Biotinylated Detection Antibodies Research Articles

B-036 Systematic Evaluation of Antibody-Antigen Interactions for Suitability in Immunoassays Using Parallel Microarray ELISA

Abstract Background A critical step in immunoassay development is the selection of the appropriate capture and detection antibodies. The use of suboptimal antibodies can drastically affect the quality of the final assay. Traditional selection methods involve a direct testing of potential antibodies in the final assay setting; a time-consuming and material-intensive exercise that is often too complex to test all possibilities. Alternatives, such as abstract K-on/K-off determinations via SPR or BLI can be costly and often show limited translatability to the ultimate assay format. Here, we describe a novel systematic approach to rapidly screen antibody-antigen interactions for key assay specifications and test for translatability of the findings. Methods Nanoliter volumes of potential capture antibodies were spotted onto the bottom of ELISA wells, generating a microarray of antibodies in each well (>11,000 spots per plate). For the analysis of capture antibody-antigen interactions, a titration series of biotinylated analyte in negative sample matrix was applied. Binding was visualized using strep-polyHRP, precipitating TMB and high resolution scanning of each ELISA well. Antibody pair performance was investigated using unlabeled analyte and biotinylated detection antibody options. Sensitivity, specificity, dynamic range, binding speed, binding strength and matrix compatibility were investigated using time courses, concentration ranges, dilutions, interferent spikes and pH/salt/detergent challenges. Signals were quantified to calculate and statistically compare each possible interaction. Results Antibody-antigen interactions were investigated via parallel microarray ELISA for two example immunoassays: the HBsAg and cTnI test. For both analytes, a panel of antibodies was screened revealing antibodies with diverse characteristics. Striking differences were observed depending on the sample matrix composition. Without optimization, antibodies could be identified showing dynamic assay ranges of 3-4 log orders. Time course experiments revealed high-affinity antibodies suitable for rapid diagnostic tests. For HBsAg, levels down to 0.01 IU/mL (∼40 pg/mL) could be reproducibly detected, which is well below the minimal requirement of 0.13 IU/mL. Reactivity against multiple virus serotypes could be confirmed. Interestingly, for the cTnI test, a strong synergistic effect was observed when specific monoclonal antibodies were combined in the capture mix, resulting in a 6-fold sensitivity enhancement compared to their separate performances. Finally, we show that the results obtained with the parallel microarray ELISA approach are highly translatable to the final assay format, ranging from bead-based dry-chemistry assays to chemiluminescent immunoassays. Conclusions The parallel microarray ELISA evaluation presents a robust and reliable method for identifying the best antibodies for an immunoassay. It allows for a systematic and quantitative screen of numerous antibody options within 1-2 weeks under real immunoassay conditions with minimal antibody amounts. This method has shown to be highly translatable and is customizable to accommodate required assay specifications.

Microfluidic Digital Immunoassay for Point-of-Care Detection of NT-proBNP from Whole Blood.

The high prevalence and economic burden of heart failure remain a challenge to global health. This lifelong disease leads to a buildup of permanent heart damage, making early detection and frequent monitoring crucial for effective treatment. N-terminal proBNP (NT-proBNP) is an important biomarker for monitoring the disease state, but current commercial and research NT-proBNP assays require phlebotomy and bulky equipment or do not satisfy clinical requirements such as sensitivity and detection thresholds. Here, we report a point-of-care (POC) compatible microfluidic digital immunoassay that can quantify the NT-proBNP concentration in a small volume of whole blood. Our automated microfluidic device takes whole blood samples mixed with biotinylated detection antibodies and passes through a plasma filter to react with a capture antibody-functionalized sensor surface. Streptavidin-coated gold nanoparticles (GNPs) are then released to mark the surface-bound single NT-proBNP immunocomplexes and recorded with bright-field microscopy. NT-proBNP concentrations in the sample are quantified via a hybrid digital/analog calibration curve. Digital counts of bound GNPs are used as readout signal at low concentrations for high sensitivity detection, and GNP pixel occupancies are used at high concentrations for extended dynamic range. With this approach, we detected NT-proBNP in the range of 8.24-10 000 pg/mL from 7 μL of whole blood in 10 min, with a limit of detection of 0.94 pg/mL. Finally, the method was validated with 15 clinical serum samples, showing excellent linear correlation (r = 0.998) with Roche's Elecsys proBNP II assay. This evidence indicates that this method holds promise for decentralized monitoring of heart failure.

Cerium dioxide-based nanostructures as signal nanolabels for current detection in the immunosensing determination of salivary myeloperoxidase

This work reports the first electrochemical immunoplatform involving the use of synthesized cerium dioxide nanoparticles (CeO2NPs) / multi-walled carbon nanotubes (MWCNTs) composites as signal nanolabels for the determination of salivary myeloperoxidase (MPO), one of the proteins suggested as diagnostic biomarkers of periodontitis. The immunosensing device profits the benefits of CeO2NPs in terms of fast electron transfer and the catalytic activity and prevents the CeO2NPs tendency to agglomerate by decoration over MWCNTs, also providing high sensitivity. After a thorough characterization of the CeO2NPs/MWCNTs using various techniques and optimization of the variables involved in the preparation of the immunoplatform, a sandwich-type immunoassay was implemented with specific MPO capture and biotinylated detection antibodies. The use of horseradish peroxidase-streptavidin (HRP-Strept) or CeO2NPs/MWCNTs-(HRP-Strept) as labels for the amperometric detection at screen printed carbon electrodes (SPCEs) at −0.20 V vs. Ag pseudo-reference electrode using the H2O2/hydroquinone (HQ) system, was compared. The method developed involving CeO2NPs/MWCNTs-(HRP-Strept) provided a linear calibration plot for MPO over the 1.0 to 100.0 ng mL−1 (R2 = 0.994) range with a limit of detection value of 0.40 ng mL−1. The immunoplatform was applied to the analysis of saliva from volunteers showing that the method allowed discrimination between healthy individuals and patients diagnosed with periodontitis through the determination of MPO in 1000-times diluted saliva. The obtained results agreed with those provided by an ELISA kit, this latter requiring a much longer assay time (4 h vs 2 h).

Disposable electrochemical immunoplatform to shed light on the role of the multifunctional glycoprotein TIM-1 in cancer cells invasion

Detecting overexpression of cancer biomarkers is an excellent tool for diagnostic/prognostic and follow-up of patients with cancer or their response to treatment. This work illustrates the relevance of interrogating the levels of T-cell immunoglobulin and mucin domain 1 (TIM-1) protein as a diagnostic/prognostic biomarker of high-prevalence breast and lung cancers by using an amperometric disposable magnetic microparticles-assisted immunoplatform. The developed method integrates the inherent advantages of carboxylic acid-functionalized magnetic beads (HOOC-MBs) as pre-concentrator support and the amperometric transduction at screen-printed carbon electrodes (SPCEs). The immunoplatform involves a sandwich-type immunoassay assembled on HOOC-MBs through the specific capture/labeling of TIM-1 using capture antibodies and horseradish peroxidase (HRP)-conjugated biotinylated detection antibodies as biorecognition elements. The magnetic immunoconjugates were confined onto the working electrode (WE) surface of the SPCEs for amperometric detection using the hydroquinone/hydrogen peroxide/HRP (HQ/H2O2/HRP) redox system. The method allows the selective detection of TIM-1 protein over the 87–7500 pg mL−1 concentration range in only 45 min, with a limit of detection of 26 pg mL−1. The developed bioplatform was successfully applied to the analysis of breast and lung cancer cell extracts, providing the first quantitative results of the target glycoprotein in these types of samples.

Simultaneous determination of procalcitonin and interleukin-6 in human serum samples with a point-of-care biosensing device

The simultaneous determination of two inflammatory diseases biomarkers, namely procalcitonin (PCT) and interleukin-6 (IL-6), in human serum samples employing a Point-of-Care device based on Multi Area Reflectance Spectroscopy is presented. Dual-analyte detection was achieved using silicon chips with two silicon dioxide areas of different thickness, one functionalized with an antibody specific for PCT and the other with an antibody specific for IL-6. The assay included reaction of immobilized capture antibodies with mixtures of PCT and IL-6 calibrators with the biotinylated detection antibodies, streptavidin and biotinylated-BSA. The reader provided for the automated execution of the assay procedure, as well as for the collection and processing of the reflected light spectrum, the shift of which is correlated to analytes concentration in the sample. The assay was completed in 35 min and the detection limits for PCT and IL-6 were 2.0 and 0.01 ng/mL respectively. The dual-analyte assay was characterized by high reproducibility (the intra- and inter-assay coefficients of variation were less than 10% for both analytes) and accuracy (the percent recovery values ranged from 80 to 113% for both analytes). Moreover, the values determined for the two analytes in human serum samples with the assay developed were in good agreement with the values determined for the same samples by clinical laboratory methods. These results support the potential of the proposed biosensing device application for inflammatory biomarkers determination at the Point-of-Need.

An ultra-sensitive SPR immunosensor for quantitative determination of human cartilage oligomeric matrix protein biomarker

This paper reports the development of a novel surface plasmon resonance (SPR) immunosensor for ultra-sensitive quantitative determination of human articular cartilage oligomeric matrix protein (COMP), a major component of the extracellular matrix and an exploratory biomarker. Capture antibodies against human COMP (anti-COMP16F12) were covalently immobilized on an 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayer (SAM)-coated SPR sensor disk and a dual sandwich-type signal amplification strategy using biotinylated detection antibodies against COMP (anti-COMP17C10-biot) and streptavidin-conjugated quantum dots (SAv‒QDs) were used for the development of an immunosensor. The binding of high-mass SAv‒QDs via biotin–streptavidin interaction to the surface of the immunosensor resulted in a drastic increase in the sensitivity. The developed immunosensor was able to detect concentrations of COMP in a range from 2.80 to 680.54 fM with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.15 and 0.50 fM, respectively. The immunosensor exhibited good repeatability (relative standard deviation (RSD) 8.05%) and reproducibility (RSD 9.88%) as well as excellent operational stability (2.14 % decrease in SPR signal after 13 days). In addition, the analysis of secretomes of human knee articular cartilage explants from patients with osteoarthritis revealed that the immunosensor has good accuracy (analytical error less than 5 %). These results indicate that the immunosensor developed may be suitable for quantitative determination of COMP derived from articular cartilage and other synovial joint tissues in clinical studies.

Development of signal multiplication system for quinone linked immunosorbent assay (Multi-QuLISA) by using poly-l-lysine dendrigraft and 1,2-naphthoquinone-4-sulfonate as enzyme-free tag

A sensitive and stable signal multiplied quinone-linked immunosorbent assay (Multi-QuLISA) was developed. In Multi-QuLISA, an oligomerized quinone linked to biotin, namely biotin-8mer-naphthoquinone (Bio8mer-NQ), is used as a signal-generating label. Bio8mer-NQ is formed from a dendrigraft poly-l-lysine generation 1 (DPLL G1), a controlled branched oligomer composed of eight lysine moieties with nine free amino groups as a backbone. One of the nine amino groups of DPLL G1 is attached to biotin moiety, while the other eight are attached to 1,2-naphthoquinone-4-sulfonate (NQS). Bio8mer-NQ labels a biotinylated detection antibody using avidin as a co-binder. Then, multi-quinones in Bio8mer-NQ undergo a redox cycle with dithiothreitol and luminol, generating strong chemiluminescence. Standard ELISA uses a label enzyme that suffers from vulnerability in different conditions and poor stability. Bio8mer-NQ showed better stability than the enzyme (biotin-HRP) under different drastic pH and temperature conditions, hydrolytic enzymes, etc. Furthermore, Bio8mer-NQ was used as both chemiluminescence and colorimetric label based on the redox cycle of quinone, and it had LODs of 1.5 and 6.5 nM, respectively. The method could detect biotinylated immunocomplex in an in-house designed immunoassay down to 0.2 nM, which is about 25 times more sensitive than biotin HRP. Eventually, Bio8mer-NQ was applied successfully in Multi-QuLISA for detecting β-casein with a sensitivity of 3.2 ng/mL, while the conventional ELISA had an LOD of 35 ng/mL. Overall, Bio8mer-NQ is a stable compound that could be used as an excellent replacement for the enzyme in immunoassay and can be used in both colorimetric and chemiluminescence assays with good sensitivity.

Malaria quantitative POC testing using magnetic particles, a paper microfluidic device and a hand-held fluorescence reader

A point-of-care (POC) device is reported for highly sensitive and selective detection of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), a biomarker of malaria infection, based on a single-step magneto-immunoassay, a single-use microfluidic paper device and a customized hand-held fluorescence reader. The single-step magneto-immunoassay consists in a single 5-min incubation of immuno-modified magnetic particles (c-MAb-MPs), biotinylated detection antibody (bd-MAb), and an enzymatic signal amplifier (Poly-HRP). After on-chip MP concentration and washing, signal generation is achieved by adding a fluorescent enzymatic substrate (QuantaRed). Fluorescence signal is measured using a low-cost customized, portable, and sensible fluorescent detector. The POC affords quantitative Pf-LDH detection in <20 min, with a detection limit of 0.92 ng mL−1 (equivalent to 4.6 parasites μL−1). Furthermore, Pf-LDH quantitation in clinical samples correlates with that provided by the reference ELISA, is more sensitive than a commercial rapid diagnostic test (RDT) and entails little user intervention. These results show that fluorescent paper-based microfluidic devices can be exploited to simplify magneto-immunoassay handling, taking this type of test closer to the requirements of POC testing.

RapidQ: A reader-free microfluidic platform for the quantitation of antibodies against the SARS-CoV-2 spike protein.

We describe RapidQ, a fast, disposable, easy-to-use microfluidic assay for the quantitation of the anti-SARS-CoV-2 spike (S) protein IgG in plasma samples. The assay utilizes antigen-coated paramagnetic microbeads, which are induced to aggregate inside the RapidQ microfluidic device in the presence of the target antibody. Aggregation occurs via interaction between the biotinylated detection antibody and polymeric streptavidin. The mobility of the beads inside the two microchannels of the device depends on their aggregation state, with larger clusters moving at higher velocities under a given liquid flow rate. One of the microchannels incorporates a permanent magnet that captures arriving beads and forms a localized constriction that retards liquid flow. Since the constriction grows faster when the beads are more aggregated, the length of the liquid column accumulated downstream from the constriction relative to that of the unconstricted control channel is proportional to the sample antibody concentration. The assay demonstrates a detection limit of 4 μg/ml of monoclonal anti-S protein antibody diluted in plasma with CV ≤ 13%, as well as negative and positive percent agreements of 100% (95% CI: 92.75%-100%) and 100% (95% CI: 80.5%-100%), respectively, when compared to a nucleic acid amplification test used to identify COVID-19 positive individuals, whose samples were collected ≥17 d from a positive PCR test. Finally, the RapidQ assay was used to monitor the kinetics of antibody responses to COVID-19 vaccination in a small study cohort.

POS-383 PLASMONIC-FLUOR ENHANCEMENT OF KIDNEY INJURY BIOMARKER MICROARRAYS

Biomolecular microarrays based on fluorescence read-out are important clinical and research tools, especially for simple, high-throughput and rapid proteomic analyses allowing miniaturization of tens to hundreds of assays on one small piece of analytical substrate. Despite advantages such as high multiplicity, rapid screening, and low sample volume, this methodology suffers from low sensitivity (even inferior to ELISA), which hinders its widespread application. Therefore, we investigated the applicability of plasmonic-fluors for enhancing the sensitivity of immunosandwich arrays A urine sample from a patient with chronic kidney disease was diluted using blocking/sample buffer, mixed with the biotinylated detection antibody cocktail, and added onto four nitrocellulose membrane arrays per the manufactures (Raybiotech) instructions for fluorescent detection using a LI-COR instrument with fluorophore streptavidin-800CW and then imaged/quantified on a LI-COR CLx (Figure left). Finally, plasmonic-fluor-800CW was added on the arrays and thoroughly rinsed to remove the unbound plasmonic nanoconstructs before reimaging. Also imaged is an array using R&D Systems human kidney array as analytical substrate and patient plasma as biospecimen source. Seven biomarkers were identifiable and quantifiable (albumin, β2-microglobulin, CCL-2/MCP-1, cystatin C, CXCL9, sTNFR1 and NGAL) (Spots D, G, L, M, P, S, T, respectively) using conventional fluor (Figure 1, left). Along with the three positive control Spots (A, B and W), the mean repeatability was 104% with a standard deviation of 4% for the four arrays Three of the arrays were then treated with plasmonic-fluor and reimaged (Figure 1, right). All 20 biomarkers (C-V) (KIM-1, albumin, osteopontin, trefoil factor-3, b2-microglobulin, clusterin, CXCL16, GPNMB, FABP-1, CCL-2/MCP-1, sTNFR1, calbindin-1, CXCL10, cystatin C, hepatocyte growth factor, CXCL9, NGAL, TIMP-1, and VCAM-1) were now quantifiable (Figure 1, right). Repeatability between the three arrays for all 46 spots either biomarker or control was a mean of 99% with a standard deviation of 5%. Comparing the relative densities of the biomarker and control spots found a 75-fold enhancement of the fluorescence by the plasmonic-fluor (red bars) over that of the standard fluor (blue bars) with a standard deviation of 8-fold. The reproducibility of the positive control spots (A, B and W) was 103% with a standard deviation of 5% without fluor and 6% with fluor. This demonstrates the reproducibility of the technique to identify biomarkers relevant to kidney injury and function and normalize biomarker levels from array to array. Similar results were obtained with kidney arrays from R&D Systems (Not Shown Here) using patient plasma. Additionally, since the plasmonic-fluor uses gold Nano rods, visible spots are quantifiable for abundant biomarkers while fluorescence-based analyses make the relative quantifiable dynamic range of all 38 biomarkers of interest of five-orders of magnitude. Use of plasmonic-fluor to enhance the sensitivity of multiplexed microarrays to detect biomarkers of interest allows relative quantification ofall analytes on the array, not just abundant markers. This technology can be adapted to analysis on smartphones extending analysis beyond central laboratories.

Evolution of “On-Barcode” Luminescence Oxygen Channeling Immunoassay by Exploring the Barcode Structure and the Assay System

The multiplexed luminescence oxygen channeling immunoassay (multi-LOCI) platform we developed recently that combines conventional LOCI and suspension array technology is capable of realizing facile “mix-and-measure” multiplexed assays without tedious washing steps. However, previous work lacks comprehensive studies of the structure–performance relationship of the host–guest-structured barcode, which may obstruct the evolution and further translation of this exciting new technology to practical applications. Accordingly, this work revealed that polyelectrolyte interlayers played a crucial role in tuning the packing density of guest acceptor beads (ABs). More interestingly, we noticed that “sparse” barcodes (barcodes with low ABs packing density) exhibited comparable assay performance with “compact” ones (barcodes with high ABs packing density). The high robustness of barcodes allows for multi-LOCI to be a more universal and flexible assay platform. Furthermore, through optimization of the assay system including the laser power, as well as the concentrations of donor beads and biotinylated detection antibodies, the multi-LOCI platform showed a significant improvement in sensitivity compared with our previous work, with the limit of detection decreasing to as low as ca. 1 pg/mL. Impressively, multi-LOCI that enabled simultaneous detection of multiple analytes exhibited comparable sensitivity with the classical single-plexed LOCI, due to the ingenious structural design of the multi-LOCI barcode and the unique “on-barcode” assay format.

Improved ELISA for linoleate-derived diols in human plasma utilizing a polyHRP-based secondary tracer.

Dihydroxyoctadecenoic acids (DiHOMEs) are cytochrome P450 pathway-derived metabolites of linoleic acid, a highly abundant dietary fatty acid. They serve thermogenic functions at low concentrations but, at high concentrations, are involved in proinflammatory and deleterious outcomes in a wide range of pathologies. Hence, the development of a reliable analytical method is critical to elucidate their potential as biomarkers of health, and enzyme-linked immunoassay (ELISA)-based approaches offer unique benefits as alternatives to traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems. Accordingly, an earlier ELISA for DiHOMEs was dramatically improved employing new secondary tracers and geared towards use in human plasma, a universal matrix in biomedical applications, as well as urine. Three ELISA formats, two utilizing polyHRP-based secondary labels for signal amplification, were compared. The best format involved a biotinylated detection antibody and a polyHRP-conjugated streptavidin tracer. Assay detectability was enhanced 20-fold, relative to the original immunoassay, and performance assessments validated precision, selectivity, and robustness. Fast and easy extraction-clean up steps yielded high analytical recovery and permitted the assay to operate in moderate concentrations (up to 20%) of plasma, expanding its practical relevance. Finally, the ELISA was applied towards detection of DiHOMEs in clinical samples and authenticated with complementary LC-MS/MS analysis. Hence, the method provides a valuable analytical tool to investigate the diverse and extensive roles of DiHOMEs in regulatory biology.

A descriptive analysis of non-human leukocyte antigens present in renal transplant donor-recipient pairs

IntroductionEnd stage renal disease (ESRD) is the irreversible deterioration of renal function requiring renal replacement therapy by dialysis or transplant. Human leucocyte antigens (HLA) have been well examined however research still is required into the non-HLA antibodies. Antibody mediated rejection (AMR) can be seen in the absence of HLA antibodies on biopsies of patients who have received identical transplants; anti-endothelial cell antibodies may explain this. Investigation into endothelial cell antigens on donor and recipient endothelium may elucidate and stratify the degree of risk of any given transplant and may guide towards the best matched donor. MethodsProtein array analysis was carried out on 8 patient pairs using nitro-cellulose membranes and biotinylated detection antibodies. The fluorescence emitted was captured by X-Ray film and results were recorded with ImageJ software. A fold increase of more than 2 was considered to be positive. Results11 proteins identified had a fold increase of increase ≥2 and were present in ≥2 patient pairs which may point to potential clinical utility. Nectin2/CD112 may be measured in order analyse graft survival time in transplant recipients. Prognosticating renal failure has clinical importance and potential markers that have been identified to aid which include MEPE, CRELD2, and TIMP-4. Novel pharmacological therapies for specific biomarkers identified in this study include JAM-A, E-Selectin, CD147, Galectin-3, JAM-C, PAR-1, and TNFR2. ConclusionProtein analysis showed differences in expression of antigens between patients with and without Chronic Kidney Disease (CKD). This information could be used at the matching stage of renal transplantation and also in the treatment of rejection episodes. The results highlight biomarkers that potentially prognosticate and pharmacological therapies that may ameliorate kidney disease and rejection in ESRD and transplant recipients.

Simultaneous determination of CXCL7 chemokine and MMP3 metalloproteinase as biomarkers for rheumatoid arthritis

This paper reports the preparation of the first dual electrochemical immunosensor for the simultaneous determination of the CXCL7 chemokine and the MMP3 metalloproteinase as relevant biomarkers for the better diagnosis and monitoring of rheumatoid arthritis derived from the multiple biomarkers measurement. The developed immunosensor involves the use of carboxylated magnetic beads (MBs) and dual screen-printed carbon electrodes (SPdCEs). Sandwich-type configurations implied the covalent immobilization of specific anti-CXCL7 (cAb1) or anti-MMP3 (cAb2) capture antibodies onto MBs and the use of biotinylated detection antibodies with further labelling with HRP-Strept conjugates. The resulting MBS bioconjugates were magnetically captured on the respective working electrode of the SPdCE and the determination of the antigens was accomplished by measuring the amperometric responses of H2O2 mediated by hydroquinone (HQ) at a potential value of −0.20 V. The dual immunosensor provided calibration plots with linear ranges between 1 and 75 ng mL−1 (CXCL7) (R2 = 0.997) and from 2.0 to 2000 pg mL−1 (MMP3) (R2 = 0.998) with detection limits of 0.8 ng mL−1 and 1.2 pg mL−1, respectively. The assay took 2 h 20 min for the simultaneous determination of both biomarkers. The dual immunosensor was successfully applied to the analysis of human serum from positive and negative RA patients.

Role of the Isoform p-53 Protein in Failed Cases of Chronic Lymphocytic Leukemia

Introduction It was discovered in the last few years that the production of some percentage of mutant p-53 proteins, with the increased stability in type B lymphocytes, leads to the carcinogenesis process. This discovery led to the identification and quantification of the p-53 protein by different methods such as immuno-histochemistry (IHC), polymerase chain reaction (PCR), single-stranded peptide microarray, (SSPMa), next-generation sequencing (NGS), and the sandwich enzyme-linked immunosorbent assay (sandwich ELISA). Method Using the ELISA technique, the frequency of p-53 protein expression in 20 representative patients diagnosed with CLL-B was analyzed, in order to investigate the relationship of the p-53 protein at the different stages of the disease and the impact on patient survival. ELISA Kit Component: Coated 96-well, Strip Plate 1, Standard (Lyophilized) 2 vials Assay, Diluent (5x) 1 vial x 15 ml, Biotinylated Detection Antibody 2 vials, HRP-Streptavidin Conjugate (800x) 1 vial x 200 µl, Wash Buffer (20x) 1 vial x 25 ml, TMB Substrate 1 vial x 12 ml, Stop Solution H2SO4 1 vial x 8 ml, Plate Sealers 4. Molecular biology technology used in the method The monoclonal p-53 antibody, PAb 240, used in the ELISA method recognizes both mutant and wild-type p-53 under denaturing conditions. Species reactivity is for human or rhesus monkey in conformity with the prospect. The monoclonal antibody PAb 240 recognizes an epitope that is structurally hidden in the wild-type conformation of p-53 and becomes exposed by denaturing the p-53 protein or the mutant conformations of p-53, where point mutations in the P-53 gene alter the terminal structure of the p-53 protein. This ELISA kit is recommended for use in serum, plasma or tissue homogenates. Using other types of sample is not supported. The sample collection protocols below were adapted from the references. 1. Full physical examination of patients diagnosed with CLL All patients were admitted to hospital with symptoms and clinical features of CLL, such as cough, night sweating, and retrosternal pain. Clinical examination and ultrasounds revealed adenopathy and/or splenomegaly, with spleen enlargement of 3 cm above the normal diameter. 2.Immunophenotyping using monoclonal antibodies (Flow Cytometry) CLL-B diagnosis was confirmed by immunophenotyping: monoclonal antibodies in CD5⁺, CD19⁺, CD20⁺, CD23⁺, CD28⁺ receptors, and with B lymphocytes expressing IgM or IgG heavy chains with kappa or lambda light chains. 3.Laboratory Exams: For each of the cases, a 5 Diff Hematology Analyzer was used to perform a haemogram, and blood smear cytology exams on peripheral blood and medullary bone marrow were carried out by May-Grunwald Giemsa staining. The leukemia cells found in the peripheral blood smear had characteristic microscopic morphology, with the small nuclei having mature lymphocytes with full or partially aggregated chromatin and lacking nucleoli. Results Of the 20 patients studied, 14 men have aged 55-85 years and 6 women aged 39-85 years. Patients were treated at the time of these investigations with cytostatic and immunotherapy specific for CLL. Male results: Protein concentration p-53 / µg / dl: 20, 15, 18, 40, 10, 12, 14, 60, 30, 10, 13, 13, 5, 10, 15, 12. Women's results; Protein concentration p-53 / µg / dl: 140, 30, 13, 10, [Graphic 1]. Statistical interpretations: The probability index (NORMDIST) p, was calculated in the value of p = 0.034. The reference interval was established between the values 10-40µg / dl, (m = 24.5 µg / dl. The pathological values in the 3 cases of highly elevated p-53, reflecting the concentration of p-53 protein mutant, were calculated in 2 men in the amount of 60 µg / dl, respectively in 50 µg / dl, and in the female case, it was calculated in the amount of 140 µg / dl, the frequency of chronic lymphocytic leukemia with mutant p-53 protein being higher in men than in women, in ratio 2/1, in accordance with the data from the specialized literature. Conclusions In the context of a heterogeneous malignant disease, such as CLL-B, a simple and inexpensive ELISA method, such as employed in this study, proves useful for identifying patients to be considered as candidates for personalized therapeutic strategies, based on the mutation of the TP- 53 gene and the presence of p-53 isoform protein. Figure Disclosures No relevant conflicts of interest to declare.

A novel low-field NMR biosensor based on dendritic superparamagnetic iron oxide nanoparticles for the rapid detection of Salmonella in milk

The growing threat of foodborne disease has made it imperative to develop rapid and efficient detection techniques. Here, we developed dendritic superparamagnetic iron oxide nanoparticles (SPIONs) combined with low-field nuclear magnetic resonance (LF-NMR) biosensor for Salmonella detection in milk. We first used a Salmonella capture antibody and biotinylated detection antibody (BT-mAb) in the form of a sandwich in a 96-well plate to immobilize target bacteria. Then streptavidin modified polyamidoamine (SA-PAMAM) was used to connect BT-mAb and functionally modified SPIONs. PAMAM-mediated SA amplified functionalized SPIONs via streptavidin-biotin amplification system, aggregated nanoparticles to form dendritic SPIONs, and achieved dual signal amplification. Finally, the dendritic SPIONs capture complex is eluted, and the lateral relaxation time T2 of the water molecules around the SPIONs was used as the output of the target bacteria capture signal. The limit of detection of Salmonella in a buffer solution and milk was 2.6 × 104 CFU/mL, and the LF-NMR biosensor showed good specificity and anti-interference properties. Therefore, this innovative detection platform can be used as a sensitive, fast, and efficient tool for detecting foodborne pathogens.

A sensitive thrombin-linked sandwich immunoassay for protein targets using high affinity phosphorodithioate modified aptamer for thrombin labeling

Thrombin and its aptamers have been well studied and widely used as models in aptamer based assays and sensors. Here we reported a thrombin-linked sandwich immunoassay for proteins to demonstrate new applications of thrombin and the aptamers, converting protein detection to analysis of thrombin label. In this assay, target protein was sandwiched by the capture antibody on a microplate and the biotinylated detection antibody. Thrombin bound to one biotinylated aptamer, and then the thrombin-labeled aptamer was attached on the sandwich complex through streptavidin-biotin interaction by using streptavidin as a linker. Thrombin catalyzed cleavage of fluorogenic peptide substrates, generating fluorescence signals for target detection. Among a few different anti-thrombin aptamers, the use of one nuclease resistant RNA aptamer having phosphorodithioate (PS2) modification on a specific backbone position enabled higher assay sensitivity due to its much higher affinity. This thrombin-linked sandwich immunoassay allowed detection of prostate-specific antigen (PSA) at 2 pM, an important protein related cancer disease, with high sensitivity and specificity. The strategy was general, and also enabled sensitive detection of botulinum neurotoxin type A (BoNTA) light chain, one toxin protein causing risk to human health. This assay combines advantages of antibody recognition, aptamer affinity labeling, high affinity of aptamers, and enzyme activity of thrombin. Labeling thrombin on the immunosandwich complex through simple affinity binding overcomes limitations of covalent conjugating enzyme on antibody in conventional immunoassay. This assay is promising in applications for protein detection.

Sensitivity and binding kinetics of an ultra-sensitive chemiluminescent enzyme-linked immunosorbent assay at arrays of antibodies

We have characterized the sensitivity and kinetics of a multiplex immunoassay system based on detection of chemiluminescence (CL) at arrays of antibodies. This enzyme-linked immunosorbent assay (ELISA) was based on the spotting of different antibodies in a circular pattern at the bottom of a well of a microtiter plate. Sandwich immunocomplexes within each spot were labeled with horse radish peroxidase, and CL was generated locally to each spot in the array from turnover of luminol substrate. CL from the arrays across the plate was collected in single images; long exposure times were used to maximize sensitivity, and short exposure times were used to extend the dynamic range at higher signals. Image analysis was used to determine the intensity of light from each spot in the array, and intensity was converted to concentration of protein via comparison to a calibration curve. To determine the intrinsic sensitivity of the CL ELISA array, streptavidin horseradish peroxidase (SA-HRP) was captured on an array spotted with biotinylated detection antibodies. The limit of detection (LOD) of SA-HRP was 105 aM, or 3200 enzymes per 50 μL. A single-plex assay for prostate specific antigen (PSA) was developed that had an LOD of 79 aM when the microtiter plate was shaken orbitally, comparable to the most sensitive immunoassays reported to date. Normalization of CL signals in the PSA assay to signal per molecule of SA-HRP showed that the efficiency of the shaken assay was ~40%. When the plates were not shaken, the efficiency was ~4.5%, i.e., ~9-fold lower than when shaken. To better understand the theoretical basis of the sensitivity of these assays, we developed COMSOL numerical models of the binding kinetics at the array for plates that were shaken orbitally and those not shaken. Experimental data from the orbitally shaken PSA assay were best modeled by inertial mixing in a three-layer system that included a 8-μm-thick concentration boundary layer. Experimental data from the unshaken PSA assay were well modeled by diffusion-limited kinetics. A single-plex assay for IL-10 was developed with an LOD of 69 aM or 1.5 fg/mL, and used to measure this cytokine in plasma and serum of 10 healthy individuals. A 5-plex assay for IL-5, IL-6, IL-10, IL-22, and TNF-α was developed with LODs of 56 aM, 237 aM, 69 aM, 88 aM, and 373 aM, respectively. The assay was used to measure these 5 cytokines in the plasma and serum of the same individuals. The correlation in concentration of IL-10 measured in single-plex and multiplex assays was good (r2 = 0.89; bias = 14.5%). The factors that result in the high sensitivity of CL ELISA arrays—mostly high signal to noise ratio of extended chemiluminescent imaging—are discussed. This multiplex CL ELISA could be used for sensitive profiling of multiple proteins for in vitro diagnostics and biomarker detection in the development of therapeutics.

Development of an Immunosensor Based on the Exothermic Reaction between H2O and CaO Using a Common Thermometer as Readout.

Thermometers, one of the most commonly used instruments at home, are normally adapted to measure temperature directly with high accuracy but rarely adopted to act as readout in the biosensors. It is necessary to find some ways to establish a relationship between the concentration of the target and the temperature change. In this study, a common thermometer was used as readout to develop a convenient immunosensor. The designed immunosensor comprises three components, including target recognition area, water flow system, and exothermic reaction bottle. The capture antibody for the target [carcinoembryonic antigen (CEA) was selected as a model target] was preloaded on the bottom of the recognition area. In the presence of CEA, a sandwich-type structure was formed between the capture antibody, CEA, and biotinylated detection antibody. Then, the streptavidin-functionalized platinum nanoparticles were labeled on the detection antibody due to biotin-avidin interaction. The captured platinum nanoparticles can effectively catalyze the decomposition of H2O2 into O2. The continuous production of gas resulted in pressure increment inside the reaction bottle and further pushed the water flow into the exothermic reaction bottle. Finally, the water reacted with calcium oxide to generate a large amount of heat in the exothermic reaction bottle; thereby the temperature inside the bottle was enhanced and recorded by a common thermometer easily. The temperature enhancement has a linear relationship with the CEA concentration in the range of 7.81-500 pg/mL with a detection limit of 0.6 pg/mL. Furthermore, by taking advantage of simplicity, compatibility, stability, and high sensitivity, our temperature-based immunoassay has been applied to detect CEA in human serum samples with satisfactory results.

Evaluation of Antibody Biotinylation Approaches for Enhanced Sensitivity of Single Molecule Array (Simoa) Immunoassays.

In this study, we evaluated the performance of Single Molecule Array (Simoa) immunoassays based on various detection antibody biotinylation approaches. Simoa immunoassays, like other sandwich ELISAs, are highly dependent on the interaction of a biotinylated detection antibody with an enzyme conjugated to streptavidin. Thus, we sought to assess whether different biotinylation reagents can improve the performance and sensitivity of Simoa assays. We selected three proteins, GM-CSF, IFNγ, and IL-2, that are present at ultralow levels in many biological samples. We compared the performance of these Simoa assays by using five different biotinylation reagents and varying the amount of molar fold excess biotin during the biotinylation process. We found that the choice of biotinylation reagent and the molar fold excess biotin can highly affect the performance of the Simoa assays, with differences of up to an order of magnitude in sensitivity. We also tested the performance of bulk ELISAs using the different biotinylated detection antibodies and observe differences greater than an order of magnitude in sensitivity. We show that evaluating different strategies for detection antibody biotinylation is a simple approach for optimizing immunoassay performance for enhanced sensitivity.