Sandwich Format Research Articles

Determination of the platelet-derived growth factor BB by a sandwich format thrombin-linked aptamer assay on a microplate

Microplate based assays have advantages such as rapid detection and high throughput analysis.

A novel colorimetric sensing platform for the detection of S. aureus with high sensitivity and specificity.

In this study, a novel colorimetric sensing platform was developed for the detection of S. aureus using dog immunoglobulin G (IgG) as the capture antibody and chicken anti-protein A immunoglobulin Y labeled with horseradish peroxidase (HRP-IgY) as the detection antibody. Dog IgG labeled with magnetic beads was used to capture S. aureus through the interaction between the Fc region of dog IgG and Staphylococcal protein A (SPA). HRP-IgY was introduced to recognize the residual SPA on the surface of S. aureus and to create a sandwich format, after which a soluble 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added. A stop solution was utilized to cease the enzymatic chromogenic reaction, and then optical density was read at 450 nm. Under optimal conditions, the proposed method displayed a low detection limit of 1.0 × 103 CFU mL−1 and a wide linear range of 3.1 × 103 to 2.0 × 105 CFU mL−1. This detection method exhibited high specificity against other foodborne bacteria. The recovery rates ranged from 95.2% to 129.2%. To our knowledge, this is the first report to employ dog IgG and chicken IgY as an antibody pair to detect S. aureus. This technique exhibits high application potential for S. aureus monitoring in various kinds of samples.

Focusing aptamer selection on the glycan structure of prostate-specific antigen: Toward more specific detection of prostate cancer

The development of chemical sensors capable of detecting the specific glycosylation patterns of proteins offers a powerful mean for the early detection of cancer. Unfortunately, this strategy is scarcely explored because receptors recognizing the glycans linked to proteins are challenging to discover. In this work, we describe a simple method for directing the selection of aptamers toward the glycan structure of the glycoproteins, with prostate-specific antigen (PSA) as a model target. Using this strategy, we identified one aptamer (PSA-1) that binds the glycan moiety of PSA with reasonable affinity (a dissociation constant of 177 ± 65 nM). Interestingly, an electrochemical sensor with a sandwich format employing the identified aptamer as a signaling receptor, provides a tool of discriminating human PSA from the unglycosylated protein, with a limit of detection of 0.66 ng/mL. The sensor responds to different levels of PSA in serum, correlating well with chemiluminescence ELISA used in hospitals even with higher potential to discriminate clinically meaningful prostate cancer. Although validation on a larger cohort is needed, this is the first demonstration of an aptamer-based sensor to detect PSA by focusing in its glycan moiety

Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies

Reliable detection and diagnosis of dengue virus (DENV) is important for both patient care and epidemiological control. Starting with a llama immunized with a mixture of recombinant nonstructural protein 1 (NS1) antigen from the four DENV serotypes, a phage display immune library of single domain antibodies was constructed and binders selected which exhibited specificity and affinity for DENV NS1. Each of these single domain antibodies was evaluated for its binding affinity to NS1 from the four serotypes, and incorporated into a sandwich format for NS1 detection. An optimal pair was chosen that provided the best combination of sensitivity for all four DENV NS1 antigens spiked into 50% human serum while showing no cross reactivity to NS1 from Zika virus, yellow fever virus, tick-borne encephalitis virus, and minimal binding to NS1 from Japanese encephalitis virus and West Nile virus. These rugged and robust recombinant binding molecules offer attractive alternatives to conventional antibodies for implementation into immunoassays destined for resource limited locals.

Developing liquid crystal-based immunoassay for melamine detection

We have developed a liquid crystal (LC)-based immunoassay for melamine detection by using direct, sandwich and competitive formats. The detection mechanism was based on the specific immunobinding of melamine with the anti-melamine immobilized on the glass substrate, which disrupted the orientation of the LCs and resulted in changes in the optical images of the LCs under polarized light. In the direct immunoassay where the melamine was bound to the anti-melamine on the substrate, no change in the optical images of LC was observed. In contrast, in the sandwich immunoassay where the immunocomplex of melamine and secondary anti-melamine linked with streptavidin were bound to the primary anti-melamine on the substrate, a dark-to-bright transition of the LC images was observed when the concentration of melamine was 2.5 ng/mL or above. Moreover, in the competitive immunoassay where both melamine and melamine-streptavidin conjugate were bound to the anti-melamine on the substrate, a bright-to-dark transition of the LC images was observed when the concentration of melamine was 5 ng/mL or above. We found that the larger the molecules bound to the substrate, the more easily was the orientation of the LCs disrupted which resulted in the change in the optical images of the LCs. This result provides a good strategy for developing LC-based immunoassays for the detection of small molecules by using a large auxiliary molecule attached to the target molecules. To the best of our knowledge, our work demonstrates for the first time the detection of small molecules by using a LC-based immunoassay.

Ionic Liquid Aqueous Two-Phase Systems for the Enhanced Paper-Based Detection of Transferrin and Escherichia coli.

Aqueous two-phase systems (ATPSs) have been widely utilized for liquid-liquid extraction and purification of biomolecules, with some studies also demonstrating their capacity as a biomarker concentration technique for use in diagnostic settings. As the limited polarity range of conventional polymer-based ATPSs can restrict their use, ionic liquid (IL)-based ATPSs have been recently proposed as a promising alternative to polymer-based ATPSs, since ILs are regarded as tunable solvents with excellent solvation capabilities for a variety of natural compounds and proteins. This study demonstrates the first application of IL ATPSs to point-of-care diagnostics. ATPSs consisting of 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4]) and sodium phosphate salt were utilized to quickly concentrate biomarkers prior to detection using the lateral-flow immunoassay (LFA). We found the phase separation speed of the IL ATPS to be very rapid and a significant improvement upon the separation speed of both polymer-salt and micellar ATPSs. This system was successfully applied to both sandwich and competitive LFA formats and enhanced the detection of both Escherichia coli bacteria and the transferrin protein up to 8- and 20-fold, respectively. This system's compatibility with a broad range of biomolecules, rapid phase separation speed, and tunability suggest wide applicability for a large range of different antigens and biomarkers.

Sensitive and Selective Immunofluorescence Assay for CA15-3 Detection Using Fluorescein Derivative A10254.

Carbohydrate antigen 15-3 (CA15-3), a specific breast cancer-related biomarker in serum, can provide direct information to monitor a patient's postoperative status and can predict recurrence and metastasis of breast cancer. The conventional Enzyme-Linked Immunosorbent Assay (ELISA) is an effective approach for CA15-3 detection, but there is a high limit of detection which is more than 1 U mL-1. Therefore, a convenient, highly sensitive and specific CA15-3 detection method is needed for earlier prognoses of breast cancer. In this work, we aim to develop a novel sensitive and selective immunofluorescence assay for the highly effective detection of CA15-3. We pre-coat the antibody I onto the solid phase to capture antigen CA15-3. The A10254- labeled antibody II identified by electrophoresis and fluorescence spectra recognized the antigen CA15-3 and formed the sandwich format. The fluorescence signal of A10254 directly relating to the amount of antigen could be detected and quantified by the microplate reader excited at 440nm. The obtained method for CA15-3 detection showed lower limits of detection (0.11 and 0.18 U mL-1) in a linear range of 0.25-14 U mL-1 in the buffer and 2% serum, respectively. This assay exhibited a good specificity and reproducibility in 2% serum with lower relative standard deviations in different tested concentrations. The developed immunofluorescence assay enhanced the performance of the conventional CA15-3 ELISA kit. Moreover, the acceptable accuracy and good reproducibility in diluted serum indicated the assay has great potential for the diagnosis of breast cancer in clinical practice.

Development of an immunochromatographic assay for the specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin

Cry1Ab has been widely used in genetically modified (GM) crops and its amino acid sequence had high identity to Cry1Ac toxin. Existing nanogold immunochromatographic strips cannot distinguish Cry1Ab from Cry1Ac toxin. In this study, a rapid (5–6 min), qualitative nanogold immunochromatographic strip was successfully developed for the specific detection of Cry1Ab toxin. The assay was based on double antibody sandwich format with the visual detection limit (vLOD) of 0.1 μg mL−1. The results of immunochromatographic assay were all positive validated against the DAS-ELISA (recoveries between 109.6 and 111.8%). In addition, 10%, 5% and 0% error probability results were found in 20 times repeated tests for Cry1Ab concentration of 0.1, 0.2, 0.5 and 1 μg mL−1, respectively, demonstrating the reproducibility of the test strip. Furthermore, the test strip could be stored for 3 months under dry conditions without significant loss of sensitivity. Furthermore, the practical sample analysis results showed that the test strip was able to detect the presence of Cry1Ab in GM materials containing as low as 0.5% MON 810 Bt maize which indicated the practical value of the test strip. To our knowledge, this is the first report on the detection of Cry1Ab by immunochromatographic assay without interference from Cry1Ac toxin.

Robust, Highly Visible, and Facile Bioconjugation Colloidal Crystal Beads for Bioassay.

High mechanical strength, highly visible, and admirable grafting molecular ability is the key challenge for colloidal photonic crystal (CPC) barcode beads in multiplex analysis fields. To achieve this goal, we proposed self-adhesion particles, polydopamine-coated SiO2 nanoparticles (PDA@SiO2), to construct CPC barcode beads by droplet-based microfluidic approach. Because of the adhesion, broad absorption of light, and "active" functional groups of PDA, the beads are endowed with high robustness, visibility, and excellent biomolecule immobilization. Ultrasonic treatment and compression experiments demonstrated that PDA@SiO2 CPC barcode beads have a high mechanical strength. Color analysis illustrated that PDA@SiO2 CPC beads exhibited a high visibility in color. The verification of fluorescent-tagged biomolecule conjugation together with the antigen detection stated that PDA@SiO2 CPC beads are capable of immobilizing biomolecule by covalent binding. With a sandwich format, the beads were applied to analyze the tumor makers including alpha fetal protein, carcinoembryonic antigen, and prostate specific antigen from practical clinical serum. The proposed suspension arrays using PDA@SiO2 CPC beads as a barcode showed acceptable accuracy and detection reproducibility.

Enhanced lateral flow assay with double conjugates for the detection of exosomes

Exosomes are promising biological biomarkers for monitoring a number of diseases, especially cancers. Here, we developed a double gold nanoparticles (GNPs) conjugates based lateral flow assay (D-LFA) for rapidly and sensitively detecting and molecular profiling of exosomes. Based on these two GNPs conjugates, the signal amplification can be achieved without any additional operation. The antibody on the 1st GNPs conjugate could recognize exosomes and form a sandwich format on the test zone. The 2nd GNPs conjugate was designed to bind to the 1st GNPs conjugate to realize signal amplification. This biosensor enabled visual and quantitative detection of exosomes by the accumulation of GNPs on the test zone and showed a low detection limit of 1.3×103 particles/μL, which has been improved 13-fold compared with the normal lateral flow assay. The D-LFA exhibited good sensitivity and reproducibility and has been successfully used for the detection of exosomes in fetal bovine serum, which proved its potential application in practical diagnostics.

Rapid Colorimetric Determination of Procalcitonin Using Magnetic Separation and Enzymatic Catalysis

Human procalcitonin is an early diagnostic biomarker for sepsis and bacterial infections and can be used in distinguishing bacterial infections from viral infections. In this study, a colorimetric sensing platform for the rapid determination of procalcitonin was developed. The approach involves the capture of procalcitonin by immunomagnetic beads, and a detection antibody labeled with horseradish peroxidase to perform sandwich format, where it catalyzes the oxidation of 3,3′,5,5′–tetramethylbenzidine to produce the colorimetric signal. Under the optimal conditions, a detection limit of 0.04 ng/mL (3σ) was obtained within the calibration range 0.1–10 ng/mL. The proposed method was performed in less than 90 min and exhibited good specificity without interferences from other biomarkers including C-reactive protein and human serum albumin. Overall, the proposed method provided a new alternative strategy for procalcitonin detection due to its sensitive, rapid, specific, and simple characteristics. This method is suitable for rapid screening of various biomedical targets.

Evaluation of the Elecsys Syphilis Immunoassay for Detection of Syphilis in Populations at Risk of Disease in the US and Argentina.

The Elecsys® syphilis immunoassay is an automated, qualitative immunoassay that uses a double-antigen sandwich format to detect antibodies to Treponema pallidum in human serum and plasma. We aimed to validate performance of the immunoassay in various populations at risk for syphilis infection in the US and Argentina. Samples were obtained for a number of study cohorts, including participants from routine syphilis testing at high or low risk for syphilis, HIV-positive patients, pregnant women, and patients in various stages of syphilis infection. The primary objective was to validate the Elecsys syphilis immunoassay by comparing it with a composite testing algorithm using US Food and Drug Administration (FDA)-approved tests, including the predicate IMMULITE 2000 syphilis screening assay, the rapid plasma reagin, and the T. pallidum particle agglutination assay. Complete algorithm testing was performed on all 2660 collected samples. Acceptable precision was demonstrated in all samples. Comparison of the Elecsys syphilis immunoassay with the final syphilis status for all samples yielded a diagnostic sensitivity of 99.5% (95% CI, 98.21-99.94) and a diagnostic specificity of 99.2% (95% CI, 98.69-99.49). Overall, the lower limit of the 95% CIs for sensitivity and specificity met the expected performance of ≥95%. This is the first study that confirms the high sensitivity and specificity of the Elecsys syphilis immunoassay in US and Argentinian cohorts and highlights the assay's usefulness as an alternative to current tests for the diagnosis of syphilis infection in a broad range of participant cohorts.

Quantitative immunoassay for mink immunoglobulin in serum and milk

BackgroundThe significance of maternal immunoglobulin G (IgG) for the resistance against a number of infections affecting the health of young mink offspring is not known. Here, we present a validated immunoassay for quantification of mink IgG in serum and milk, using a commercially available polyclonal goat anti-ferret IgG antibody cross-reactive with mink IgG as both the catching and the detection antibody, in a sandwich format enzyme linked immunosorbent assay (ELISA). Using this ELISA, serum IgG concentrations was analyzed over time in both mothers and kits in order to establish a correlation between maternal IgG serum concentrations and those of the offspring.ResultsIntra-assay coefficient of variation (CV) for a serum sample ranged from 2.15 to 5.97% depending on the dilution, while the inter-assay CV ranged from 5.17 to 17.78%. In addition, the range of milk intra-assay CV was 2.71–5.92%, while the range of the inter-assay CV was 4.20–16.03%. Calibrating the ELISA with purified mink IgG (an in-house preparation purified from mink serum) the lower limit of detection was found to be 5 ng/mL for serum and 1 ng/mL for milk. Both serum and milk showed high precision and good linearity over a two-log dilution range. When comparing the serum IgG concentrations of the mink kits a clear within litter effect was seen, while the mean serum IgG concentrations of litters differed significantly between some of the litters (P = 0.0013). Mean maternal serum IgG concentrations correlated positively with the IgG serum concentration of the corresponding offspring sampled over a 3 week period (R2 = 0.63).ConclusionsA calibrated and reproducible sandwich ELISA for quantifying mink IgG concentrations in both milk and serum with high analytical sensitivity was developed and validated. The results in this study corroborate previous investigations supporting the usability of the ELISA, paving the way for investigations into the importance of maternal IgG in milk and in serum for the welfare and health of the offspring.

Determination of IgG by electron spin resonance spectroscopy using Fe3O4 nanoparticles as probe

A novel electron spin resonance (ESR) spectroscopy method for determination of rabbit IgG was presented. Anti-rabbit IgG antibody was bound to Fe3O4 nanoparticles (FeNPs). The FeNPs were used as probe to give ESR signal. After the optimization of the assay, a molar ratio 500:1 of antibody to FeNPs was selected. The sepharose beads were used as solid phase and used to separate the analyte from the sample solution. Two typical immunoassay formats, sandwich and competitive formats, were applied to the determination of IgG. The analytical sensitivities obtained by sandwich and competitive formats were 0.13 μg mL−1 and 0.56 μg mL−1, respectively. The two formats were applied to the determination of the rabbit IgG in rabbit serum. The diluted sample was directly analyzed and other additional sample treatment was not required. The analytical results of the spiked samples indicated that the recoveries of the analytes were acceptable. Furthermore, the sample preparation time used in this method is shorter than most existing immunoassay methods.

Development of two ELISA formats to determine glycinin. Application to detect soy in model and commercial processed food

Indirect competitive and sandwich ELISA formats were developed to determine glycinin and applied to detect soy in model and commercial processed food. Antiserum to glycinin was used in the competitive format and specific purified antibodies in the sandwich format. Results obtained showed that the sandwich format could detect as low levels as 0.005% and 0.05% of incurred soy protein isolate in sausages and bread, respectively; whereas indirect competitive format could only detect levels of 0.25% and 0.5% of soy protein in the same incurred foods. However, soy protein could not be detected in pâté using both formats even at the highest level assayed of 1%. Furthermore, the sandwich ELISA could detect glycinin in samples of UHT milk spiked with 0.05% of soy drink and in cookies spiked with 0.005% of soy seeds. The sandwich format could also discriminate between commercial foods with declared or not soy proteins or derivatives as ingredients, although glycinin could not be detected in some soy labelled foods. A prototype of the sandwich format was prepared and validated, obtaining adequate results in terms of recovery, precision and robustness. The results of this study indicate that the concentration of immunoreactive glycinin decreased with the intensity of heat processing applied to food. Furthermore, the antibody population used and the ELISA format applied influence greatly the determination of glycinin in processed food.

Nanozyme-based lateral flow assay for the sensitive detection of Escherichia coli O157:H7 in milk

Lateral flow assay (LFA) has been applied in many fields due to its relative ease of use and cost-effectiveness. However, it has low sensitivity and its applications are limited. Probe materials play a significant role in improving the detection efficiency and sensitivity of LFA. In this study, by using concave palladium-platinum (Pd-Pt) nanoparticles as a nanozyme probe, we developed a sensitive LFA based on the sandwich format for qualitative and quantitative detection of Escherichia coli O157:H7. The sensitivity of the LFA was improved by applying the 3,3',5,5'-tetramethylbenzidine (TMB) substrate onto the test line where the nanozyme was accumulated in the presence of analytes. The nanozyme showed high catalytic performance toward TMB and greatly enhanced the signal intensity of the test line. The sensitivity of the nanozyme-based LFA was 9.0 × 102 cfu/mL in milk, which was 111-fold higher than that of traditional colloidal gold-based LFA. The proposed method has remarkable potential in the detection of various pathogens in real samples.

Towards an Electrochemical Immunosensor System with Temperature Control for Cytokine Detection.

The cytokine interleukin-13 (IL-13) plays a major role in airway inflammation and is a target of new anti-asthmatic drugs. Hence, IL-13 determination could be interesting in assessing therapy success. Thus, in this work an electrochemical immunosensor for IL-13 was developed and integrated into a fluidic system with temperature control for read-out. Therefore, two sets of results are presented. First, the sensor was set up in sandwich format on single-walled carbon nanotube electrodes and was read out by applying the hydrogen peroxide–hydroquinone–horseradish peroxidase (HRP) system. Second, a fluidic system was built up with an integrated heating function realized by Peltier elements that allowed a temperature-controlled read-out of the immunosensor in order to study the influence of temperature on the amperometric read-out. The sensor was characterized at the temperature optimum of HRP at 30 °C and at 12 °C as a reference for lower performance. These results were compared to a measurement without temperature control. At the optimum operation temperature of 30 °C, the highest sensitivity (slope) was obtained compared to lower temperatures and a limit of detection of 5.4 ng/mL of IL-13 was calculated. Taken together, this approach is a first step towards an automated electrochemical immunosensor platform and shows the potential of a temperature-controlled read-out.

Electrochemical genoassays on gold-coated magnetic nanoparticles to quantify genetically modified organisms (GMOs) in food and feed as GMO percentage

The integration of nanomaterials in the field of (bio)sensors has allowed developing strategies with improved analytical performance. In this work, ultrasmall core-shell Fe3O4@Au magnetic nanoparticles (MNPs) were used as the platform for the immobilization of event-specific Roundup Ready (RR) soybean and taxon-specific DNA sequences. Firstly, monodisperse Fe3O4 MNPs were synthesized by thermal decomposition and subsequently coated with a gold shell through reduction of Au(III) precursor on the surface of the MNPs in the presence of an organic capping agent. This nanosupport exhibited high colloidal stability, average particle size of 10.2 ± 1.3 nm, and spherical shape. The covalent immobilization of ssDNA probe onto the Au shell of the Fe3O4@Au MNPs was achieved through a self-assembled monolayer (SAM) created from mixtures of alkane thiols (6-mercapto-1-hexanol and mercaptohexanoic acid). The influence of the thiols ratio on the electrochemical performance of the resulting electrochemical genoassays was studied, and remarkably, the best analytical performance was achieved for a pure mercaptohexanoic acid SAM. Two quantification assays were designed; one targeting an RR sequence and a second targeting a reference soybean gene, both with a sandwich format for hybridization, signaling probes labelled with fluorescein isothiocyanate (FITC), enzymatic amplification and chronoamperometric detection at screen-printed carbon electrodes (SPCE). The magnetogenoassays exhibited linear ranges from 0.1 to 10.0 nM and from 0.1 to 5.0 nM with similar detection limits of 0.02 nM and 0.05 nM for the event-specific (RR) and the taxon-specific (lectin) targets, respectively. The usefulness of the approach was demonstrated by its application to detect genetically modified organisms (GMOs) in feed and food.

Simultaneous competitive and sandwich formats multiplexed immunoassays based on ICP-MS detection

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) based immunoassay method has been proposed in multiple immunoassays but has not been used in competitive and sandwich formats immunoassay simultaneously. The two immunoassays were usually conducted separately in clinical field depending on the size and the amount of binding sites of targets. We proposed an immunoassay method based on magnetic beads and ICP-MS detection that could be suitable for both small and large molecules. The functionalized magnetic beads were added to capture the immune complex after immune reaction. In this way, thyrotropin and free thyroid hormone can be captured, separated and then detected according to the elemental tags by ICP-MS simultaneously. The new method was evaluated by comparing the results with time resolved fluorescence immunoassays (TRFIA). The dynamic ranges of TSH and FT4 were 0.16–105 mU/L and 3.5–65 pmol/L, respectively. The limits of detections were 0.06 mU/L for TSH and 1.59 pmol/L for FT4. And the relative standard deviations (RSD) of TSH and FT4 were 4.64% at 2.5 mU/L and 1.87% at 5.85 pmol/L. This immunoassay method enables the determination of small and large biomolecules simultaneously via competitive and sandwich immunoassay formats.

Heteroassembled gold nanoparticles with sandwich-immunoassay LSPR chip format for rapid and sensitive detection of hepatitis B virus surface antigen (HBsAg)

This study aimed to develop a more sensitive method for the detection of hepatitis B surface antigen (HBsAg) using heteroassembled gold nanoparticles (AuNPs). A single layered localized surface plasmon resonance (LSPR) chip format was developed with antigen-antibody reaction-based detection symmetry using AuNPs, which detected HBsAg at 10 pg/mL. To further improve the detection limit, a modified detection format was fabricated by fixing a secondary antibody (to form a heteroassembled sandwich format) to the AuNP monolayer, which enhanced the detection sensitivity by about 100 times. The developed heteroassembled AuNPs sandwich-immunoassay LSPR chip format was able to detect as little as 100 fg/mL of HBsAg within 10–15 min. In addition, the heteroassembled AuNPs sandwich-immunoassay LSPR chip format did not show any non-specific binding to other tested antigens, including alpha fetoprotein (AFP), C-reactive protein (CRP), and prostate-specific antigen (PSA). These findings confirm that the proposed detection strategy of heteroassembled AuNPs sandwich-immunoassay LSPR chip format may provide a new platform for early diagnosis of various human diseases.