Nitrocellulose Strips Research Articles

Serological profile of voluntary blood donors in a blood bank of tertiary health care centre- An institution-based study

Background: The safe transfusion of blood facilities is an essential component of an efficient, excellent healthcare sector. Unsafe blood transfusions can be a cause of transfusion-transmitted infections and can be lethal. The present study was conducted to find out the seroprevalence of HBsAg, HCV, HIV, malarial parasites, and syphilis in voluntary blood donors in a tertiary care center. Materials and methods: This prospective study was conducted at the blood bank of the Shri Mahant Indiresh Hospital, Dehradun. 16413 samples from all blood donations were screened for HBsAg, HCV, HIV 1 and 2, malaria, and syphilis. Samples were collected in vacutainers at the time of blood donation and screened for HIV 1and2, HBsAg, and HCV using the fourth-generation enzyme-linked immunosorbent assay technique. Syphilis was tested by Treponema antibodies using the VITROS Syphilis TPA Reagent Pack. Malaria was tested by pan malaria card, monoclonal anti-pan specific parasite lactate dehydrogenase antibody conjugated to colloidal gold, and another monoclonal anti-pan specific parasite lactate dehydrogenase antibody immobilized on a nitrocellulose strip in a thin line. Results: Out of 16413 blood donors 342(2.1%) were found to be seropositive and 16071 (97.9%) were found to be seronegative. The total number of donors, who were found positive for HBsAg/Anti HCV was 1 (0.3%). Out of the seropositive cases, HBsAg, HCV, HIV, and Syphilis infections constituted 28.9%, 27.5, 14.6, and 28.7% cases respectively. Conclusions: There is a need for the development and implementation of stringent donor selection and sensitive screening tests to reduce the risk of acquiring transfusion-transmitted illnesses.

Paper-based isotachophoretic preconcentration technique for low-cost determination of glyphosate.

Electrophoretic microfluidic paper-based analytical devices (e-µPADs) are promising for low-cost and portable technologies, but quantitative detection remains challenging. In this study, we develop a paper-based isotachophoretic preconcentration and separation method for the herbicide glyphosate as a model analyte. The device, consisting of two electrode chambers filled with leading and terminating electrolytes and a nitrocellulose strip as the separation carrier, was illuminated by a flat light source and operated with a voltage supply of 400V. Detection was accomplished using a simple camera. Colorimetric detection was optimized through competitive complexation between glyphosate, copper ions, and pyrocatechol violet as a dye. The buffer system was optimized using simulations, (i) ensuring the pH was optimal for the demetallation of the blue pyrocatechol violet-copper complex [PV] to the yellow free dye and (ii) ensuring the electrophoretic migration of glyphosate into the slower [PV] for the colorimetric reaction. A new data evaluation method is presented, analyzing the RGB channel intensities. The linear range was between 0.8 and 25µM, with a LOD of approximately 0.8µM. The ITP separation preconcentrated glyphosate by a factor of 820 in numerical simulations. The method may be applied to control glyphosate formulations, especially in developing countries where herbicide sales and applications are poorly regulated.

Portable surface acoustic wave device platform coupled with a paper-based capillary fluidics for real-time biosensing applications

Surface acoustic wave (SAW) sensors have emerged as prominent real-time, label-free biosensors with diverse applications in immunoassays and nucleic acid detection. However, widespread adoption in diagnostics has been impeded by challenges such as miniaturization of instrumentation, microfluidics fabrication and high attenuation in liquid environments. In this study we address these limitations by presenting a portable platform capable of real-time monitoring of a SAW microarray chip comprising up to four sensing channels, combined with a novel paper-based capillary flow channel. For the fabrication of a portable, automated and versatile sensor platform, we integrated a microcontroller, RF components, and a micropump for flow control. Calibration procedures ensured accurate measurements for both amplitude and phase, with a low drift rate (0.72 mdB/h and 1.00 mdeg./h, respectively) and high resolution (2.10 mdB and 6.10 mdeg.), indicating stable operation with minimal interface electronics. The capillary flow channel, implemented by confining the fluid on the sensing surface using a nitrocellulose strip, facilitated laminar and continuous sample flow with minimum damping of the acoustic signal. The sensor system was evaluated with two SAW devices at 100 and 200 MHz using glycerol dilutions within the range of 1 % and 70 %, demonstrating real-time monitoring capabilities and linear correlations between amplitude and phase changes. To prove the biosensing and clinical validity of the SAW newly developed system, DNA adsorption on a poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) functionalized SAW-surface was demonstrated with a detection limit of 0.001 μg/mL. Our study demonstrates the feasibility of a portable SAW sensor system coupled with a low-cost and replaceable capillary flow channel for real-time monitoring and biomolecular detection.

Evaluation of the Performance of Rapid Diagnostic Tests for Malaria Diagnosis and Mapping of Different Plasmodium Species in Mali.

The first-line diagnosis of malaria in Mali is based on the use of rapid diagnostic tests (RDT) that detect the Histidin Rich Protein 2 (HRP2) antigen specific to Plasmodium falciparum. Our study, based on a real-time polymerase chain reaction (qPCR) gold standard, aimed to describe the distribution of the Plasmodium species in each administrative region of Mali and to assess the performance of RDTs. We randomly selected 150 malaria-negative and up to 30 malaria-positive RDTs in 41 sites distributed in 9 regions of Mali. DNA extracted from the RDT nitrocellulose strip was assayed with a pan-Plasmodium qPCR. Positive samples were then analyzed with P. falciparum-, P. malariae-, P. vivax-, or P. ovale-specific qPCRs. Of the 1496 RDTs, 258 (18.6%) were positive for Plasmodium spp., of which 96.9% were P. falciparum. The P. vivax prevalence reached 21.1% in the north. RDT displayed acceptable diagnostic indices; the lower CI95% bounds of Youden indices were all ≥0.50, except in the north (Youden index 0.66 (95% CI [0.44-0.82]) and 0.63 (95% CI [0.33-0.83]. Overall, RDT diagnostic indices are adequate for the biological diagnosis of malaria in Mali. We recommend the use of RDTs detecting P. vivax-specific antigens in the north.

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

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

Capillary flow-driven immunoassay platform for COVID-19 antigen diagnostics

Over the last few years, the SARS-CoV-2 pandemic has made the need for rapid, affordable diagnostics more compelling than ever. While traditional laboratory diagnostics like PCR and well-plate ELISA are sensitive and specific, they can be costly and take hours to complete. Diagnostic tests that can be used at the point-of-care or at home, like lateral flow assays (LFAs) are a simple, rapid alternative, but many commercially available LFAs have been criticized for their lack of sensitivity compared to laboratory methods like well-plate ELISAs. The Capillary-Driven Immunoassay (CaDI) device described in this work uses microfluidic channels and capillary action to passively automate the steps of a traditional well-plate ELISA for visual read out. This work builds on prior capillary-flow devices by further simplifying operation and use of colorimetric detection. Upon adding sample, an enzyme-conjugated secondary antibody, wash steps, and substrate are sequentially delivered to test and control lines on a nitrocellulose strip generating a colorimetric response. The end user can visually detect SARS-CoV-2 antigen in 15–20 min by naked eye, or results can be quantified using a smartphone and software such as ImageJ. An analytical detection limit of 83 PFU/mL for SARS-CoV-2 was determined for virus in buffer, and 222 PFU/mL for virus spiked into nasal swabs using image analysis, similar to the LODs determined by traditional well-plate ELISA. Additionally, a visual detection limit of 100 PFU/mL was determined in contrived nasal swab samples by polling 20 untrained end-users. While the CaDI device was used for detecting clinically relevant levels of SARS-CoV-2 in this study, the CaDI device can be easily adapted to other immunoassay applications by changing the reagents and antibodies.

Graphene oxide coatings enhance fluorescence signals in a lateral flow immunoassay for the detection of UCH-L1, a marker for trauma brain injury

We present a lateral flow immunoassay (LFA) for the quantitative, fluorescence-based detection of the trauma brain injury (TBI) biomarker ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) that features a layer of graphene oxide (GO) particles over the test zone of nitrocellulose (NC) strips. The introduction of 80 ng of GO at test lines increased fluorescence signals 2–3-fold. This was attributed to an increase in the immobilization of capture antibodies in the top layer of NC, which increased the density of recognition complexes at the surface and hence the signals reaching the reader used. A linear concentration–response relationship was observed in the 0–200 pg/mL UCH-L1 range with an inter assay CoV of 2.65 %. The LOD and LOQ values of 11.1 pg/mL and 33.5 pg/mL were compatible with threshold levels of UCH-L1. Finally, mock plasma samples with UCH-L1 levels characteristic of TBI patients with negative (60 pg/mL) and positive (130 pg/mL) CT scans were successfully analyzed. Storage stability testing further showed that GO did not affect the biological activity of the capture antibody. In summary, we demonstrate that the use of GO particles in the top layers of the test zone of NC strips improves signal intensity, which could potentially enhance the accuracy and efficiency of LFA-based diagnosis.

Development of paper-immobilized molecularly imprinted polymers by laser pointer activation for methamphetamine extraction with analysis by ion mobility spectrometry

A fast, simple, cheap, and versatile strategy has been proposed for the synthesis of paper-immobilized molecularly imprinted polymers (MIPs) by photoactivated bulk polymerization over a piece of nitrocellulose using a 405 nm laser pointer. Polymerization was carried out using a mixture of methacrylic acid and ethylene glycol dimethacrylate, using methamphetamine as template molecule and bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide as radical initiator. After investigation of different polymerization parameters, the following experimental conditions were found to give best results: size of nitrocellulose strip (13.5 × 4.0 × 0.8 mm), type of porogen (acetonitrile), polymerization mixture volume (75 µL), and irradiation times (10 min). Experimental conditions (such as sample pH, extraction and desorption time, and type and volume of desorption solvent) were also adjusted for the extraction of methamphetamine using the proposed paper-MIP. Methamphetamine determination was carried out by ion mobility spectrometry providing a limit of detection of 14 µg L−1 and quantitative recoveries from 81 to 95% using spiked urine and oral fluid samples. The proposed paper-immobilized MIP device allows a simple and selective sample extraction procedure for the determination of methamphetamine in oral fluids and urine with a high portability, minimal solvent consumption, and reduced costs compared to other conventional approaches.

Development of a lateral flow device for rapid simultaneous multiple detections of some common bacterial causes of bovine mastitis.

This work was conducted for the development of a 5-combi lateral flow immunochromatographic kit (LFK) for rapid and simultaneous identification of the common bacterial causes of bovine mastitis. The following pathogens are the identification targets of this kit: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes in milk samples from suspected bovine mastitis cases. The conventional microbiological identification of these agents is not only time-consuming and requires a fully equipped laboratory but also requires experienced personnel. Rabbit polyclonal antibodies (PAbs) specific to the antigenic components of the selected pathogens were prepared, and the pathogen-specific IgG was separated, purified, and conjugated with nanogold that was laid on the conjugate pad. Guinea pig PAbs specific to the microbial antigens of the selected pathogens were prepared, and their IgG content was separated, purified, and used as a capture antibody in the test (T) line on the nitrocellulose (NC) strips. Goat anti-rabbit IgG antibodies were used to capture the rabbit antibodies in the control (C) line of NC strips. The kit was held in a device comprising five strip-holding channels for the above-mentioned five bacterial species antigens. The developed LFK was evaluated, and its sensitivity and specificity were determined. The developed kits were applied for the examination of bovine milk samples from suspected mastitis cases, and the average sensitivity, specificity, and accuracy of 5-combi LFK for the detection of the five selected bacterial species compared to bacteriological examination (gold standard test) were 93.90%, 80.83%, and 90.53%, respectively. The minimal microbial count that gave positive results using the developed LFK was 103 colony forming unit/ml. Treatment of the milk samples with an application buffer and its pre-incubation in trypticase soy broth for 6 h at 37°C before testing significantly increased the sensitivity of the prepared LFK. The developed kit proved simple and convenient, and the results could be obtained in less than 10 min.

Development and Validation of a Rapid Screening Test for HTLV-I IgG Antibodies.

Initial diagnosis of human T cell lymphotropic virus (HTLV) infections is mainly based by detecting antibodies in plasma or serum using laboratory-based methods. The aim of this study was to develop and evaluate a rapid screening test for HTLV-I antibodies. Our rapid screening test uses HTLV-I p24 antigen conjugated to gold nanoparticles and an anti-human IgG antibody immobilized to a nitrocellulose strip to detect human HTLV-I p24-specific IgG antibodies via immunochromatography. Performance of the rapid screening test for HTLV-I was conducted on a total of 118 serum specimens collected in Salvador, Bahia, the epicenter for HTLV-1 infection in Brazil. Using a Western blot test as the comparator, 55 serum specimens were HTLV-I positive, 5 were HTLV-I and HTLV-II positive, and 58 were negative. The sensitivity of the rapid screening test for HTLV-1 was 96.7% and the specificity was 100%. The rapid screening test did not show cross-reaction with serum specimens from individuals with potentially interfering infections including those caused by HTLV-II, HIV-I, HIV-II, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, Epstein-Barr virus, SARS-CoV-2, Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Toxoplasma gondii, and Plasmodium falciparum. The rapid screening test also did not show cross-reaction with potentially interfering substances. Strategies for HTLV diagnosis in non- and high-endemic areas can be improved with low-cost, rapid screening tests.

Paper-based monolith extraction of psychoactive substances from biological fluids

A monolith of poly(methacrylic acid-co-ethylene glycol dimethacrylate) has been immobilised to a nitrocellulose strip by radical photopolymerisation to be used in the extraction of psychoactive substances in biological fluids. Codeine, methylone, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, butylone, norketamine, ketamine, heroin, cocaine, lysergic acid diethylamide and fentanyl were employed as model drugs and final extracts were analysed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Polymerisation parameters were adjusted in order to obtain a stable and homogeneous layer of monolith onto the nitrocellulose strip. The resulting sorptive phase was characterized by Fourier-transform infrared spectroscopy and scanning electron microscopy. Extraction conditions were investigated by the evaluation of sample pH, extraction and desorption times and desorption solvent volume, providing enrichment factor values ranging from 5.3 to 39.9. The proposed methodology provided limit of quantification values from 0.013 μg L−1 for methylone to 0.057 μg L−1 for amphetamine, and recoveries from 64 to 120%. Urine and serum certified reference materials were employed in the validation of the proposed methodology, providing results statistically comparable. The developed approach is simple and straightforward for the determination of psychoactive substances in urine and serum samples.

Exploring carbohydrate binding module fusions and Fab fragments in a cellulose-based lateral flow immunoassay for detection of cystatin C

This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the kidney biomarker cystatin C that features conjugates of capture antibodies and fusions of carbohydrate binding modules (CBM) with ZZ domains anchored on cellulose deposited over nitrocellulose (NC). The ZZ-CBM3 fusion provides a biomolecular interface between the cellulose layer and the Fc portion of the capture antibodies. By resorting to detection Fab fragments that lack the Fc portion we overcome the observed interference of full-length detection antibodies with the ZZ-CBM3 fusion at the test lines. Using the new LFA architecture, a linear concentration–response relationship was observed in the 0–10 ng/mL cystatin C concentration range, which is compatible with the clinically normal (5–120 ng/mL) and abnormal (> 250 ng/mL) levels of cystatin C, as long as proper dilutions are made. An inter assay CoV of 0.72% was obtained. Finally, mock urine samples characteristic of normal (100 ng/mL) and kidney tubular disease (4000 ng/mL) patients were successfully analyzed. Overall, we demonstrate an innovative LFA architecture that combines NC strips with layered cellulose, ZZ-CBM3 fusions and fluorescently labeled Fab fragments.

A New Rapid and Quantitative Assay to Determine the Phytase Activity of Feed.

The fortification of animal feed with enzymes in order to optimize feed utilization has become a standard for the meat production industry. A method for measuring levels of active enzymes that can be carried out quickly would ensure that feed has been supplemented with the appropriate amount of enzyme. Phytase is the most widely used feed enzyme and is routinely quantified with an activity assay in a limited number of specialized laboratories. As an alternative, we report here the development of a rapid and easy method to perform a quantitative assay for the phytase from Citrobacter braakii. The method is suitable for use at local sites with a minimum lab setup and will reduce delays and potential interferences due to improper sample storage and shipment. The new assay is based on a lateral flow immunoassay that utilizes magnetic immune-chromatographic test (MICT) technology to quantify the phytase content of a feed extract. After extraction of the phytase from the feed, the sample is simply diluted and added to a reaction tube containing a specific anti-phytase antibody coupled to superparamagnetic particles. The mixture is then applied on an assay cassette, where the formed particle–antibody–phytase complexes are captured by immobilized antibodies on a nitro-cellulose strip housed in a cassette. The cassette is placed in the MICT reader that measures the magnetic signal of the captured particles. Using the calibration information stored in the cassette barcode, the signal is converted to a phytase concentration, given as phytase activity (FYT) per kilogram of feed. The accuracy and robustness of the assay compared to the ISO phytase activity assay were demonstrated through a large validation study including real feed samples from different compositions and origins. The MICT assay is the first quantitative assay for feed enzymes that is fast, reliable, and simple to use outside of a specialized reference laboratory and that is suitable for use in place of the current ISO assay.

(Invited) Enhancing Sensitivity of Lateral Flow Assays By Immobilization of Affinity Molecules in Hydrogel Polymer Embedded Test Lines

Rapid tests of COVID-19 have played a critical role in controlling the virus spread during the pandemic. Lateral flow assays (LFA), a paper-based technique, are widely adopted as a platform for detecting SARS-CoV-2 antigens, anti-SARS-CoV-2 antibodies, and viral RNA in combination with other technologies such as CRISPR. Due to its rapidness, cheapness, and visual readout, LFA is especially suitable for the at-home test. However, LFA has manifested a low sensitivity for antigen-based COVID-19 test. It has been reported that the sensitivity was as low as 12–18% when Ct values of RT-PCR were between 30 and 37, which limits LFA as a diagnostic tool.We intend to develop a sensitive LFA for at-home use, like a pregnancy test strip, with a configuration shown in Figure 1a. Our first efforts are to lower its limit of detection (LoD) by enhancing the efficiency of molecular capture on the nitrocellulose membrane. As shown in Figure 1b, a test line on the strip comprises a hydrogel polymer, which would provide a biologically friendly environment for molecular interactions. We have fabricated a polyacrylamide test line containing streptavidin on a nitrocellulose strip using photolithography. Thus, a test strip can be produced by printing biotinylated affinity molecules on the test line. We have used gold nanoparticles as a colorimetric readout of LFA. Our results show that the immobilization of an antibody in the hydrogel polymer would enhance the sensitivity of LFA’s detection. Compared to those by physical adsorption of antibodies directly to the nitrocellulose membrane, our LFA reduced the LoD by ~ 2 orders of magnitude. We are currently applying our LFA to detect neutralizing antibodies and variants of concern (VOC) of SARS-CoV-2. In this presentation, we will report the current progress of our project. Figure 1. Illustration of (a) general configuration of lateral flow strips (b) a lateral flow strip with hydrogel polymer embedded in the test line for antibody testing Figure 1

Phacoemulsification and nucleic acid-enriched aerosol: considerations from an eye model of cataract simulation.

PurposeThe purpose of this study is to investigate whether phacoemulsification can generate aerosolized single-stranded RNA (ssRNA) and retain sequence integrity using an artificial eye model for experimental cataract surgery.MethodsA simulation of cataract surgery was performed using an anterior chamber eye model filled with an ssRNA probe at different scalar dilutions (kanamycin positive control ssRNA). A plastic conical cage was built over the artificial eye surface of the mock-up. A total of 24 tests (twice reproduced) were performed, and five nitrocellulose strips were placed 15 cm from the artificial surface of the mock-up and used to collect aerosol particles, from each experiment. Phaco-activity was mimicked using a phacoemulsification equipped with a 2.75-mm tip, and strips were removed at the end of the procedure. RNA extraction, reverse transcription, and agarose gel electrophoresis were performed and compared.ResultsStrips collected aerosol droplets enriched with ssRNA, mainly at the higher concentrations tested, compared to related untouched standard solutions. Complementary DNA (cDNA) synthesis confirmed the presence of intact ssRNA fragments. As observed from densitometric analysis of resolved RNA in extracted samples and cDNA bands after retro-transcription, lower concentrations of ssRNA were also detected.ConclusionsAs the main output of the study, the phaco-generated aerosol can deliver an intact ssRNA sequence. Since the aerosol can potentially reach the operator’s face, any biological agent (virus/bacteria) potentially inside the anterior chamber of a patient undergoing cataract surgery, eventually escaping from biomolecular checks, can be potentially infective for operators. The data reported herein suggest that collective versus individual protective countermeasures should always be encouraged in ocular surgery and should not be restricted to coronavirus disease emergencies.

Continuous capillary-flow sensing of glucose and lactate in sweat with an electrochemical sensor based on functionalized graphene oxide

We describe an electrochemical device for the simultaneous monitoring of glucose and lactate in sweat, based on enzymatic sensors exploiting capillary flow to induce continuous, stable sensing. The enzymes, namely glucose oxidase and lactate oxidase, were anchored to a graphene oxide and chitosan composite (GO-Ch) of original synthesis, to achieve stable deposition of the bioreceptors on the electrochemical platform. We tested both biosensors on a realistic device architecture: they were embedded in a nitrocellulose strip, to exploit capillary force to induce a continuous flux of sweat on the sensor platform, ensuring the constant renewal of sample. We could achieve good sensitivity at potentials close to zero by using Prussian Blue as redox mediator, thus avoiding interference from other chemical species present in the complex matrix. The sensing signal was stable and linear over two hours in a concentration range of glucose and lactate between the limit of quantification (32 and 68 nM, respectively) and the upper limit of linearity (3.8 and 50.0 mM, respectively). The device is simple, robust, stable, and can be easily worn without the direct contact of the active part with the skin, making it suitable for simultaneous monitoring of glucose and lactate in human sweat.

A Cellulose Paper-Based Fluorescent Lateral Flow Immunoassay for the Quantitative Detection of Cardiac Troponin I.

This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the cardiac biomarker troponin I (cTnI) that features an analytical strip made of cellulose filter paper. The results show that the wicking and test time are comparable to those obtained with conventional nitrocellulose (NC)-based LFAs. Further, the cellulose paper provides an excellent background with no auto-fluorescence that is very adequate in detecting fluorescent lines. While fluorescence that was generated with cellulose strips was lower when compared to that generated in NC strips, signals could be improved by layering carbon nanofibers (CNF) on the cellulose. A nonlinear behavior of the concentration–response relationship was observed for the LFA architectures with NC, cellulose, and cellulose-CNF in the 0 to 200 ng/mL cTnI concentration range. The measurements were consistent and characterized by coefficients of variation lower than 2.5%. Detection and quantitation limits that were in the range 1.28–1.40 ng/mL and 2.10–2.75 ng/mL were obtained for LFA with cellulose and cellulose CNF strips that are equivalent to the limits obtained with the standard NC LFA. Overall, we showed that commercially available filter paper can be used in the analytical strip of LFA.

Applications of Recombinant Monoclonal Antibodies against Filarial Antigen Proteins.

This study investigated the applications of recombinant monoclonal antibodies (rmAbs) produced against two recombinant filarial proteins of diagnostic value. Ab5B and Ab3A were produced against recombinant BmSXP, and Ab4 and Ab4-fragment crystallizable (Fc) against recombinant BmR1. Ab5B and Ab4-Fc were found to be useful as quality control (QC) reagents for two commercial rapid test kits, such as Brugia RapidTM and BLF Rapid® (Reszon Diagnostics International Sdn. Bhd., 47600 Subang Jaya, Selangor, Malaysia), respectively. The two rmAbs reacted positively with the corresponding recombinant proteins lined on the nitrocellulose strips of the cassette tests, thus may replace or reduce the need for patient serum samples as positive controls for QC of the commercial kits. They were also successfully conjugated to gold nanoparticles and reacted positively with the test lines containing the corresponding recombinant proteins when directly applied to the cassette tests. The gold-conjugated reagents can be used to confirm the antigenicity of test lines after the storage of the rapid tests for a prolonged period or under unfavorable conditions. Furthermore, Ab5B and Ab3A were shown to be able to capture the target recombinant proteins through immunoaffinity purification, enabling their use for applications that need very highly purified proteins. In conclusion, this study demonstrated several potential uses of rmAb proteins produced against recombinant filarial proteins.

Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small epitopes and Their Use in Protein-Lipid Overlay Assays.

We adapted an efficient cell-free protein synthesis-based protocol for the production of lipid-binding proteins. The experimental procedures are based on the following steps: (1) cell-free synthesis of soluble, lipid-binding proteins fused to small tags; (2) analysis by dot blot of the accessibility of antibodies to the small tags. (3) protein lipid overlay assay with, immunodetection of bound protein by either chemiluminescence or fluorescence. We also provide a fast and inexpensive protocol for homemade lipid nitrocellulose strips spotted with acidic lipids (mostly phosphoinositides) extracted from plant tissues. These homemade lipid strips can be used for preliminary screen and characterization of putative phosphoinositide-binding proteins.

Analysis of humoral immune responses to recombinant Chlamydia pneumoniae antigens

ObjectivesChlamydia pneumoniae is a difficult to diagnose respiratory pathogen. This study was performed to systematically characterize humoral immune responses to selected C. pneumoniae antigens in order to provide novel serodiagnostic perspectives for clinical and epidemiological issues. MethodsBased on a literature search, gene library screening, and serological proteome analysis, 15 immunogenic surface-associated, virulence-associated, and hypothetical C. pneumoniae antigens were selected, recombinantly expressed, and lined on a nitrocellulose strip. Specific IgM and IgG reactivity was measured in a total of 172 PCR- and micro-immunofluorescence testing (MIF)-characterized serum samples from patients with respiratory infections. A theoretical model was conceived to approximate a putative course of C. pneumoniae antigen expression and assess the potential of early and late antigens. ResultsWhile surface antigens performed poorly, the virulence-associated TARP was a reliable antigen for IgM detection, with a sensitivity of 80.0% and a diagnostic specificity of 90.2%. The hypothetical protein YwbM proved powerful for IgG detection with MIF-correlative sensitivities of up to 94.4% and a diagnostic specificity of 95.1%. ConclusionsThis study provides new insights into antibody profiles to immunogenic proteins in C. pneumoniae infection. The study findings offer antigen candidates for more reliable and standardized serological investigations of C. pneumoniae infections, including studies on seroprevalence and epidemiology.