Linear Standard Curve Research Articles

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3).

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

CALUX bioassay: a cost-effective rapid screening technique for screening dioxins like compounds.

Xenobiotic detection systems-chemically activated luciferase expression (XDS-CALUX) bioassay in determining the toxic equivalency (TEQ) of PCDD/Fs from contaminated sites reported in several papers has been discussed in this study. CALUX bioassay method has been validated by an effective combined column clean-up system followed by addition of samples to monolayer cell cultures of H1L6.1c3 cell line in 96 well plates. Cultures are then examined under microscope after 24 h incubation followed by rinsing with 75 μL phosphate buffer saline and 30 μL of cell culture lysis. The response is observed in the luminometer and expressed in relative light unit (RLUs). CALUX-TEQ is estimated from a TCDD standard curve for unknown samples. Quality control in CALUX is done by selecting the range of CALUX values falling in the center of the linear standard curve. For developing nations CALUX biossay can be used as a cost effective and rapid screening technique for screening xenobiotic compounds from the hotspots like open solid waste burning sites, informal e-waste recycling workshops and industrial zones where constant monitoring for such compounds is required.

Malachite Green and Leuco-Malachite Green Detection in Fish Using Modified Enzyme Biosensor

A modified enzyme-based biosensor was developed for amperometric measurement of total Malachite Green (sum of MG and LMG) in tilapia fish. The 4 UmL-1 butyrylcholinesterase enzyme (BuChE) was used and immobilized onto the surface of carbon paste electrode during the electro-polymerization of polypyrrole (PPy). It is a simple, rapid and sensitive technique used for determination of total MG using 0.1M phosphate buffer pH 8.0. In this study, butyrylthiocholine iodide (0.3 Mm BTCi) was used as a substrate which produced the thiocholine due to the enzymatic hydrolysis. Then, it was oxidized at 0.4V against silver-silver chloride electrode for 100 s. The modified enzyme biosensor provided a high sensitivity and linear concentration range from 0.25 to 10 ppb with a limit of detection (LOD) at 0.25 ppb. The results showed the linear standard curve of total MG standard solution (based on the current output in microampere) with the equation of y = - 0.9113x + 10.84, R2 = 0.9445.

Quantitative Real-Time PCR Analysis of Gene Transcripts of Mosquito Follicles.

Real-time (quantitative) PCR, or QPCR, has become an indispensible tool for characterizing gene expression. Depending on the experimental design, researchers can use either the relative or absolute (standard curve) method to quantify transcript abundance. Characterizing the expression of genes in mosquito ovaries will require use of the standard curve method of quantification. Here, I describe reagents and equipment necessary to run standard curve QPCR. I also provide details on the construction of the standard linear curve and calculations required to determine transcript abundance.

Development and validation of a UHPLC-MS/MS bioanalytical method to quantify in plasma the analgesic candidate PT-31 following a preliminary pharmacokinetic study in rats.

A selective and sensitive UHPLC-MS/MS bioanalytical method to determine PT-31, an analgesic drug candidate, in rat plasma was developed and validated. Analyses were performed using a UHPLC-MS/MS system equipped with an electrospray ionization interface operating in the positive ionization mode using a C18 reversed-phase column with a mobile phase of water:acetonitrile (68:31, v/v) containing 0.1% acetic acid eluting in a gradient mode with a flow rate of 0.3 mL/min. Plasma samples were deproteinized with cold acetonitrile containing 0.01% TFA (1:2, v/v) and 50 μL of the supernatant were injected into the system. PT-31 and phenytoin (internal standard) retention times were roughly 1.0 and 1.5 min, respectively. Linear standard curves were plotted for the 0.01-10 µg/mL concentration range, with a coefficient of determination > 0.99. The method's precision was over 88%. Maximum intra- and inter-day relative standard deviations were 14.6% and 11.6%, respectively. Interfering substances were not detected in the chromatogram, indicating that the method was specific. PT-31 stability was assessed under different temperature and storage settings. The method was used to characterize PT-31 plasma pharmacokinetics following administration of 5 mg/kg i.v. to Wistar rats. Therefore, the method described is sensitive, linear, precise and specific enough to determine PT-31 in preclinical pharmacokinetic investigations. Copyright © 2015 John Wiley & Sons, Ltd.

Calebin A: Analytical Development for Pharmacokinetics Study, Elucidation of Pharmacological Activities and Content Analysis of Natural Health Products.

To develop a bioanalytical assay using RP-HPLC to quantify the curcuminoid calebin A, to characterize its pharmacokinetics in rats after intravenous (IV) and oral (PO) administration, to identify its pharmacological activities and to evaluate its content in natural health products. An RP-HPLC method was developed for the detection of calebin A. Separation was carried out using a Phenomenex® Kinetex® C18 column with UV detection at 339 nm. An isocratic mobile phase consisting of acetonitrile and water with 10 mM ammonium formate (pH 7.0) (40:60, v/v) at a flow rate of 0.8 mL/min was employed. Linear standard curves were established and applied in the pharmacokinetic study. Calebin A was administered to male Sprague-Dawley (CD) rats IV (20 mg/kg) or PO (500 mg/kg) (n=4/route of administration). Serum and urine samples were collected for 72 h post dose. In vitro antioxidant activity, anti-inflammatory activity (cyclooxygenase and lipoxygenase inhibition), dipeptidyl peptidase-4 (DPP-4) inhibition and cytochrome P450 inhibitory activties of calebin A were examined using commercial assay kits. Content analysis of calebin A in 14 natural health products advertised to contain turmeric were carried out using methanolic extraction. The HPLC method was successfully applied to a pharmacokinetic study of calebin A in rats. After IV and PO administration of calebin A, the compound was detected as the aglycone and a glucuronidated metabolite. Oral bioavailabitily was found to be ~0.5%, serum half-life was ~1-3 h. Calebin A appears to be primarily excreted via non-renal routes. Calebin A possessed concentration-dependent antioxidant activity and DPP-4 inhibition. Calebin A appears to be a non-selective cyclooxygenase inhibitor and also a poor lipoxygenase inhibitor. The curcuminoid displayed in vitro interactions with CYP2D6 and CYP1A2. Content analysis of 14 natural health products that claimed to contain turmeric showed that concentration of calebin A was inconsistent among the products. A successful assay was developed for the detection of calebin A using RP-HPLC. Preliminary pharmacokinetic studies indicate that an unoptimised formulation of calebin A has poor oral bioavailability. Calebin A exhibits various pharmacological activities. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

The method of estimating bisulfite conversion rate in DNA methylation analysis.

To establish an effective method to estimate the conversion rate of bisulfite-treated genomic DNA, TaqMan qPCR assay was performed using probes and primers that are specific for bisulfite-converted or -unconverted DNA standard samples separately. Then two linear standard curves were generated by plotting Ct values against logarithm of absolute DNA amount with serial dilutions of the bisulfite-converted or unconverted DNA samples. Based on two standard curves, the unknown bisulfite-treated genomic DNA sample was analyzed using the same TaqMan probes and the bisulfite conversion rate was precisely estimated. This method was further verified to be reliable using known mixed bisulfite-converted and -unconverted DNA templates as well as DNA samples treated with different bisulfite kits. These results showed that this method can effectively estimate bisulfite conversion rate of genomic DNA and thus provides a reliable and quick method for accurate analyses of DNA methylation.

Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration.

NG,NG‐dimethyl‐l‐arginine (asymmetric dimethylarginine, ADMA),NG‐monomethyl‐l‐arginine (l‐NMMA) and NG,N G’‐dimethyl‐l‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l‐NMMA inhibit nitric oxide synthase by competing with l‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd.

Applied circular dichroism: a facile spectroscopic tool for configurational assignment and determination of enantiopurity.

In order to determine if electronic circular dichroism (ECD) is a good tool for the qualitative evaluation of absolute configuration and enantiopurity in the absence of chiral high performance liquid chromatography (HPLC), ECD studies were performed on several prescriptions and over-the-counter drugs. Cotton effects (CE) were observed for both S and R isomers between 200 and 300 nm. For the drugs examined in this study, the S isomers showed a negative CE, while the R isomers displayed a positive CE. The ECD spectra of both enantiomers were nearly mirror images, with the amplitude proportional to the enantiopurity. Plotting the differential extinction coefficient (Δε) versus enantiopurity at the wavelength of maximum amplitude yielded linear standard curves with coefficients of determination (R 2) greater than 97% for both isomers in all cases. As expected, Equate, Advil, and Motrin, each containing a racemic mixture of ibuprofen, yielded no chiroptical signal. ECD spectra of Suphedrine and Sudafed revealed that each of them is rich in 1S,2S-pseudoephedrine, while the analysis of Equate vapor inhaler is rich in R-methamphetamine.

Fluorescent immunoassay for determination of penicillin and sulphonamide residues in milk using carboxylic magnetic nanoparticles

A fluorescent immunoassay for separate and simultaneous determination of penicillin (PEN) and sulphadimethoxine (SDM) in milk was developed. Monoclonal antibodies immobilised on carboxylic magnetic nanoparticles (MNPs) were used. It was proved that the protein A‐oriented immobilisation method (OI) provided a 1.5‐times more sensitive assay than the random immobilisation one (RI). The activation of MNPs with a mixture of N‐(3‐dimethylaminopropyl)‐N‐ethylcarbodiimide (EDC) and N‐hydroxysuccinimide (NHS) ensured a five‐times higher sensitivity of immunoassay than the activation using EDC. The linearity of standard curves in milk was: 3/10 ng/mL PEN (OI and RI), 50/250 ng/mL SDM (OI), and 50/500 ng/mL SDM (RI).

A validated liquid chromatography tandem mass spectrometry method for simultaneous determination of pioglitazone, hydroxypioglitazone, and ketopioglitazone in human plasma and its application to a clinical study.

A simple and rapid high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of pioglitazone and its active metabolites hydroxypioglitazone and ketopioglitazone in human plasma. Samples were processed by protein precipitation with acetonitrile and selective phospholipid depletion in a 96-well plate format. The method used deuterated internal standards for each analyte. Chromatographic separation was achieved with gradient elution on a Hypersil GOLD C18 column. The mass spectrometer was operated in electrospray positive ion mode with detection by selected reaction monitoring using the transitions m/z 357.1>134.0 for pioglitazone, m/z 373.1>150.0 for hydroxypioglitazone, and m/z 371.0>148.0 for ketopioglitazone. A linear standard curve was established for the range of 10-1800ng/mL for all three analytes. Intra-run and inter-run precision and accuracy (relative error) were less than 15%, and the mean extraction recoveries of all analytes were more than 87.8%. The validated method is sensitive and selective and was successfully applied to analyze clinical samples obtained from patients with nonalcoholic fatty liver disease taking pioglitazone.

Simultaneous determination of cyromazine, melamine and their biodegradation products by ion-pair high-performance liquid chromatography

An ion-pair high-performance liquid chromatography with ultraviolet detection method for the determination of cyromazine, melamine and its biodegradation products (ammeline, ammelide, cyanuric acid and biuret) was developed. C18 column was utilised to separate the six analytes with a mobile phase consisting of perchloric acid-ammonia solution and acetonitrile, under gradient elution and variable flow rate. The detection wavelengths were 205 nm for cyanuric acid and biuret and 222 nm for cyromazine, melamine, ammeline and ammelide. For analysis of sediment samples, the extraction solution containing acetonitrile, ammonia and water (80:10:10 by volume) was used to extract the analytes from sediment matrix. Using the extraction method for the spiked sediment sample, high linearity of matrix-matched standard curve could be obtained for the six analytes. The method detection limit was 0.1 μg g−1 for melamine and cyromazine, 0.2 μg g−1 for ammeline and ammelide, 1.2 μg g−1 for cyanuric acid and 1.0 μg g−1 for biuret in sediment matrix. The recoveries of these compounds were 70.1–98.3% and the relative standard deviations were 0.5–4.4%. Finally, the proposed method was successfully applied to the analysis of the sediment sample near the wastewater outlet of a melamine-producing factory.

Development of GC‐MS method for quantification of phenolic acids after in vitro fecal fermentation (1044.4)

Unabsorbed polyphenols are subject to metabolism catalyzed by colonic microbiota into a wide range of phenolic acids (PA) with implications for gut health. The aim of this study was to develop a reliable, sensitive method for quantification of PA generated from in vitro anaerobic fecal fermentation. Fecal ferment containing 2% fecal matter (w/v) were purified by solid‐phase extraction (SPE) using a Phenomenex Strata SDB‐L cartridge. PA in the resulting SPE eluate were derivatized sequentially by halogenation using 30% pentafluorobenzyl bromide and 10% diisopropylethylamine and by silylation using 10% trymethylchlorosilane in BSTFA. This 2‐step protocol diminished the impact of ambient humidity on derivatization reproducibility compared to silylation alone. SPE recovery averaged 70% for 25 quantified PA. The derivatized products were separated by a 0.25 mm x 0.25 µm GC column and determined by an Agilent GC‐MS operated in an electron ionization and SIM modes. The internal standard was 4’‐hydroxy‐3’‐methoxyacetophenone. The limit of detection ranged from 1 to 10 ng/mL and the limit of quantification from 10 to 100 ng/mL. The linearity of standard curves constructed from PA spiked into the fecal ferment was r² >0.99. The intra‐day coefficient of variation for authenticated PA standards was <9% and for standards spiked into the fecal ferment was <14%. (Funded by Department of Army, CONACYT, and USDA)Grant Funding Source: Supported by Department of Army, CONACYT, and USDA

Quantitative determination of enzalutamide, an anti‐prostate cancer drug, in rat plasma using liquid chromatography–tandem mass spectrometry, and its application to a pharmacokinetic study

This report details a method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that allows one to determine the concentration of an atypical anticancer drug, enzalutamide, in rat plasma. Specifically, this method involves the addition of an acetonitrile and bicalutamide (internal standard) solution to plasma samples. Following centrifugation of this mixture, an aliquot of the supernatant was directly injected into the LC-MS/MS system. Separation was achieved using a column packed with octadecylsilica (5 µm, 2.1 × 50 mm) with 10 mM ammonium acetate in acetonitrile as the mobile phase; detection was accomplished using MS/MS by multiple-reaction monitoring via an electrospray ionization source. This method demonstrated a linear standard curve (r = 0.997) over a concentration range of 0.001-1 µg/mL, as well as an intra- and inter-assay precision of 2.7 and 5.1%, respectively, and an accuracy range from 100.8 to 105.6%. The lower limit of quantification was 1.0 ng/mL in 50 μL of rat plasma sample. We also demonstrated that this analytical method could be successfully applied to the pharmacokinetic study of enzalutamide in rats.

Rapid quantitative analysis of microcystins in raw surface waters with MALDI MS utilizing easily synthesized internal standards

The freshwater cyanotoxins, microcystins (MCs), pose a global public health threat as potent hepatotoxins in cyanobacterial blooms; their persistence in drinking and recreational water has been associated with potential chronic effects in addition to acute intoxications. Rapid and accurate detection of the over 80 structural congeners is challenged by the rigorous and time consuming clean up required to overcome interference found in raw water samples. MALDI-MS has shown promise for rapid quantification of individual congeners in raw water samples, with very low operative cost, but so far limited sensitivity and lack of available and versatile internal standards (ISs) has limited its use. Two easily synthesized S-hydroxyethyl-Cys(7)-MC-LR and -RR ISs were used to generate linear standard curves in a reflectron MALDI instrument, reproducible across several orders of magnitude for MC-LR, -RR and -YR. Minimum quantification limits in direct water samples with no clean up or concentration step involved were consistently below 7 μg/L, with recoveries from spiked samples between 80 and 119%. This method improves sensitivity by 30 fold over previous reports of quantitative MALDI-TOF applications to MCs and provides a salient option for rapid throughput analysis for multiple MC congeners in untreated raw surface water blooms as a means to identify source public health threats and target intervention strategies within a watershed. As demonstrated by analysis of a set of samples from Uruguay, utilizing the reaction of different MC congeners with alternate sulfhydryl compounds, the m/z of the IS can be customized to avoid overlap with interfering compounds in local surface water samples.

Comparison of salivary collection and processing methods for quantitative HHV‐8 detection

Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accuracy of viral load determinations is unknown. Stabilising fluids impede nucleic acid degradation, compared with collection onto ice and then freezing, and we have shown that the DNA Genotek P-021 prototype kit (P-021) can produce high-quality DNA after 14 months of storage at room temperature. Here we evaluate the quantitative capability of 10 collection/processing methods. Unstimulated whole mouth fluid was spiked with a mixture of HHV-8 cloned constructs, 10-fold serial dilutions were produced, and samples were extracted and then examined with quantitative PCR (qPCR). Calibration curves were compared by linear regression and qPCR dynamics. All methods extracted with commercial spin columns produced linear calibration curves with large dynamic range and gave accurate viral loads. Ethanol precipitation of the P-021 does not produce a linear standard curve, and virus is lost in the cell pellet. DNA extractions from the P-021 using commercial spin columns produced linear standard curves with wide dynamic range and excellent limit of detection. When extracted with spin columns, the P-021 enables accurate viral loads down to 23 copies μl(-1) DNA. The quantitative and long-term storage capability of this system makes it ideal for study of salivary DNA viruses in resource-poor settings.

Determination of Amaranth in Beverage by Indirect Competitive Enzyme-linked Immunosorbent Assay (ELISA) Based on Anti-amaranth Monoclonal Antibody

Amaranth (E 123) is a member of azo dyes, and it is allowed to use in various foods. The acceptable maximum addition of amaranth is strictly fixed because of its potential risk to physical health. The objective of this study was to prepare a specific anti-amaranth monoclonal antibody and develop an indirect competitive ELISA for amaranth quantification analysis. The immunogen and the coating antigen were designed by introducing a carboxyl group into amaranth for the conjugation with carrier proteins. Based on the immunogen, the monoclonal antibody exhibits satisfactory performances and the proposed ELISA shows an IC50 of 20.33 ng mL−1. The limit of detection is as low as 3.35 ng mL−1, and the linear standard curve of the method ranges from 3.0 to 243.0 ng mL−1. Additionally, the antibody reflects minimal cross-reactivity (<1 %) with six related food dyes (erythrosine, ponceau 4R, allura red, tartrazine, sunset yellow FCF, and brilliant blue). The recoveries of amaranth spiked beverage samples are in the range of 85.8–100.7 % with low coefficient of variation values (<11.5 %). The data shows that the developed ELISA provides a simple, sensitive, specific, and accurate alternative for amaranth determination and monitoring. Furthermore, it is the first time that icELISA of amaranth is developed based on monoclonal antibodies.

Simultaneous analysis and monitoring of thirteen preservatives in cosmetics by liquid chromatography

The content of 13 preservatives was investigated for 107 sold in retail markets. A simultaneously analytical method of liquid chromatography, LC, for the preservatives in cosmetics was developed using an AvQuityTM Ultra performance LC System, Waters, BEH C18 column, 150mmx2.1mm,1.7 ㎛, and photo Diode Array, PDA, detector λ;220 nm for quantitative analysis, λ;190∼400nm for qualitative analysis. The developed instrumental method was validated using limit of detection, LOD, limit of quantification, LOQ, precision, accuracy, and linearity of standard curves. LOD and LOQ wre lower than 0.23mg/l and 0.75mg/l, respectively. When standard compounds were analyzed seven times consecutively at the LOQ level, precision and accuracy were lower than 2.49% and, 2.04%, respectively. Good linear relationships, R2 ⊇0.9998, were obtained for all compounds. Recoveries ranged between 96.03% and 102.61%and the overall variability was below 3%, RSD, The type of cosmetics showing the lowest detection rate of target compounds was sunscreen lotion, with a detection rate of 42.9%, followed by baby lotions and mask packs with the same detection rate at 69.2% At least one of the target preservatives was detected in 82.4% of creams, 93.3% of skin toners, 95.2% of lotions, and 92.9% of body lotions. Overall 79.4% of all samples contained at least one of the target compounds. The most frequently detected compound was phenoxyethanol. Methyl and propyl paraben also had a higher detection rate than the others, except, phenoxyethanol. Although the target preservatives in cosmetics sold in retail markets were detected by the simultaneous analysis of LC, the content of the detected preservatives was within the maximum allowed amount established by the Korean FDA.

Detection of HIV-1 p24 Antigen Using a Simple and Highly Sensitive Chemiluminescence Immunoassay

Aim: Our study aimed to improve the p24 antigen assay, which is a well-established and widely adopted method for the early detection of HIV-1 infections, in order to increase its sensitivity and efficiency and reduce its cost if possible. Materials &amp; methods: We tested HIV-1 standard p24-positive samples and an HIV-1 p24 antigen panel using a new luminol-based chemiluminescence immunoassay (CLIA) based on a typical ‘sandwich’ p24 ELISA with two antibodies, but utilizing luminol as the luminescence compound, which is oxidized by hydrogen peroxide in the presence of horseradish peroxidase in an alkaline environment. A standard curve for the p24 antigen CLIA was generated and fitted a third-order polynomial regression model very well. To quantify p24 antigen, we investigated the linear standard curve fitting by measuring and comparing the effective assay range, r2 value and error of each function. Results: The p24 antigen CLIA showed a detection limit of 0.5 pg/ml and a detection range of 0.5–5 × 103 pg/ml. The specificity and sensitivity of this assay were further evaluated, and were both found to be 100% for the HIV-1 p24 antigen panel. The luminescence reaction of the CLIA p24 assay takes only 2 min to perform, which greatly reduced the diagnosis time. Additionally, the CLIA p24 assay is as affordable as the normal ELISA method. Conclusion: In our studies, the p24 antigen CLIA shows improved sensitivity, a wider range of response and was less time-consuming than ELISA, as traditional ELISA uses chromogen or a substrate that changes color when cleaved by the enzyme attached to a second antibody. This seems to be an easy and affordable method for the diagnosis of HIV-1 infection and monitoring of disease progression in developing countries. Although more advantages have been seen in CLIA than in ELISA, additional studies are still needed to confirm this.

Application of Selected Reaction Monitoring for Multiplex Quantification of Clinically Validated Biomarkers in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.