optimal conditions for TsAPase assays. In addition, the potential for application of TsAPase was evaluated using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) active staining method. Results. Recombinant TsAPase was identified as having a dimeric structure and a molecular mass of 109 kDa. Sequence alignment analysis of known thermophilic APases with TsAPase and E. coli APase revealed that catalytic and binding residues were highly conserved. This finding suggested that the catalytic mechanism of TsAPase is the same as that of other APases. TsAPase activity was inhibited by glycerol and SDS but enhanced by NaCl and Tris-HCl buffer. The kinetic parameters Km, kcat, and Vmaxwere determined to be 81 µM, 6.08 s-1, and 6.76 U·mg-1, respectively. The optimum temperature of TsAPase was 80 °C, and TsAPase was stable at room temperature for more than 10 days. Moreover, TsAPase could catalyze the dephosphorylation of BCIP and could elicit blue color development by the BCIP/NBT reaction kit at room temperature. Conclusions. These results illustrate that TsAPase has potential for application in medical or basic research.

Expression, purification and characterization of human cytosolic sulfotransferase (SULT) 1C4.

Human cytosolic sulfotransferase 1C4 (hSULT1C4) is a dimeric Phase II drug-metabolizing enzyme primarily expressed in the developing fetus. SULTs facilitate the transfer of a hydrophilic sulfonate moiety from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto an acceptor substrate altering the substrate's biological activity and increasing the compound's water solubility. While several of the hSULTs' endogenous and xenobiotic substrates have been identified, the physiological function of hSULT1C4 remains unknown. The fetal expression of hSULT1C4 leads to the hypothesis that the function of this enzyme may be to regulate metabolic and hormonal signaling molecules, such as estrogenic compounds, that may be generated or consumed by the mother during fetal development. Human SULT1C4 has previously been shown to sulfonate estrogenic compounds, such as catechol estrogens; therefore, this study focused on the expression and purification of hSULT1C4 in order to further characterize this enzyme's sulfonation of estrogenic compounds. Molecular modeling of the enzyme's native properties helped to establish a novel purification protocol for hSULT1C4. The optimal activity assay conditions for hSULT1C4 were determined to be pH 7.4 at 37°C for up to 10 min. Kinetic analysis revealed the enzyme's reduced affinity for PAPS compared to PAP. Human SULT1C4 sulfonated all the estrogenic compounds tested, including dietary flavonoids and environmental estrogens; however, the enzyme has a higher affinity for sulfonation of flavonoids. These results suggest hSULT1C4 could be metabolizing and regulating hormone signaling pathways during human fetal development.

QUALITY BY DESIGN-BASED OPTIMIZATION AND VALIDATION OF NEW REVERSE PHASE-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF LEVOFLOXACIN HEMIHYDRATE AND AMBROXOL HYDROCHLORIDE IN BULK AND ITS PHARMACEUTICAL DOSAGE FORM

ABSTRACTObjective: Innovative application of quality by design (QbD) technique for simultaneous estimation of levofloxacin and ambroxol hydrochloride (HCL)in bulk and its pharmaceutical dosage form using reverse phase-high-performance liquid chromatography (RP-HPLC) method.Method: A method has been developed for the separation of levofloxacin and ambroxol HCL using RP-HPLC on C18 column (250 4.6 mm, 5 ml) withultraviolet detection at 306 nm. Experimental designs were applied for multivariate optimization of the experimental conditions of RP-HPLC method.Three independent factors: Acetonitrile content in the mobile phase composition, buffer pH, and flow rate were used to design mathematical models.Here, central composite design (CCD) experimental design was used to study the response surface technique and to study in depth the effects ofthese independent factors. Derringer’s desirability function was applied to simultaneously optimize the retention time of last eluting peak (ambroxolhydrochloride) and resolution between levofloxacin and ambroxol hydrochloride.Result and Discussion: The predicted optimum assay condition consisted of acetonitrile, potassium dihydrogen phosphate buffer (pH 5.00;potassium dihydrogen phosphate), and methanol in a proportion of 20:70:10% v/v, respectively, as the mobile phase at a flow rate of 1.2 ml/minute.Using this optimum condition, baseline separation of both drugs with good resolution and a run time of &lt;5 minutes were achieved. The optimizedassay condition was validated according to the ICH guidelines to confirm specificity, linearity, accuracy, and precision.Keywords: Levofloxacin, Ambroxol hydrochloride, Experimental design, Response surface methodology, Derringer’s desirability, Quality by designapproach.

Modified carbon black as label in a colorimetric on-chip immunoassay for histamine

The present study demonstrates the modification of carbon black and its usage as a detection label in colorimetric immunoassays on a microarray format. We show the feasibility using a binding inhibition assay for histamine, which is a marker of food freshness and quality. The modified carbon nanoparticles (CNPs) were characterized by determining functional surface groups, elemental composition, size, surface charge and dispersion stability to investigate the influence of modification on the assay performance with the overall goal to optimize assay conditions and enhance signal strength. Carbon nanoparticles were oxidized and further modified with silanes with different functional end groups (thiol, epoxy, amine) 3-glycidoxypropyltrimethoxysilane being the most effective modification. Additionally, 1-pyrenebutanoic acid N-succinimidyl ester (PSE) was tested as conjugation agent allowing semi-quantitative read-out by naked eye over a broad histamine range (0-600 μg/mL), similar to just oxidized CNPs. The dispersion stability was enhanced with acrylic polymer Atlox and dispersion agent xanthan. However, no concentration dependent response towards histamine was observed. With oxidized CNPs read-out could be done by naked eye; otherwise an office scanner was needed for detection. Lower zeta potential was recorded after oxidation, resulting in better colloidal stability. Electrostatic stabilization was enough to create stable suspensions, and further modification was not necessary for the antibody coupling and stability in the assay.

Loop-mediated isothermal amplification based on the mitochondrial COI region to detect Pratylenchus zeae

Pratylenchus zeae parasitizes various crops and damages the host roots, resulting in decreased yield and quality of the host plants. Alignments of mitochondrial DNA (mtDNA) Cytochrome Oxidase I (COΙ) sequences revealed the genetic variation among Pratylenchus species. The results indicated 0.2–2.4% intraspecific variations for mtDNA COI sequences among eight P. zeae populations, and 25.4–35.1% interspecific variations between P. zeae and other Pratylenchus species. Based on the mtDNA COΙ region, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and specific detection of P. zeae. The optimal conditions for the LAMP assay were 64 °C for 40 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR), analysis with the restriction enzyme Bam HI and visual inspection by adding SYBR Green I to the products. The LAMP assay could detect P. zeae populations from different hosts and different geographical origins specifically. The LAMP assay was also sensitive, detecting 0.1 individual P. zeae, which was 10 times more sensitive than conventional PCR. This is the first report of the detection of Pratylenchus spp. using LAMP. In addition, the results also suggested that use of the COI gene might allow for good resolution at the Pratylenchus species level.

Immunoanalytical characteristics of fecal calprotectin.

Besides structural and physiological data of calprotectin, this paper points out the optimal conditions for sampling, fecal assays and interpretation of results.

Fluorescence polarization immunoassay of ractopamine

A technique was developed for fluorescence polarization immunoassay (FPIA) of ractopamine, a toxic low molecular weight nonsteroidal growth regulator belonging to the most controlled contaminants of food products of animal origin. The assay is based on the competition between a sample containing ractopamine and ractopamine–fluorophore conjugate for binding to antibodies. The competition is monitored via changes in the degree of fluorescence polarization for plane-polarized excitation light, which differs for the free and antibody-bound forms of the conjugate. The optimal assay conditions were established, ensuring a high accuracy and minimal detection limit. The developed assay demonstrated a detection limit of 1 ng/mL and a range of detectable concentrations of 2.3–50 ng/mL, which met the requirements of sanitary control. The duration of the analysis was 10 min. The possible application of the developed FPIA was demonstrated with testing of turkey meat. The speed and simplicity of the proposed assay define its efficiency as a screening tool for safety of foods.

Enhanced detection of RNA by MMLV reverse transcriptase coupled with thermostable DNA polymerase and DNA/RNA helicase.

Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA polymerase with RT activity, K4polL329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.

Application of 2,4-Dinitrophenylhydrazine (DNPH) in High-Throughput Screening for Microorganism Mutants Accumulating 9α-Hydroxyandrost-4-ene-3,17-dione (9α-OH-AD).

To develop a quick method for the preliminarily screening of mutant strains that can accumulate 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD), a high-throughput screening method was presented by applying the principle that 2,4-dinitrophenylhydrazine (DNPH) can react with ketones to produce precipitation. The optimal color assay conditions were the substrate androst-4-ene-3,17-dione (AD) concentration at 2.0 g/L, the ratio of AD to DNPH solution at 1:4, and the sulfuric acid and ethanol solution percentages in DNPH solution at 2% and 35%, respectively. This method was used to preliminarily screen the mutants of Rhodococcus rhodochrous DSM43269, from which the three ones obtained could produce more 9α-OH-AD. This DNPH color assay method not only broadens screening methods and increases screening efficiency in microbial mutation breeding but also establishes a good foundation for obtaining strains for industrial application.

Designing of a specific fluorogenic peptide substrate for HIV-1 protease activity assay

Based on the specific hydrolysis sequence in the Gag-Pol protein substrate of HIV-1 protease, we designed a fluorescence resonance energy transfer (FRET) substrate (designated peptide HF) for the enzyme. peptide HF has the sequence of QXL 520-GABA-SFNFPQITK-HiLyte Flour 488-NH2. HiLyte Fluor 488/QXL 520 as a FRET pair because of high excitation/emission and good activity at the optimal pH of HIV-1 protease (pH&lt;5) was chosen. QXL 520 was more stable when conjugated with serine residue at N-terminus via GABA group while leucine at the C-terminus was replaced with a lysine residue-HiLyte Fluor 488 conjugate. The optimal reaction conditions for HIV-1 protease activity assay were found to include 100-200 ng HIV-1 protease, 2 µM peptide HF, 100 mM CH3COONa buffer at pH 4.7, containing 1 M NaCl, 1 mM EDTA, 1 mM DTT, 5% DMSO and 0.5 mg/mL BSA. Peptide HF could be stored at the concentration of 0.1 mg/mL in DMSO and at -80oC until use. Under the above mentioned conditions, HIV-1 protease showed the high affinity and the effective hydrolysis for the substrate with the kinetic parameters Vmax of 4.45 nM/s, Kcat/Km = 10.89 (mM.s)-1, which were 5 times higher than the Vmax and Kcat/Km when the commercial SensoLyte® 520 substrate (Anaspec-America) was used. Citation: Nguyen Thị Hong Loan, Tran Thi Thu Huyen, Dang Thi Lieu, Phan Thi Lam Hong, Phan Tuan Nghia, 2017. Designing of a specific fluorogenic peptide substrate for HIV-1 protease activity assay. Tap chi Sinh hoc, 39(1): 115-121. DOI: 10.15625/0866-7160/v39n1.8291. *Corresponding author: loannhbio@gmail.com Received 1 May 2016, accepted 20 December 2016

Development of a flow cytometry-based potency assay for measuring the in vitro immunomodulatory properties of mesenchymal stromal cells

Human bone marrow-derived mesenchymal stromal/stem cells (MSC) have well-documented modulatory effects on multiple immune cell types. Although these effects are linked to their therapeutic benefit in diverse diseases, a reliable, quantitative assay of the immunomodulatory potency of individual human MSC preparations is lacking. The aims of this study were to develop an optimised rapid turnaround, flow cytometry-based whole-blood assay to monitor MSC potency and to validate its application to MSC immunomodulation. A protocol for short-term LPS stimulation of anti-coagulated whole blood samples followed by combined surface CD45/CD14 and intracellular TNF-α staining was initially developed for analysis on a 4 colour desktop cytometer. Optimal monocyte activation was dependent on the presence of extracellular calcium ions thereby precluding the use of EDTA and sodium citrate as anticoagulants. Optimal assay conditions proved to be 1ng/mL ultrapure-LPS added to 10-fold diluted, heparin anti-coagulated whole blood incubated for 6h at 37°C. Under these conditions, addition of human bone marrow-derived MSC (hBM-MSC) from multiple donors resulted in a reproducible, dose-dependent inhibition of LPS-stimulated monocyte TNF-α expression. We conclude that this protocol represents a practical, quantitative assay of a clinically relevant functional effect of hBM-MSCs as well as other immunomodulatory agents.

Bioenergetic profiling of Trypanosoma cruzi life stages using Seahorse extracellular flux technology

Energy metabolism is an attractive target for the development of new therapeutics against protozoan pathogens, including Trypanosoma cruzi, the causative agent of human Chagas disease. Despite emerging evidence that mitochondrial electron transport is essential for the growth of intracellular T. cruzi amastigotes in mammalian cells, fundamental knowledge of mitochondrial energy metabolism in this parasite life stage remains incomplete. The Clark-type electrode, which measures the rate of oxygen consumption, has served as the traditional tool to study mitochondrial energetics and has contributed to our understanding of it in T. cruzi. Here, we evaluate the Seahorse XFe24 extracellular flux platform as an alternative method to assess mitochondrial bioenergetics in isolated T. cruzi parasites. We report optimized assay conditions used to perform mitochondrial stress tests with replicative life cycle stages of T. cruzi using the XFe24 instrument, and discuss the advantages and potential limitations of this methodology, as applied to T. cruzi and other trypanosomatids.

Porcine antigen-specific IFN-γ ELISpot as a potentially valuable tool for monitoring cellular immune responses in pig-to-non-human primate islet xenotransplantation.

Recent progress in xenotransplantation of porcine islets to non-human primates (NHPs) gives hope for human clinical trials in the near future. Thus, implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen-specific cellular immune responses is necessary. The enzyme-linked immunospot (ELISpot) assay has been widely used to monitor antigen-specific alloreactive T-cell responses in humans; however, the utility of porcine islet-specific ELISpot assay has not yet been thoroughly evaluated for pig-to-NHPs intraportal islet xenotransplantation. The optimal ELISpot assay conditions, including the number of responder and stimulator cells and the provision of costimulation, were determined. Then, ELISpot assays were conducted on serial stocks of peripheral blood mononuclear cell (PBMC) samples previously isolated from NHP recipients transplanted with porcine islets. Either splenocytes from donor pigs or pancreatic islets from third-party pigs were used for antigen stimulation. At the same time, the ratio of CD4(+) /CD8(+) T cells and the percentage of CD4(+) FoxP3(+) T cells in the peripheral blood were evaluated. Finally, liver biopsy samples were evaluated to assess the immunopathology of the grafts. The optimal conditions for the ELISpot assay were defined as 2.5 × 10(5) responder cells incubated with 5.0 × 10(5) stimulator cells in 96-well, flat-bottom plates without further costimulation. Using donor splenocytes as stimulators, a serial interferon-gamma (IFN-γ) ELISpot assay with PBMCs from the monkeys with prolonged porcine islet grafts (>180 days) demonstrated that the number of donor antigen-specific IFN-γ-producing cells significantly increased upon overt graft rejection. However, use of third-party porcine islets as stimulators did not reflect graft rejection, suggesting that the use of donor-specific PBMCs, and not tissue (porcine islet)-specific cells, as stimulators could better serve the purpose of this assay in adult porcine islet transplantation. IFN-γ spot number was neither influenced by the peripheral blood CD4(+) /CD8(+) T-cell ratio nor the percentage of CD4(+) FoxP3(+) T cells. Finally, in cases of overt graft rejection, the number of IFN-γ spots and the graft-infiltrating T cells in biopsied liver samples increased simultaneously. Use of PBMCs in a porcine antigen-specific IFN-γ ELISpot assay is a reliable method for monitoring T-cell-mediated rejection in pig-to-NHP islet xenotransplantation.

Development of novel bienzymatic assay for quantification of glucose in human serum

Aim: The main objective of this investigation was to design and to develop a simple, sensitive, and convenient assay for quantification of glucose in human serum sample. Materials and Methods: A novel bienzymatic assay for the quantification of glucose by spectrophotometry has been developed. The method is based on the coupling of 4-aminoantipyrine and iminodibenzyl in the presence of bienzyme peroxidase/glucose oxidase (GOx) to form a blue colored product which is monitored for absorbance at 620 nm at 23°C in 100 mmol/L KH2PO4/NaOH buffer of pH 6.3. Results and Discussion: Under optimized assay conditions, glucose could be quantified in a linear range from 1.15 to 562 μmol/L and 2.31 to 370 μmol/L by kinetic and fixed time method. The limit of detection and limit of quantification were measured. Michaelis–Menten constant for glucose was found to Km = 357 μmol/L and maximum rate of the reaction (Vmax) was 0.117 × 10−6 mol/L/min. The proposed method was applied for the quantification of glucose in human serum samples, which has a good regression coefficient of 0.9992 with the enzymatic kit method. The inter-day precision was 0.53-2.9% (n = 10) and intra-day precision was 1.5-3.03% (n = 10). The accuracy range for glucose was fond to be 90-104%. The glucose recovery ranged from 91.92% to 108.5%. Conclusion: The present assay was rapid, sensitive, and convenient for quantification of glucose in human serum sample with good recovery, high accuracy, and minimum interference.

A qPCR aptasensor for sensitive detection of aflatoxin M1.

Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, imposes serious health hazards. AFM1 had previously been classified as a group 2B carcinogen [1] and has been classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) [2]. Determination of AFM1 thus plays an important role for quality control of food safety. In this work, a sensitive and reliable aptasensor was developed for the detection of AFM1. The immobilization of aptamer through a strong interaction with biotin-streptavidin was used as a molecular recognition element, and its complementary ssDNA was employed as the template for a real-time quantitative polymerase chain reaction (RT-qPCR) amplification. Under optimized assay conditions, a linear relationship (ranging from 1.0 × 10(-4) to 1.0μgL(-1)) was achieved with a limit of detection (LOD) down to 0.03ngL(-1). In addition, the aptasensor developed here exhibits high selectivity for AFM1 over other mycotoxins and small effects from cross-reaction with structural analogs. The method proposed here has been successfully applied to quantitative determination of AFM1 in infant rice cereal and infant milk powder samples. Results demonstrated that the current approach is potentially useful for food safety analysis, and it could be extended to a large number of targets.

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18

High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies.