Nitrocellulose Membrane Strip Research Articles

The crucial role of immunoblots in distinguishing lyme disease from tick-borne relapsing fever.

Lyme disease (LD) and tick-borne relapsing fever (TBRF), both consequences of Borrelia burgdorferi infection, pose significant public health threats with overlapping symptomatic presentations. The study by Shah et al., explores the potential of line immunoblots (IBs) as a transformative diagnostic tool for distinguishing between LD and TBRF. By employing strategically designed recombinant proteins on nitrocellulose membrane strips, IBs offer enhanced accuracy in identifying antibodies in patient samples. While serological assays for LD have seen advancements, the diagnostic landscape for TBRF lags behind, necessitating comprehensive diagnostic approaches capable of addressing both conditions. Immunoblots, with their ability to detect antibodies with exceptional precision, not only facilitate accurate diagnosis but also enable tailored patient treatment and monitoring of treatment response. The study underscores the importance of immunoblots in improving diagnostic accuracy and patient outcomes for tick-borne diseases. In conclusion, as research progresses in understanding LD and TBRF, immunoblots emerge not only as diagnostic tools but also as valuable resources for advancing research and refining patient treatment strategies.

Nonsteady-State Electric Circuit in Electrophoresis on Paper: Thermal Consideration of Electrophoretic Lateral-Flow Assays.

Nonsteady-state behaviors are not expected in electric circuits that lack significant capacitance, inductivity, and/or active feedback. Here, we report that electrophoresis on paper─used, e.g., to electrophoretically driven lateral-flow immunoassays (LFIA)─can create a nonsteady-state electric circuit. We studied electrophoresis on 50 × 4 mm nitrocellulose membrane strips utilized in LFIA. The voltage was applied to strip termini immersed in reservoirs with a running buffer. If the electric power of this circuit exceeded approximately 0.5 W, neither the electric current nor the temperature map reached their steady states on a multiminute time scale. The current grew slowly to its maximum and then slowly decreased. The temperature map evolved slowly, with one or more hot spots appearing and disappearing gradually in different positions on the strip. The slow evolution of a temperature map led to the occurrence of a terminal hot spot in which the strip burned. No chaotic behavior was observed, i.e., time dependences of both the current and temperature map were reproducible. We analyzed major processes involved in paper-based electrophoresis and explained the nonsteady-state behavior. Unlike ordinary electric circuits with metal conductors, paper-based electrophoresis involves two slow processes: (i) intense buffer evaporation from hot spots and (ii) buffer supply from the reservoirs by an interplay of the capillary penetration and the electroosmotic flow. These processes affect heat generation and/or dissipation on the strip and, accordingly, the resistivity profile. The slow evolution of the resistivity profile is responsible for the nonsteady-state behavior. The results of our computer modeling support this explanation. The hot spots may have a destructive effect on electrophoretically driven LFIA. To avoid denaturation of immunoreagents, experimentalists should empirically confirm that spatiotemporal temperature maps are compatible with the developed assay.

Skin Morphology and Proteins Associated With Chronic Venous Insufficiency

Objective: Skin changes in CVI are clinically observable, but have now been quantified using non-invasive techniques including the The DermaLab Combo (Cortex Technology ApS), the Skin-pH-Meter (Courage + Khazaka electronic GmbH), and skin blotting (University of Tokyo). Skin blotting is a non-invasive technique using moistened nitrocellulose membrane strips to quantify specific proteins on intact skin and has been previously used to identify proteins associated with skin tears,1 pressure injuries,2–5 and itch.6

Recombinant protein immunoblots for differential diagnosis of tick-borne relapsing fever and Lyme disease.

Lyme disease (LD) is caused by a group of tick-borne bacteria of the genus Borrelia termed Lyme disease Borreliae (LDB). The detection of serum antibodies to specific LDB antigens is widely used to support diagnosis of LD. Recent findings highlight a need for serological tests that can differentiate LD from tick-borne relapsing fever (TBRF) caused by a separate group of Borrelia species termed relapsing fever Borreliae. This is because LD and TBRF share some clinical symptoms and can occur in overlapping locations. The development of serological tests for TBRF is at an early stage compared with LD. This article reviews the application of line immunoblots (IBs), where recombinant proteins applied as lines on nitrocellulose membrane strips are used to detect antibodies in patient sera, for the diagnosis and differentiation of LD and TBRF.

Immunochromatographic thread-based test platform for diagnosis of infectious diseases

Patterning is an important step in fabrication of multiplexed microfluidic devices. Various approaches including cutting, photolithography, wax-printing, plotting and etching have been developed and tested. Recently, using threads has emerged as a convenient and low-cost approach for fabrication of microfluidic devices. We explored the application of threads in combination with nitrocellulose membrane to fabricate multi-channel immunochromatographic diagnostic devices. Microfluidic channels were made using hydrophilic threads and nitrocellulose membrane strips. Household sewing needle was used to weave hydrophilic thread into desired patterns through a double-sided mounting tape. Glass fibre discs were used as conjugate pads while nitrocellulose membrane was used for immobilisation of capture antibodies. Patterned threads were linked to nitrocellulose membrane strips by overlapping so that reagents flowing through threads were eventually transferred to the membrane. The design was tested using IgG, H. pylori and Hepatitis B surface antigen. Continuous flow was observed from hydrophilic threads to the nitrocellulose membrane, and a positive signal was visualised on the membrane within 5 min of sample application. The observed limit of detection ranged between 30 and 300 ng/ml for H. pylori and Hepatitis B, respectively. Using thread and tape offers a promising alternative for patterning of simple, low-cost multiplexed microfluidic diagnostic devices with potential point-of-care applications in resource-limited settings.

COVALENT CONJUGATION OF ANTIBODY AND GOLD NANOPARTICLE FOR DEVELOPMENT OF LATERAL FLOW IMMUNOASSAY TEST STRIP

Nanotechnology is one of the fastest growing technologies in this era. Gold nanoparticle (AuNP) based immunoassays have been performed on the basis of antigen-antibody interaction using AuNP antibody conjugates. Lateral flow immunoassays(LFA) which are also based on AuNP antibody conjugates are useful innovation in nanotechnology and widely applied in medicine and research fields. However, there are some limitations of the present LFA kits such as sensitivity and stability. In the study, we showed the result of covalent conjugation of anti-rotavirus antibody and AuNP for generating a lateral flow immunoassay strip to detect rotavirus in fecal samples. The suitable conditions for coating polyethylene glycol (PEG) on the surface of AuNP were 10.0 mM PEG for 3 hours at room temperature (25 oC). Optimized conditions for covalent conjugation of antibody and AuNP were pH 4.0, 0.1 mg antibody/conjugate, 0.01 mM reactant EDC/NHS [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/(N-hydroxy sulfosuccinimide]. The coupling reaction was carried out at room temperature for 90 min. The conjugate pad, antibody immobilized nitrocellulose membrane strip were created with investigated conditions for generating an LFA test strip. The limit of detection of LFA test strip was determined by 1.6 × 105 virus particles/ml, three times lower than that of Rotaclone kit (UK). The generated strip could be used to detect rotavirus in fecal sample of patient.

Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia

A rapid and reliable screening test for thalassemia carrier couples is the most effective strategy to decrease the risk of conceiving fetuses with severe thalassemia. We present an approach based on the isothermal strand-displacement polymerase reaction and the use of a lateral flow strip for the visual detection of an α-thalassemia Southeast Asian-type deletion. This assay was used to evaluate 86 clinical samples (72 cases of Southeast Asian-type deletions and 14 cases of other types of thalassemia), and the results obtained were 100% consistent with those obtained using conventional gap-PCR. The approach thus provides a simple, sensitive, rapid, and cost-effective method for the diagnosis of thalassemia genotypes.

Simple and rapid detection of infectious myonecrosis virus using an immunochromatographic strip test

A strip test was developed for detection of infectious myonecrosis virus (IMNV) using a pair of monoclonal antibodies (MAbs), called IMN7 and IMC6, that are specific for the N and C fragments, respectively, of the IMNV capsid protein. The test strips were placed in plastic cassettes and stored desiccated in sealed plastic bags. In detection assays using the test-strip cassettes, 100-μl samples of application buffer containing homogenates from muscles or pleopods of normal or IMNV-infected shrimp were applied to the cassette sample chamber. Subsequent flow through the glass-fiber pad and the nitrocellulose membrane strip led to the development of visible antibody-protein complexes within 15min. In samples containing IMNV, viral capsid protein bound to gold-labeled IMN7 in the glass-fiber pad and the complex was subsequently captured by MAb IMC6 at the T line to form a reddish-purple band. Any unbound gold-labeled IMN7 migrated past the T line to be captured by the GAM antibody to form a band at the C line. Samples without IMNV or containing it below the test detection limit gave reddish-purple bands only at the C line. The sensitivity of the test was comparable to that of dot blot tests using single MAbs but was ~300-fold less sensitive than a one-step RT-PCR test for IMNV. Despite this lower sensitivity, the strip test has advantages of low cost, speed and simplicity (i.e., no sophisticated equipment or specialized skills required), and it is appropriate for use by farmers for pathogen confirmation when IMNV is suspected in diseased shrimp.

A strip-based immunoassay for rapid determination of fenpropathrin

We report the generation of monoclonal antibodies against fenpropathrin, originating from a BALB/c mouse immunized with a conjugate of hapten 1 [(RS)-cyano-3-phenoxybenzyl-2,2,3,3-tetramethylcyclopropane-carboxylic acid)] and keyhole limpet hemocyanin and the establishment of a monoclonal antibody-based immunochromatographic test strip. A coating antigen (test band) and a goat anti-mouse antibody (control band) were separately immobilized on nitrocellulose membrane strips as capture reagents. The anti-fenpropathrin monoclonal antibody was labeled with gold nanoparticles as a detection probe. This test strip had a wide linear range (15.6–250 μg L−1) with a low detection limit of 62 ± 6 μg L−1 for fenpropathrin, evaluated using a strip reader, and could be read within 10 min. This novel test strip was used to test fenpropathrin in fortified samples and found to have a high recovery rate (>80%). In summary, our monoclonal antibody-based immunochromatographic strip offers a rapid screening tool for fenpropathrin detection in agricultural commodities.

Detection of Deoxyribonucleic Acids on A Membrane Strip

A lateral-flow membrane strip was developed to detect the signal of deoxyribonucleic acids. The signal amplification was demonstrated by the detection of the model sample, biotinlyated Avian-Influenza (AI) genetic sequence, with a sandwich configuration of the target-RCA template complex captured between a RCA (Rolling Circle Amplification) template and streptavidin protein immobilized on a nitrocellulose membrane strip.

Nanocolloidal gold-based immuno-dip strip assay for rapid detection of Sudan red I in food samples

A semiquantitative dip strip assay was developed using nanocolloidal gold-labelled monoclonal antibody (Mab) 8A10 for the rapid detection of Sudan red I in food samples. A protein–Sudan red I conjugate was coated on a nitro cellulose membrane strip in a defined test line. In flow of the complex of nanocolloidal gold labelled-Mab and Sudan red I along the strip, intensive red colour that was formed in the test line reflected the Sudan red I concentration. The test required 10min and had a visual limit of detection of 10ng/g Sudan red I in tomato sauce and chilli powder samples. The results of the strip assay agreed well with those of a high performance liquid chromatography method for both spiked and real commercial samples. The strip was stable for at least 2months at 4°C. The strip assay offers the potential as a useful rapid and simple method for screening of Sudan red I in food samples.

Development of dipstick‐based immuno‐chemiluminescence techniques for the rapid detection of dichlorodiphenyltrichloroethane

The occurrence of organochlorine pesticides in the environment has been a major concern, due to their high persistence and the possible impacts of their exposure to humans. Dichlorodiphenyltrichloroethane (DDT) is most hazardous and one of the most widely used organochlorine insecticides. DDT and its main metabolites are highly stable to physical, chemical and biological degradation and are therefore still being detected in many parts of the world. The present study describes dipstick-based immuno-chemiluminescence method for the detection of DDT with high sensitivity. Anti-DDT antibodies raised in chicken (IgY) were used as the biological sensing elements by immobilizing onto nitrocellulose membrane strips in a chemiluminescence (CL)-based dipstick technique. The photons generated during the biochemical interaction were directly proportional to the DDT concentration. A mean recovery of 81.2-95.6% was obtained for DDT-spiked fruit juice samples with 2.8-4.6% relative standard deviation (RSD). Using the proposed dipstick-based immuno-CL method, DDT was detected with linearity in the range 0.05-1 ng/mL, having a limit of detection (LOD) of 0.05 ng/mL. This method can be used for the rapid, reliable detection of DDT pesticide.

Multiplexed dot immunoassay using Ag nanocubes, Au/Ag alloy nanoparticles, and Au/Ag nanocages

We report the first application of Ag nanocubes, Au/Ag alloy nanoparticles, and Au/Ag nanocages in a multiplexed dot immunoassay. The assay principle is based on the staining of analyte drops on a nitrocellulose membrane strip by using multicolor nanoparticles conjugated with biospecific probing molecules. Nanoparticles were prepared by a galvanic replacement reaction between the Ag atoms of silver nanocubes and Au ions of tetrachloroauric acid. Depending on the Ag/Au conversion ratio, the particle plasmon resonance was tuned from 450 to 700 nm and the suspension color changed from yellow to blue. The particles of yellow, red, and blue suspensions were functionalized with chicken, rat, and mouse immuno gamma globulin (IgG) molecular probes, respectively. The multiplex capability of the assay was illustrated by a proof-of-concept experiment involving simultaneous one-step determination of target molecules (rabbit anti-chicken, anti-rat, and anti-mouse antibodies) with a mixture of fabricated conjugates. Under naked eye examination, no cross-colored spots or nonspecific bioconjugate adsorption were observed, and the low detection limit was about 20 fmol. Open image in new window

Simultaneous and rapid detection of white spot syndrome virus and yellow head virus infection in shrimp with a dual immunochromatographic strip test

A strip test for the dual detection of white spot syndrome virus (WSSV) and yellow head virus (YHV) was developed using monoclonal antibodies (MAbs) specific to the WSSV major envelope protein VP28 (W1 and W30) and the YHV nucleocapsid protein p20 (Y19 and Y21). The MAbs W30 and Y19 were conjugated with colloidal gold and sprayed onto a glass fiber pad that was placed adjacent to a sample chamber. The MAbs W1 and Y21 and the goat anti-mouse immunoglobulin G (GAM) antibody were sprayed onto a nitrocellulose membrane in strips at positions designated W, Y and C, respectively. These test strips were placed in plastic cases and stored desiccated in a plastic bag. The test strips were assessed for their ability to detect WSSV and YHV simultaneously using pleopods sampled from shrimp. A pleopod homogenate in application buffer 100 μl was applied to the sample chamber to flow through the nitrocellulose membrane strip, and antibody–protein complexes could be observed within 15 min. In sample from shrimp infected with WSSV and/or YHV, viral protein bound to the colloidal gold-conjugated MAbs. These complexes were captured by the MAbs at the W and/or Y test lines, resulting in the appearance of reddish-purple coloured bands. Any unbound colloidal gold-conjugated MAbs migrated pass the W and Y lines would be captured by the GAM antibody, forming a band at position C. When samples not containing WSSV and YHV proteins or containing viral proteins at below the detection limit of the test, only the band at position C was observed. The sensitivity of the test was comparable to dot blot tests using single MAbs, and ∼500-fold less sensitive than a 1-step PCR test for WSSV and 1000-fold less sensitive than an RT-PCR test for YHV. Despite this lower sensitivity, the dual strip test has advantages in speed and simplicity in not requiring sophisticated equipment or specialized skills. The ability to co-detect WSSV and YHV provides simultaneously cost savings.

Development of colloidal carbon-based immunochromatographic strip for rapid detection of carbaryl in fruit juices

A simple immunochromatographic assay for sensitive detection of insecticide carbaryl in fruit juices was developed. The test is based on inhibition format on a nitrocellulose membrane strip. The strip was separately coated with rabbit anti-swine antibody (control line) and carbaryl hapten-OVA conjugate (test line). Colour intensity on the test line was possible to recognize visually from that of negative sample within 10 min, with the detection limit of 5 ng mL−1. All characteristics of the visually evaluated strips were also measured quantitatively, and then the detection limit was 1.5 ng mL−1. Cross-reactions with other carbamate pesticides were not found (<1%). No matrix effects were observed when fruit juices (orange, apple, pear, banana) were diluted tenfold times before analyses. The results from immunochromatographical assay were in a good agreement with those obtained by enzyme-linked immunosorbent assay as reference method. All these facts indicate the potential of the immunochromatographic strip for the quality control of fruit juices.

Strip-based immunoassay for rapid detection of thiabendazole

There is increased interest in the investigation and implementation of rapid screening methods for detection of pesticide residues. This study reports development of an immunostrip test for thiabendazole detection based on indirect competitive principle using carbon particles as a label. Nitrocellulose membrane strip was coated with a thiabendazole-protein conjugate in the defined test zone. In flow of an antibody-carbon complex and thiabendazole along the strip, the intensity of black colour formed in the test line reflected the thiabendazole concentration and semi-quantitative estimation could be carried out visually. The optimized test was accomplished within 10 min and the visual detection limit was achieved 0.25 ng mL −1 of standard sample. Moreover, immunostrip was evaluated quantitatively using scanning densitometry. Based on standard curve, the detection limit of the proposed test was as low as 0.08 ± 0.03 ng mL −1 with an IC 50 value of 0.60 ± 0.08 ng mL −1 and a linear working range of 0.11–4.13 ng mL −1. Results of testing precision, stability, and specificity demonstrated that the assay provided a reliable performance. This immunostrip was applied to analysis of spiked fruit juices in range of 0.05–5 mg L −1. Matrix interferences were avoided by simple dilution of samples. Both visual and instrumental evaluations indicated a good agreement with results obtained by ELISA. Recoveries from juices were from 81.9 to 123.6% and relative standard deviations ranged from 9.9 to 19.3%. The developed strip offers potential as a useful rapid and simple method for screening of thiabendazole in fruit juices at levels far below the maximum residue limits.

Time-resolved luminescent lateral flow assay technology

We here report a detection technology that integrates highly sensitive time-resolved luminescence technique into lateral flow assay platform to achieve excellent detection performance with low cost. We have developed very bright, surface-functionalized and mono-dispersed phosphorescent nanoparticles of long lifetime under ambient conditions. The phosphorescent nanoparticles have been used to conjugate with monoclonal antibody for C-reactive protein (CRP), an inflammatory biomarker. Lateral flow immunoassay devices have been developed using the conjugate for highly sensitive detection of CRP. The CRP assay can achieve a detection sensitivity of <0.2 ng mL −1 in serum with a linear response from 0.2 to 200 ng mL −1 CRP. We have also developed a low cost time-resolved luminescence reader for the lateral flow immunoassay (LFIA) devices. The reader does not use expensive band pass filter and still provide very low detection background and high detection sensitivity on solid substrates such as nitrocellulose membranes. The reader can detect less than 2.5 ng phosphorescent particles captured on a nitrocellulose membrane strip with more than three orders of magnitude linear detection dynamic range. The technology should find a number of applications, ranging from clinical diagnostics, detection of chemical and biological warfare agents, to food and environmental monitoring.

Development of a simple and rapid immunochromatographic strip test for diarrhea-causative porcine rotavirus in swine stool

A rapid and simple immunochromatography (IC) strip test, for specific detection of porcine rotavirus (PRV) in stool specimen, was developed. Monoclonal antibodies (mAbs) to the OSU strain of PRV have been produced in mice. Among them, two hybridoma clones that generate mAb-1 and mAb-2, respectively, specific for VP6 protein of PRV, have been selected. In the IC configuration, mAb-1, one of the selected mAbs was used to the designed coat microparticles (MP), while another mAb-2 was used to fix it on the nitrocellulose membrane strip to form a result line. The control line was formed on the same membrane strip past the result line by fixing anti-mouse IgG antibody. The IC test was capable of detecting 1000 plaque-forming units of PRV/ml in less than 5 min, and the binding capacity was demonstrated by specific recognition of PRV only, but not other porcine diarrhea viruses, transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). The IC test produced positive results with all the nine PRV-positive stool specimens and negative results with five different non-PRV specimens, which were identified previously by the polymerase chain reaction (PCR) test, respectively. The results indicate an excellent concordance between the two methods, suggesting a potential application of the three combinated IC tests (PRV, TGEV and PEDV) for the on-site, rapid screening of porcine diarrhea cases.

Development of colloidal gold-based immumochromatographic assay for the rapid detection of medroxyprogesterone acetate residues in biological materials

A competitive colloidal gold-based immunoassay in lateral-flow format for the rapid detection of medroxyprogesterone acetate (MPA) in biological materials was developed. A nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and MPA hapten-OVA conjugate (test line). Anti-MPA polyclonal antibody labelled with colloidal gold particles was first incubated with MPA. The limit of detection for lateral flow was 5 ng g−1 for detecting an MPA standard solution, and the limit of detection was 10 ng mL−1 for detecting the MPA spiked in pig urine and 10 ng g−1 for spiked in pig liver. The results were confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) and indicated that there was a good agreement between both methods (R 2 = 0.976). The assay time for the test was less than 5 min, suitable for rapid testing on site.

A novel generic dipstick-based technology for rapid and precise detection of tetracycline, streptogramin and macrolide antibiotics in food samples

Excessive use of antibiotics in veterinary medicine and as growth promoters in stock farming has been associated with the dramatically increasing prevalence of multidrug-resistant human pathogenic bacteria. European community legislators have therefore restricted the veterinary use of antibiotics and banned them as growth-promoting food additives in stock breeding (1831/2003/EC). The monitoring of such legislation requires technology for precise and straightforward on-site quantification of antibiotics in farm samples and food products without the need for extensive laboratory equipment and trained personnel. Capitalizing on bacterial transcriptional regulators (TetR, PIP, E), which are dose-dependently released from their cognate operators (tetO, PIR, ETR) upon binding of specific classes of antibiotics (tetracycline, streptogramins, macrolides) we have designed an easy-to-handle dipstick-based assay for detection of antibiotic levels in serum, meat and milk whose detection limits are up to 40-fold below licensed threshold values. The generic dipstick consists of either nitrocellulose, nylon or polyvinylidenfluorid (PVDF) membrane strips coated with streptavidin and immobilized biotinylated operator DNA, which acts as capture DNA to bind hexa-histidine (His(6))-tagged bacterial biosensors. Antibiotics present in specific samples triggered the dose-dependent release of the capture DNA-biosensor interaction, which, after dipping into two different solutions, results in a correlated conversion of a chromogenic substrate by a standard His(6)-targeted enzyme complex. This can be quantified by comparison of the dipstick to a standardized color scale or by assessing the terminal solution at 450nm. As demonstrated using serum, meat and milk samples spiked with 14 different antibiotics, the dipstick technology provided sensitive detection in a rapid assay format, and could be employed to monitor non-authorized use of antibiotics and to discover novel antibiotics.