Journal articles: 'Sandwich assays'

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Sanders, Emily C., Alicia M. Santos, Eugene K. Nguyen, Aidan A. Gelston, Sudipta Majumdar, and Gregory A. Weiss. "Phage vs. Phage: Direct Selections of Sandwich Binding Pairs." Viruses 15, no. 3 (March 22, 2023): 807. http://dx.doi.org/10.3390/v15030807.

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The sandwich format immunoassay is generally more sensitive and specific than more common assay formats, including direct, indirect, or competitive. A sandwich assay, however, requires two receptors to bind non-competitively to the target analyte. Typically, pairs of antibodies (Abs) or antibody fragments (Fabs) that are capable of forming a sandwiching with the target are identified through a slow, guess-and-check method with panels of candidate binding partners. Additionally, sandwich assays that are reliant on commercial antibodies can suffer from changes to reagent quality outside the researchers’ control. This report presents a reimagined and simplified phage display selection protocol that directly identifies sandwich binding peptides and Fabs. The approach yielded two sandwich pairs, one peptide–peptide and one Fab–peptide sandwich for the cancer and Parkinson’s disease biomarker DJ-1. Requiring just a few weeks to identify, the sandwich pairs delivered apparent affinity that is comparable to other commercial peptide and antibody sandwiches. The results reported here could expand the availability of sandwich binding partners for a wide range of clinical biomarker assays.

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Zweig, M. H., G. Csako, C. C. Benson, B. D. Weintraub, and B. B. Kahn. "Interference by anti-immunoglobulin G antibodies in immunoradiometric assays of thyrotropin involving mouse monoclonal antibodies." Clinical Chemistry 33, no. 6 (June 1, 1987): 840–44. http://dx.doi.org/10.1093/clinchem/33.6.840.

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Abstract "Sandwich"-type assays are subject to positive interference by the patient's "heterophile" antibodies. If present, these bind to the animal immunoglobulins in the assay reagents, forming artefactual sandwiches indistinguishable from those formed with the analyte itself. Immunoglobulins from non-immunized animals, added to the assay reagents, can diminish this effect by blocking the patient's antibodies. Elsewhere, we studied several patients with anti-mouse immunoglobulin activity, whose serum gave spuriously high results for thyrotropin (TSH) concentrations. Here we have studied this phenomenon by adding, to pooled zero-TSH serum, antibodies to mouse, goat, and horse immunoglobulins and then assaying TSH by several other sandwich-type assays involving mouse monoclonal antibodies. Assays not supplemented with blocking immunoglobulins from mice or other animals were more susceptible to this effect. When large amounts of antibody were added, the antibody excess diminished the interference. However, the presence of blocking immunoglobulins could reverse such antibody excess, actually enhancing, instead of diminishing, the positive interference. Users should be aware that blocking immunoglobulins may diminish but not necessarily eliminate this problem with such assays.

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Prudom, Catherine, Jianhua Liu, James Patrie, Bruce D. Gaylinn, Karen E. Foster-Schubert, David E. Cummings, Michael O. Thorner, and H. Mario Geysen. "Comparison of Competitive Radioimmunoassays and Two-Site Sandwich Assays for the Measurement and Interpretation of Plasma Ghrelin Levels." Journal of Clinical Endocrinology & Metabolism 95, no. 5 (May 1, 2010): 2351–58. http://dx.doi.org/10.1210/jc.2009-2407.

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Abstract Context: Ghrelin, an endogenous ligand for the GH secretagogue receptor, is an orexigenic peptide hormone produced primarily by the stomach. Recent studies suggest significant differences in the specificity of currently available ghrelin assays. Objective: The aim of the study was to compare four ghrelin assays (two commercially available and two developed by our group) of differing specificity, each used on the same set of more than 800 plasma samples from a human study. Design: Thirteen volunteers were sampled every 20 min for 6 h after consumption of one of three isocaloric drinks consisting of either 80% fat, 80% carbohydrate, or 80% protein. The samples were assayed by RIA for total and active ghrelin, as well as by sandwich assays for acyl and des-acyl ghrelin. The ghrelin profiles for each individual were smoothed using a statistical algorithm to lessen the effects of pulsatility and noise. Results: The sandwich assays for acyl and des-acyl ghrelin yielded ghrelin values that were lower than those from the corresponding RIAs. The ghrelin profiles after nutrient ingestion were similar, yet key differences among the four assays were apparent; in particular, percentage changes were significantly greater in the sandwich assays. Conclusions: The lower levels and greater relative changes in ghrelin values reported by the sandwich assays are consistent with greater assay specificity. When applied to the nutrient study, the sandwich assays were better able to distinguish the different responses to different nutrients than were the RIAs.

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Kang, J., P. Kaladas, C. Chang, S. Chen, R. Dondero, A. Frank, S. Huhn, P. Lisi, D. Monchnal, and J. Nasser. "A highly sensitive immunoenzymometric assay involving "common-capture" particles and membrane filtration." Clinical Chemistry 32, no. 9 (September 1, 1986): 1682–86. http://dx.doi.org/10.1093/clinchem/32.9.1682.

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Abstract This highly sensitive immunoenzymometric method involves monoclonal antibodies, a common-capture microsphere, and a rapid, membrane-filtration separation step. The common-capture solid phase is monoclonal anti-fluorescein antibody convalently attached to 6.5 micron-diameter latex particles. In sandwich-type assays for large-molecule analytes, the capture antibody is conjugated with fluorescein isothiocyanate and the probe antibody is conjugated with beta-galactosidase (EC 3.2.1.23). In competitive assays for small analytes, the analyte-beta-galactosidase conjugate competes with the analyte in the clinical samples for the fluoresceinated capture antibody. After simultaneous incubation of the reagents for 2 h, the bound and unbound reagents are separated by filtration through the bottom of each well of a 96-well plate. Substrate (4-methylumbelliferyl-beta-D-galactopyranoside) is then added to the wells, and the rate of product formation is determined kinetically for 12 min. The rate is proportional to the concentration of analyte in the sandwich assays and inversely proportional in the competitive assays. The assay results for choriogonadotropin, thyrotropin, digoxin, and thyroxin show the assay to be sensitive, rapid, and applicable to any size analyte. With this system, several different sandwich and (or) competitive-type assays can be performed simultaneously on the same plate.

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Nielsen, Ulrik B., and Bernhard H. Geierstanger. "Multiplexed sandwich assays in microarray format." Journal of Immunological Methods 290, no. 1-2 (July 2004): 107–20. http://dx.doi.org/10.1016/j.jim.2004.04.012.

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Saviranta, Petri, Ryan Okon, Achim Brinker, Masaki Warashina, Joerg Eppinger, and Bernhard H. Geierstanger. "Evaluating Sandwich Immunoassays in Microarray Format in Terms of the Ambient Analyte Regime." Clinical Chemistry 50, no. 10 (October 1, 2004): 1907–20. http://dx.doi.org/10.1373/clinchem.2004.037929.

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Abstract Background: Conceptionally, antibody microarrays are simply multiplexed sandwich immunoassays in a miniaturized format. However, from the amounts of capture antibodies used, it is not apparent whether such assays are ambient analyte (Ekins. Clin Chem 1998;44:2015–30) or mass-sensing devices (Silzel et al. Clin Chem 1998;44:2036–43). We evaluated multiplexed microarray sandwich assays for 24 mouse serum proteins in these terms within the boundaries of our experimental setup and based on theoretical considerations of the law of mass action. Methods: Capture antibodies for 24 mouse serum proteins were printed on planar microarray substrates. After incubation with mixtures of purified antigens for 1 or 18 h, mixtures of biotinylated detection antibodies were used. High assay sensitivity was achieved by use of resonance-light-scattering particles for signal generation. Titration curves were generated for assay volumes of 20, 40, and 80 μL, and detection limits were calculated and compared. The assays were modeled theoretically based on the amounts of capture antibodies and the assay volumes used. Results: As predicted, experimental variations of the assay volume by up to fourfold did not appreciably affect detection. Even for the most sensitive assay, <2% of the analyte molecules present in the sample were captured and generated signal at the detection limit. However, increasing the sample incubation time from 1 to 18 h on average lowered the detection limit threefold. Conclusions: In our experimental setup, all 24 sandwich microarray assays fulfill the criteria of the “ambient analyte” regime because depletion of analyte molecules from the assay volume is insignificant.

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Neupane, Dharmendra, and Keith J. Stine. "Electrochemical Sandwich Assays for Biomarkers Incorporating Aptamers, Antibodies and Nanomaterials for Detection of Specific Protein Biomarkers." Applied Sciences 11, no. 15 (July 31, 2021): 7087. http://dx.doi.org/10.3390/app11157087.

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The development of sensitive and selective assays for protein biomarkers and other biological analytes is important for advancing the fields of clinical diagnostics and bioanalytical chemistry. The potential advantages of using aptamers in electrochemical sandwich assays are being increasingly recognized. These assays may include an aptamer as both capture and detection agent or a combination of an aptamer with a different partner such as an antibody, a lectin or a nanomaterial. The second binding partner in the sandwich structure is typically conjugated to a redox marker, a catalyst or an enzyme that can be used to generate the signal needed for electrochemical detection. Nanoparticles and other nanostructures can be used as the carriers for multiple molecules of the detection partner and thereby increase the signal. Nanostructured surfaces can be used to increase surface area and improve electron transfer. Sensitive electrochemical methods including impedance, differential and square-wave voltammetry and chronocoulometry have been used for electrochemical signal read-out. Impressive results have been achieved using electrochemical sandwich assays in terms of limit of detection and linear range for a growing range of analytes. The recent progress for this type of assay for proteins and other biomarkers is the subject of this review.

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Zhang, Yuan, Gang Xu, Lu Zhang, Jiakai Zhao, Pinpin Ji, Yaning Li, Baoyuan Liu, et al. "Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus." Applied Microbiology and Biotechnology 104, no. 24 (November 7, 2020): 10725–35. http://dx.doi.org/10.1007/s00253-020-10997-y.

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Abstract Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores. Key points • Three CDV mAbs that recognized different epitopes and bound to virion were generated. • The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed. • The sandwich ELISA is an ideal method for detecting CDV infections in the field.

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Ezzell, Carol. "Hybritech wins court injunction over sandwich assays." Nature 327, no. 6117 (May 1987): 5. http://dx.doi.org/10.1038/327005a0.

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Frohnmeyer, Esther, Nadine Tuschel, Tobias Sitz, Cornelia Hermann, Gregor T. Dahl, Florian Schulz, Antje J. Baeumner, and Markus Fischer. "Aptamer lateral flow assays for rapid and sensitive detection of cholera toxin." Analyst 144, no. 5 (2019): 1840–49. http://dx.doi.org/10.1039/c8an01616j.

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Garringer, Todd O., L. Joseph Wheat, and Edward J. Brizendine. "Comparison of an Established Antibody Sandwich Method with an Inhibition Method of Histoplasma capsulatumAntigen Detection." Journal of Clinical Microbiology 38, no. 8 (2000): 2909–13. http://dx.doi.org/10.1128/jcm.38.8.2909-2913.2000.

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The Histoplasma antigen immunoassay utilizes an antibody sandwich method that provides a rapid and reliable means of diagnosing the more severe forms of histoplasmosis. Inhibition assays have been developed for antigen detection and offer at least one potential advantage, namely, reduced antibody requirements. We have developed an inhibition assay using the polyclonal antibody employed in our standard sandwich assay. Urine and serum specimens from patients with culture-proven histoplasmosis and controls were tested using both methods. The two methods had similar sensitivities for detection of antigen in urine (antibody sandwich = 92.5% versus inhibition = 87.5%, P = 0.500) and serum (82.5% versus 80.0%, P = 1.000). With serum, the specificities of both methods were similar (antibody sandwich assay = 95.0% versus inhibition assay = 92.5%,P = 1.000), and with urine, the specificity of the antibody sandwich method was superior (97.5% versus 80.0%,P = 0.039). While the overall reproducibility of both methods was excellent (with urine, antibody sandwich assay intraclass correlation coefficient = 0.9975 and with serum = 0.9949; correlation coefficient of the inhibition assay with urine = 0.9736 and with serum = 0.9850), that of the inhibition method was only fair to poor for the controls: urine = −0.0152, serum = 0.5595. Reproducibility was good for the controls using the sandwich method: urine = 0.7717, serum = 0.9470. Cross-reactivity was observed in specimens from patients infected with Blastomyces dermatitidis,Paracoccidioides brasiliensis, and Penicillium marneffei. In conclusion, the decreased specificity and inferior reproducibility with control specimens suggest that the inhibition assay has poorer precision toward the lower end of the detection range.

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Omi, Kazuya, Tsuyoshi Ando, Takuya Sakyu, Takashi Shirakawa, Yoshiaki Uchida, Asako Oka, Nobuyuki Ise, Katsumi Aoyagi, and Katsutoshi Goishi. "Noncompetitive Immunoassay Detection System for Haptens on the Basis of Antimetatype Antibodies." Clinical Chemistry 61, no. 4 (April 1, 2015): 627–35. http://dx.doi.org/10.1373/clinchem.2014.232728.

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Abstract BACKGROUND Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies. METHODS We generated antimetatype monoclonal antibodies against a hapten–antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH)D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated. RESULTS The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH)D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%–12.6% (intraassay) and 6.2%–21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH)D were 1.0%–2.3% (intraassay) and 1.9%–3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH)D showed correlations of r = 0.99 with both LC-MS/MS and a commercially available 125I RIA. CONCLUSIONS Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins.

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Bhandari, Devendra, Fur-Chi Chen, and Roger C. Bridgman. "Magnetic Nanoparticles Enhanced Surface Plasmon Resonance Biosensor for Rapid Detection of Salmonella Typhimurium in Romaine Lettuce." Sensors 22, no. 2 (January 9, 2022): 475. http://dx.doi.org/10.3390/s22020475.

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Salmonella is one of the major foodborne pathogens responsible for many cases of illnesses, hospitalizations and deaths worldwide. Although different methods are available to timely detect Salmonella in foods, surface plasmon resonance (SPR) has the benefit of real-time detection with a high sensitivity and specificity. The purpose of this study was to develop an SPR method in conjunction with magnetic nanoparticles (MNPs) for the rapid detection of Salmonella Typhimurium. The assay utilizes a pair of well-characterized, flagellin-specific monoclonal antibodies; one is immobilized on the sensor surface and the other is coupled to the MNPs. Samples of romaine lettuce contaminated with Salmonella Typhimurium were washed with deionized water, and bacterial cells were captured on a filter membrane by vacuum filtration. SPR assays were compared in three different formats—direct assay, sequential two-step sandwich assay, and preincubation one-step sandwich assay. The interaction of flagellin and MNPs with the antibody-immobilized sensor surface were analyzed. SPR signals from a sequential two-step sandwich assay and preincubation one-step sandwich assay were 7.5 times and 14.0 times higher than the direct assay. The detection limits of the assay were 4.7 log cfu/mL in the buffer and 5.2 log cfu/g in romaine lettuce samples.

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Tamm, Natalia N., Karina R. Seferian, Alexander G. Semenov, Kadriya S. Mukharyamova, Ekaterina V. Koshkina, Mihail I. Krasnoselsky, Alexander B. Postnikov, et al. "Novel Immunoassay for Quantification of Brain Natriuretic Peptide and Its Precursor in Human Blood." Clinical Chemistry 54, no. 9 (September 1, 2008): 1511–18. http://dx.doi.org/10.1373/clinchem.2007.100545.

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Abstract Background: Brain natriuretic peptide (BNP) is an unstable molecule that can rapidly lose immunologic activity in blood. Conventional sandwich BNP immunoassays use 2 antibodies specific to 2 different epitopes. Larger distances between epitopes are associated with a greater probability of proteolysis sites being located between the antibody-binding sites, and thus such assays have an increased susceptibility to underdetect BNP because of the increased likelihood of proteolytic degradation. The purpose of our study was to develop a sandwich immunoassay for the precise quantification of BNP and BNP precursor (proBNP) in human blood that is not susceptible to proteolysis. Methods: Mice were immunized with an immune complex consisting of monoclonal antibody (MAb) 24C5 (specific for BNP peptide 11–22) and the entire BNP molecule. The MAb used in our assay (Ab-BNP2) recognizes the immune complex but neither free BNP nor MAb 24C5. Results: We used MAbs 24C5 and Ab-BNP2 to develop a new type of sandwich BNP assay (the “single-epitope sandwich assay”), which requires only a short BNP fragment (fragment 11–22) for immunodetection. This assay recognizes both BNP and proBNP with the same efficiency and sensitivity and demonstrates both considerably less susceptibility to antigen degradation and greater stability of the measured antigen than conventional sandwich BNP immunoassays. Conclusions: We have developed this sensitive single-epitope sandwich assay for detecting BNP, proBNP, and their fragments in human blood. This assay appears promising for use in clinical studies to assist in triage, management, and outcomes assessment in heart failure patients.

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Bruno, John G., Kevin Francis, Milada Ikanovic, Poornima Rao, Sulatha Dwarakanath, and Walter E. Rudzinski. "Reovirus Detection Using Immunomagnetic-Fluorescent Nanoparticle Sandwich Assays." Journal of Bionanoscience 1, no. 2 (December 1, 2007): 84–89. http://dx.doi.org/10.1166/jbns.2007.015.

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Csordas, Andrew T., Anna Jørgensen, Jinpeng Wang, Emily Gruber, Qiang Gong, Elizabeth R. Bagley, Margaret A. Nakamoto, Michael Eisenstein, and H. Tom Soh. "High-Throughput Discovery of Aptamers for Sandwich Assays." Analytical Chemistry 88, no. 22 (November 4, 2016): 10842–47. http://dx.doi.org/10.1021/acs.analchem.6b03450.

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Yang, Li-Min, Liang-Zhen Zhao, Rong-Liang Hu, Zhen-Sheng Shi, and Wen-Jun Liu. "A Novel Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Measurement of Antibodies against Rabies Virus." Clinical and Vaccine Immunology 13, no. 8 (August 2006): 966–68. http://dx.doi.org/10.1128/cvi.00102-06.

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ABSTRACT A novel double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) was developed to measure rabies antibodies in dogs. In contrast to the 4 days required for detecting rabies antibody with conventional rabies antibody virus neutralization assays, this ELISA can be completed in hours, without using live virus, in routine laboratories.

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Kai, Junhai, Nelson Santiago, Aniruddha Puntambekar, Se Hwan Lee, David Sehy, Randy Durnell, Ron Schultheis, Jungyoup Han, and Chong Ahn. "Optimiser™ microplate and OptiMax™ reagent systems enable femtogram/ml level sensitivity employing standard ELISA equipment and protocols (65.32)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 65.32. http://dx.doi.org/10.4049/jimmunol.186.supp.65.32.

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Abstract Optimiser™ Microfluidic ELISA Plates dramatically enhance ELISA sensitivity through the incorporation of microfluidic channels into a standard ELISA plate configuration. Validation studies with Optimiser™ microfluidic ELISA plates demonstrate the Optimiser™’s ability to utilize available sandwich ELISA reagents, equipment, and protocols to attain unparalleled levels of sensitivity. Limits of Detection and Limits of Quantification are demonstrated for a variety of assays in the range of 30-100 fg/ml, representing a 20-1000 fold improvement over the same assays run in conventional ELISA plates. OptiMax™ ELISA Reagent protocols on Optimiser™ Microfluidic ELISA Plates employ the similar reagents, but abbreviated assay steps because, unlike conventional sandwich ELISAs, no traditional plate washing is required. Optimiser™ Microfluidic ELISA Plates are compatible with existing pipetting systems (manual or automated) and ELISA plate readers. Furthermore, the surface area to volume ratio of each microfluidic reaction chamber represents a 50-fold increase when compared to the well of a conventional 96-well immunoassay plate. The improvement in surface to volume ratio increases binding kinetics dramatically, enabling assays to be run in less than half the time of traditional ELISA assays and providing researchers with the flexibility to use dramatically smaller sample and reagent volumes (e.g., 5 ul).

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Klee, George G., and William E. Schreiber. "MUC1 Gene–Derived Glycoprotein Assays for Monitoring Breast Cancer (CA 15-3, CA 27.29, BR): Are They Measuring the Same Antigen?" Archives of Pathology & Laboratory Medicine 128, no. 10 (October 1, 2004): 1131–35. http://dx.doi.org/10.5858/2004-128-1131-mggafm.

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Abstract Context.—There are 2 general types of assays measuring MUC1 gene–derived glycoprotein: assays for cancer antigen (CA) 15-3, which are sandwich assays, and assays for CA 27.29, which are competitive assays. These 2 types of assays measure slightly different parts of this tandem-repeat molecule. Across-method assay differences hinder the exchange of patient test values among integrated health care networks and among countries. Objective.—This report evaluates the method differences among these assays to determine if the differences between these assays are mainly related to variations in calibration or differences in analyte specificity. Design.—Data from 22 College of American Pathologists survey challenges were analyzed to compare 10 commercial assay methods for these 2 related analytes. In addition, data from 58 patient samples were analyzed to compare 3 of these assays. Results.—The linear correlation coefficients comparing the within-method medians of these proficiency test distributions were very high (>0.99) for all of the methods; however, the regression slopes varied from 0.836 to 1.095. The regression slopes for the patient specimens varied similarly, but the correlation coefficients were lower. Conclusions.—This study indicates that many of the test value differences for these measurements are due to differences in assay calibration rather than differences in the specificity of the assay measurement systems. Survey test data potentially could be used to help harmonize these assay differences.

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Koppelman, Stef J., Gülsen Söylemez, Lynn Niemann, Ferdelie E. Gaskin, Joseph L. Baumert, and Steve L. Taylor. "Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame Seed in Foods." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/853836.

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Small amounts of sesame can trigger allergic reactions in sesame-allergic patients. Because sesame is a widely used food ingredient, analytical methods are needed to support quality control and food safety programs in the food industry. In this study, polyclonal antibodies against sesame seed proteins were raised, and an enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of sesame seed residue in food. A comparison was made between this ELISA and other assays, particularly focusing on recovery of sesame seed residue from different food matrices. The developed ELISA is sensitive with a lower limit of quantification of 0.5 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested). The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter, outperforming one other test. In a baked bread matrix, the ELISA has a low recovery, while two other assays perform better. We conclude that a sensitive and specific ELISA can be constructed based on polyclonal antibodies, which is suitable for detection of small amounts of sesame seed relevant for highly allergic patients. Furthermore, we conclude that different food products may require different assays to ensure adequate quantification of sesame.

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RODBARD, David. "Radioimmunoassays and 2-site immunoradiometric "sandwich" assays: Basic principles." RADIOISOTOPES 37, no. 10 (1988): 590–94. http://dx.doi.org/10.3769/radioisotopes.37.10_590.

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Ye, Zhuo, Qingwei Wang, Jiantong Qiao, Yuanyuan Xu, and Gaiping Li. "In situ synthesis of sandwich MOFs on reduced graphene oxide for electrochemical sensing of dihydroxybenzene isomers." Analyst 144, no. 6 (2019): 2120–29. http://dx.doi.org/10.1039/c8an02307g.

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Sung, Ki-Joo, Yara Jabbour Al Maalouf, Quinlan R. Johns, Eric A. Miller, and Hadley D. Sikes. "Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins." Analyst 145, no. 7 (2020): 2515–19. http://dx.doi.org/10.1039/d0an00299b.

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Börmer, Ole P. "Standardization, specificity, and diagnostic sensitivity of four immunoassays for carcinoembryonic antigen." Clinical Chemistry 37, no. 2 (February 1, 1991): 231–36. http://dx.doi.org/10.1093/clinchem/37.2.231.

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Abstract Four "sandwich"-type immunoassays for carcinoembryonic antigen (CEA) based on monoclonal antibodies (Abbott CEA-RIA Monoclonal, Pharmacia/Wallac Delfia CEA kit, Roche CEA EIA Duomab 60, and our in-house immunoradiometric assay) were compared for 357 samples from colorectal cancer patients and samples from 48 patients with chronic liver disease or with acute, irrelevant diseases. Relative to a common 5 micrograms/L reference limit, all four assays agreed regarding classification in 92% to 94% of the samples in the colorectal cancer samples, with the Abbott and Roche assays giving slightly more "normal" values. Cross-testing of CEA standards and the 1st International Reference Preparation 73/601 showed that calibration differences could not be eliminated by the use of a common standard. It has been earlier demonstrated that the monoclonal antibodies in the Roche assay have an epitope group specificity slightly different from those of the other three assays. This can explain why the correlations involving the Roche assay were weaker (r = 0.84-0.87) than those obtained with the other assays (r = 0.94-0.97). I conclude that agreement between the assays studied is as good as can be expected when different antibodies are used; nevertheless, consistent discrepancies between assays in several patients still necessitate the use of the same assay during follow-up.

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Apostolov, Eugene O., Sudhir V. Shah, Ercan Ok, and Alexei G. Basnakian. "Quantification of Carbamylated LDL in Human Sera by a New Sandwich ELISA." Clinical Chemistry 51, no. 4 (April 1, 2005): 719–28. http://dx.doi.org/10.1373/clinchem.2004.044032.

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Abstract Background: We previously suggested that increased carbamylated LDL (cLDL), a product of nonenzymatic modification of LDL in human serum by urea-derived cyanate, may cause cardiovascular complications in patients with chronic renal insufficiency. An assay for precise measurement of cLDL in serum was not previously available. Methods: Polyclonal antibodies against human cLDL and nonmodified, native LDL (nLDL) were raised in rabbits and extensively purified by affinity chromatography. New sandwich ELISAs to measure cLDL and nLDL with use of these antibodies were developed. Serum concentrations of cLDL and nLDL were measured by the sandwich ELISAs in 41 patients with end-stage renal disease (ESRD) and 40 healthy controls. Results: Both assays showed satisfactory reproducibility, linearity, and recovery. The assays could detect 2.7 mg/L cLDL with a linear detection range of 5–1000 mg/L and 5 mg/L nLDL with a linear detection range of 50–1000 mg/L. These measurements showed that patients with ESRD have significantly increased serum cLDL [281.5 (46.9) mg/L compared with 86.1 (29.7) mg/L in a control group; P <0.001]. There was no significant difference in nLDL concentrations between the groups. Conclusions: These assays are a potentially valuable tool for cardiovascular research in renal patients and healthy individuals. The cLDL concentration appears to be the highest among all previously described modified LDL isoforms in both controls and ESRD patients.

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Lee, Jae Kwon. "Development of Enzyme-Linked Immunosorbent and Immunochromatography Assays for Diagnosing Nosema ceranae Infection in Honey Bees." Insects 15, no. 1 (January 13, 2024): 59. http://dx.doi.org/10.3390/insects15010059.

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Nosema ceranae (N. ceranae) infection is prevalent globally, causing a decline in bee populations and significant economic losses to apiarists. Although several methods have been proposed for diagnosing Nosema infections, limitations in these methods have hindered their broad applications. Therefore, this current study aimed to develop a specialized method for diagnosing Nosema infections. To achieve this, a sandwich enzyme-linked immunosorbent assay (ELISA) and immunochromatography assay (ICG) were developed, and their effectiveness in screening and diagnosing Nosema infection was assessed. In sandwich ELISA, the combination of the monoclonal antibodies (mAb) 19B2 and biotinylated-19B2 exhibited stronger binding affinity to the antigen than did other combinations of mAbs that were tested. Furthermore, the antigen detection limit achieved with the sandwich ELISA surpassed that previously reported with Western blotting. The ICG was designed using the same antibody combination as that used in sandwich ELISA; however, the assay exhibited a lower diagnostic ability for Nosema infection than the ELISA. The diagnostic models developed in this study offer practical applications for conducting rapid nosemosis detection tests. These innovative techniques will help to improve the timely identification and management of nosemosis.

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Bukasov, Rostislav, Alisher Sultangaziyev, Zhanar Kunushpayeva, Alisher Rapikov, and Dina Dossym. "Aluminum Foil vs. Gold Film: Cost-Effective Substrate in Sandwich SERS Immunoassays of Biomarkers Reveals Potential for Selectivity Improvement." International Journal of Molecular Sciences 24, no. 6 (March 14, 2023): 5578. http://dx.doi.org/10.3390/ijms24065578.

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The first application of aluminum foil (Al F) as a low-cost/high-availability substrate for sandwich immunoassay using surface-enhanced Raman spectroscopy (SERS) is reported. Untreated and unmodified Al F and gold film are used as substrates for sandwich SERS immunoassay to detect tuberculosis biomarker MPT64 and human immunoglobulin (hIgG) in less than 24 h. The limits of detection (LODs) for tuberculosis (TB) biomarker MPT64 on Al foil, obtained with commercial antibodies, are about 1.8–1.9 ng/mL, which is comparable to the best LOD (2.1 ng/mL) reported in the literature for sandwich ELISA, made with fresh in-house antibodies. Not only is Al foil competitive with traditional SERS substrate gold for the sandwich SERS immunoassay in terms of LOD, which is in the range 18–30 pM or less than 1 pmol of human IgG, but it also has a large cost/availability advantage over gold film. Moreover, human IgG assays on Al foil and Si showed better selectivity (by about 30–70% on Al foil and at least eightfold on Si) and a nonspecific response to rat or rabbit IgG, in comparison to the selectivity in assays using gold film.

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Bever, Candace S., Miles Scotcher, Luisa W. Cheng, Robert M. Hnasko, and Larry H. Stanker. "Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E." Toxins 11, no. 7 (July 13, 2019): 407. http://dx.doi.org/10.3390/toxins11070407.

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Botulism is a devastating disease caused by botulinum neurotoxins (BoNTs) secreted primarily by Clostridium botulinum. Mouse bioassays without co-inoculation with antibodies are the standard method for the detection of BoNTs, but are not capable of distinguishing between the different serotypes (A–G). Most foodborne intoxications are caused by serotypes BoNT/A and BoNT/B. BoNT/E outbreaks are most often observed in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of BoNT/E3. Monoclonal antibodies (mAbs) were generated against BoNT/E3 by immunizing with recombinant peptide fragments of the light and heavy chains of BoNT/E3. In all, 12 mAbs where characterized for binding to both the recombinant peptides and holotoxin, as well as their performance in Western blots and sandwich ELISAs. The most sensitive sandwich assay, using different mAbs for capture and detection, exhibited a limit of detection of 0.2 ng/ml in standard buffer matrix and 10 ng/mL in fish product matrices. By employing two different mAbs for capture and detection, a more standardized sandwich assay was constructed. Development of sensitive and selective mAbs to BoNT/E would help in the initial screening of potential food contamination, speeding diagnosis and reducing use of laboratory animals.

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Letellier, M., A. Lévesque, F. Daigle, and A. Grant. "Performance evaluation of automated immunoassays on the Technicon Immuno 1 system." Clinical Chemistry 42, no. 10 (October 1, 1996): 1695–701. http://dx.doi.org/10.1093/clinchem/42.10.1695.

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Abstract We performed an immunoassay evaluation for various analytes on a fully automated random-access analyzer, the Technicon Immuno 1 system from Bayer Corp. This system involves latex agglutination, magnetic separation sandwich, and magnetic separation competitive immunoassay configurations. The evaluated analytes were thyrotropin (TSH), triiodothyronine, thyroxine, free thyroxine, follitropin, lutropin, prolactin, beta subunit of human chorionic gonadotropin, cortisol, ferritin, alpha-fetoprotein, carcinoembryonic antigen, and prostate-specific antigen. We tested the assay precision, linearity, and correlation with comparison methods for these analytes. The functional sensitivity of the TSH assay and the sample-to-sample carryover were also studied. Excellent results were obtained for within-run and between-day precision studies, with most assays showing within-run CVs <4% and between-day CVs <6%. The linearity for all assays was acceptable and the correlation between Immuno 1 assays and comparison methods showed satisfactory results. The functional sensitivity of the TSH assay was estimated at 0.04 mU/L. No sample-to-sample carryover was detected.

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de LUIS, R., M. D. PÉREZ, L. SÁNCHEZ, M. LAVILLA, and M. CALVO. "Development of Two Immunoassay Formats To Detect β-Lactoglobulin: Influence of Heat Treatment on β-Lactoglobulin Immunoreactivity and Assay Applicability in Processed Food." Journal of Food Protection 70, no. 7 (July 1, 2007): 1691–97. http://dx.doi.org/10.4315/0362-028x-70.7.1691.

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Milk proteins are commonly used as ingredients in the food industry because of their functional properties, but they can cause severe reactions in milk-allergic individuals. In this work, two enzyme-linked immunosorbent assay (ELISA) formats were developed to detect bovine β-lactoglobulin. The indirect competitive ELISA involved the use of anti–β-lactoglobulin antisera, and the sandwich ELISA involved the use of specific antibodies isolated using a β-lactoglobulin immunosorbent material. The effect of heat treatment on immunoreactivity of the protein in buffer and in milk was determined with both assays. The amount of immunoreactive protein in buffer and in milk decreased as determined by the sandwich ELISA, whereas the amount increased when measuring with the competitive ELISA. Several food products, including meat, bakery products, sauces, and snacks, were analyzed. With both assays, 10 of 11 products in which the ingredient list included the terms “powdered milk” or “milk proteins” contained β-lactoglobulin. However, the β-lactoglobulin concentration in these products obtained with the competitive ELISA were much higher than those obtained with the sandwich ELISA. These differences could be explained by the fact that the determination of β-lactoglobulin concentration by immunoassay is influenced by differences in antibody recognition of the protein present in highly processed foods. Therefore, the antigen-binding properties of antibodies used in a particular immunoassay are important for a correct interpretation of results obtained in food processed at high temperature.

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Newman, E. S., L. A. Moskie, R. N. Duggal, D. M. Goldenberg, and H. J. Hansen. "Murine monoclonal antibody adsorbed onto vinylidene fluoride floccules used to eliminate antibody interference in "sandwich"-type immunoassays." Clinical Chemistry 35, no. 8 (August 1, 1989): 1743–46. http://dx.doi.org/10.1093/clinchem/35.8.1743.

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Abstract We have previously reported that human anti-mouse IgG antibody (HAMA) can cause false-positive and false-negative results in "sandwich"-type monoclonal antibody (MAb) assays. To eliminate HAMA interference in "sandwich"-type MAb assays, we investigated the use of MAb on solid-phase, vinylidene fluoride floccules, which we have previously used as a solid-phase second antibody for RIA. The simple procedure effectively removes greater than 95% of HAMA from the most positive serum we have obtained from patients hyperimmunized to murine MAb, and it allows for accurate quantification of carcinoembryonic antigen. The solid-phase complex, added to blood, effectively removes HAMA and (or) "HAMA-type" heterophilic antibody from the sera or plasma.

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Vieira, Jose Gilberto H. "PTH Assays: Understanding What We Have and Forecasting What We Will Have." Journal of Osteoporosis 2012 (2012): 1–5. http://dx.doi.org/10.1155/2012/523246.

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Parathyroid hormone (PTH) assays have evolved continuously for the last 50 years. Since the first radioimmunoassay was described in 1963, several assays based on immunological identification have been published (first generation assays). The routine assays used nowadays are immunometric “sandwich-type”. They are based on two different monoclonal antibodies, one amino-terminal and the other carboxyl terminal specific. These second generation assays are widely available and adapted to most of the automation platforms. The specificity of the amino terminal antibody defines if the immunometric assay measures only the bioactive PTH circulating form (including the first amino terminal amino acids) or the “intact” PTH, which includes, besides bioactive PTH, other “long” carboxyl-terminal forms, for example, 7–84-PTH. Assays for “intact” PTH are the most commonly available and the potential advantage of the bioactive PTH assays is still debatable. Next generation of assays will be based on different principles, mainly mass spectrometry in samples submitted to a prior purification and fragmentation steps. These assays will provide information about the whole spectra of PTH peptides in circulation, with a significant increase of the information regarding this biologically important peptide hormone.

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Csako, G., B. D. Weintraub, and M. H. Zweig. "The potency of immunoglobulin G fragments for inhibition of interference caused by anti-immunoglobulin antibodies in a monoclonal immunoradiometric assay for thyrotropin." Clinical Chemistry 34, no. 7 (July 1, 1988): 1481–83. http://dx.doi.org/10.1093/clinchem/34.7.1481.

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Abstract Heterophile antibodies in patients' serum may produce false increases in apparent analyte concentrations in "sandwich"-type immunoassays. Using three patients with endogenous anti-mouse IgG antibodies and a two-site mouse monoclonal assay for thyrotropin, we studied the ability of IgG fragments to block this positive interference. Mouse whole IgG and IgG Fc fragment blocked the interference virtually completely; IgG F(ab')2 and Fab fragments did not. Rat and horse immunoglobulin fragments gave variable results. We suggest that sandwich assays formulated with IgG Fab or F(ab')2 fragments may be less susceptible to positive interference by heterophile antibodies. Unlike sera from the three patients, simulated human specimens containing heterologous anti-IgG antibodies showed little or no selectivity in inhibition by IgG fragments, and therefore are not useful to study this phenomenon.

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Brock, Kenny V., and Leon N. D. Potgieter. "Rapid Fluorescence Detection of in Situ Hybridization with Biotinylated Bovine Herpesvirus-1 DNA Probes." Journal of Veterinary Diagnostic Investigation 1, no. 1 (January 1989): 34–38. http://dx.doi.org/10.1177/104063878900100111.

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The use of biotinylated DNA hybridization probes for clinical detection of bovine herpesvirus-1 was investigated. Biotinylated DNA hybridization probes were prepared from bovine herpesvirus-1 DNA purified from infected cell cultures. The viral DNA was nick translated in the presence of biotin-dUTP with DNA polymerase to incorporate biotin into the newly synthesized strand. The probe was tested for specificity in in situ hybridization assays with bovine herpesvirus-1 DNA. Hybridization was detected using avidin-fluorescein single sandwich systems and an avidin-globulin with anti-globulin-fluorescein double sandwich system. Hybridization was detected by specific fluorescence of infected cells. Fluorescence was present only in bovine herpesvirus-1-infected cell culture and not in noninfected cell culture or cell cultures infected with several other viruses. The assay was performed in 6 hr.

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Liu, Liqiang, Dezhao Kong, Changrui Xing, Xun Zhang, Hua Kuang, and Chuanlai Xu. "Sandwich immunoassay for lactoferrin detection in milk powder." Anal. Methods 6, no. 13 (2014): 4742–45. http://dx.doi.org/10.1039/c4ay00321g.

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Börmer, O. P. "Major disagreements between immunoassays of carcinoembryonic antigen may be caused by nonspecific cross-reacting antigen 2 (NCA-2)." Clinical Chemistry 37, no. 10 (October 1, 1991): 1736–39. http://dx.doi.org/10.1093/clinchem/37.10.1736.

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Abstract Four "sandwich"-type immunoassays of carcinoembryonic antigen (CEA) based on the use of monoclonal antibodies (Abbott CEA-RIA Monoclonal, Pharmacia/Wallac Delfia CEA kit, Roche CEA EIA Duomab 60, and our in-house immunoradiometric assay) were tested for cross-reaction with nonspecific cross-reacting antigen 2 (NCA-2) in meconium. The Roche assay did not react with NCA-2, but the other three assays cross-reacted strongly and to about the same extent with this antigen. NCA-2 is the CEA gene family member that is structurally most similar to CEA, and other authors have found by indirect evidence that this antigen also is increased in the serum of many cancer patients. In a previous study we found that the Roche assay yielded lower mean values than the other assays for CEA in serum from colorectal cancer patients. Furthermore, the correlation between the Roche CEA values and those of each of the other three assays was weaker than the correlation between these NCA-2 cross-reactive assays. We conclude that these results can be explained by the different cross-reactivity with NCA-2 of the antibodies used by the various assays, as described here. Our findings raise interesting questions regarding the possible role of NCA-2 as a tumor marker.

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Ho, Man Yi, Natasha D’Souza, and Piero Migliorato. "Electrochemical Aptamer-Based Sandwich Assays for the Detection of Explosives." Analytical Chemistry 84, no. 10 (May 2012): 4245–47. http://dx.doi.org/10.1021/ac300606n.

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Edwards, Katie A., Yang Wang, and Antje J. Baeumner. "Aptamer sandwich assays: human α-thrombin detection using liposome enhancement." Analytical and Bioanalytical Chemistry 398, no. 6 (July 3, 2010): 2645–54. http://dx.doi.org/10.1007/s00216-010-3920-4.

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Chumphukam, O., T. T. Le, S. Piletsky, and A. E. G. Cass. "Generation of a pair of independently binding DNA aptamers in a single round of selection using proximity ligation." Chemical Communications 51, no. 43 (2015): 9050–53. http://dx.doi.org/10.1039/c5cc02314a.

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Arvedson, Tara L., George Doellgast, Hossein Salimi-Moosavi, Chadwick King, Ian Foltz, Ching Chen, Hongyan Li, and Barbra J. Sasu. "Development of a Sandwich ELISA to Detect Hepcidin in Human Serum." Blood 114, no. 22 (November 20, 2009): 2000. http://dx.doi.org/10.1182/blood.v114.22.2000.2000.

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Abstract Abstract 2000 Poster Board I-1022 Hepcidin is a 25 amino acid peptide that is the central mediator of iron metabolism. Iron excess, deficiency and maldistribution have been implicated in the etiology of many diseases including atherosclerosis, diabetes, neurodegeneration and the anemia of inflammation. Determination of hepcidin levels may be useful in diagnosis and treatment decisions for some or all of these diseases. Serum hepcidin measurement has so far been limited to a prohepcidin (60 amino acid hepcidin precursor) ELISA, mass spectrometry (MS)-based assays or competition ELISAs using polyclonal anti-hepcidin antibodies. The current work describes the generation of a sandwich ELISA using monoclonal antibodies to detect human hepcidin (hHepc) and optimization of assay conditions to resolve inconsistencies between MS- and ELISA-based detection. The ability of two anti-hHepc antibodies to sandwich (bind simultaneously) with hHepc was demonstrated using a rabbit polyclonal antibody preparation from hHepc-immunized animals. The same polyclonal antibody preparation was used for both hHepc capture and detection. The limit of detection achieved with this assay was O.D.450<1, suggesting that only a small proportion of the total antibodies could bind concurrently. To improve hHepc detection, a panel of monoclonal antibodies was screened for the ability to sandwich. Antibody epitope characterization studies using purified antibodies and >1000 hybridoma supernatants identified three classes of antibodies: classes 1 and 2 each recognized epitopes found in both full length mature hHepc (hHepc 25) and a shorter version (hHepc 20); class 3 bound a different epitope and demonstrated an increased affinity for hHepc 25 over hHepc 20. The majority of antibodies characterized were in class 1 while antibodies in classes 2 and 3 were rare (∼1% of antibody panel) highlighting the difficulty in achieving a sandwiching event. Antibodies 19D12 (class 1) and 23F11 (class 2) were identified as the optimal sandwich pair with a detection range of approximately 0.2-1000 ng/ml using synthetic hHepc. Initial comparisons of data generated using the sandwich ELISA and a fully-quantitative MS-based assay demonstrated a lack of consistent agreement. This issue was somewhat addressed by introduction of an alkaline treatment step to dissociate any protein/hHepc complexes in serum. Subsequent comparison of the two assays using sera from several different patient populations (anemia of cancer, chemotherapy-induced anemia, kidney disease) as well as healthy donors demonstrated good correlation (R2 range = 0.83-0.92; n=237). This sandwich ELISA may represent a tool for aligning the MS and ELISA-generated results in a format that has the potential to be high throughput and widely available. Disclosures: Arvedson: Amgen: Employment. Doellgast:Amgen: Employment. Salimi-Moosavi:Amgen: Employment. King:Amgen: Employment. Foltz:Amgen: Employment. Chen:Amgen: Employment. Li:Amgen: Employment. Sasu:Amgen: Employment.

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Li, Juanjuan, Xiaochun Hu, Shuo Shi, Yiwei Zhang, and Tianming Yao. "Three label-free thrombin aptasensors based on aptamers and [Ru(bpy)2(o-mopip)]2+." Journal of Materials Chemistry B 4, no. 7 (2016): 1361–67. http://dx.doi.org/10.1039/c5tb02032h.

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NYQUIST-BATTIE, CYNTHIA, LAURA E. FRANK, DEANNA LUND, and DANIEL V. LIM. "Optimization of a Fluorescence Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Escherichia coli O157:H7 in Apple Juice." Journal of Food Protection 67, no. 12 (December 1, 2004): 2756–59. http://dx.doi.org/10.4315/0362-028x-67.12.2756.

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Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 104 to 106 CFU/ml, whereas that for spiked phosphate-buffered saline was 106 to 108 CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.

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Iverson, A. J., A. Bianchi, A. C. Nordlund, and L. A. Witters. "Immunological analysis of acetyl-CoA carboxylase mass, tissue distribution and subunit composition." Biochemical Journal 269, no. 2 (July 15, 1990): 365–71. http://dx.doi.org/10.1042/bj2690365.

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Changes in the mass and subunit structure of liver acetyl-CoA carboxylase (ACC) accompany altered nutrition in vivo. Enzyme activity in different tissues and cell lines is also, in part, determined by variations in both total mass and ACC isoenzyme composition. ACC isoenzyme mass and hetero/homo-isoenzyme association were quantified by three sandwich e.l.i.s.a. assays, i.e. an avidin-based assay that measured total isoenzyme mass and two antibody-sandwich assays which measure polypeptide association. Results from the avidin-based assay reveal that the two major isoenzymes, of molecular mass 265 kDa (ACC 265) and 280 kDa (ACC 280), are present in markedly variable concentration in several rat and mouse tissues and in cell lines of rat and mouse origin. Hepatic ACC mass has been reported to be distributed between mitochondrial and cytosolic fractions and to undergo only a change in subcellular distribution without alteration in total mass on induction/repression of activity in vivo [Roman-Lopez, Shriver, Joseph & Alfred (1989) Biochem. J. 260, 927-930]. However, in the present study, immunoblotting and e.l.i.s.a. analysis reveals that, in rat liver, the mass of both isoenzymes is predominantly cytosolic in distribution, is markedly diminished on fasting and rises 6-8-fold on refeeding of a high-carbohydrate diet. These data support the results of several other investigations of hepatic ACC mass, and are consistent with known nutritionally altered changes in ACC mRNA content. By the two antibody-sandwich e.l.i.s.a. assays, isoenzyme complexes either composed of both ACC 280 and 265 or with multiple copies of ACC 265 are detectable in rat liver enzyme; their concentration varies independently of total ACC mass with the nutritional state of the rat, being lowest in fasting and highest on fasting/refeeding. E.l.i.s.a. analysis, applicable to crude tissue/cell extracts, provides a simple, sensitive and quantitative measurement of ACC mass and subunit composition. Its use may permit needed quantitative insight into the role of variable total ACC and isoenzyme mass and of alterations in ACC subunit composition that occur in vivo or in isolated cells in response to a variety of hormonal and nutritional influences.

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Hansen, H. J., G. LaFontaine, E. S. Newman, M. K. Schwartz, A. Malkin, K. Mojzisik, E. W. Martin, and D. M. Goldenberg. "Solving the problem of antibody interference in commercial "sandwich"-type immunoassays of carcinoembryonic antigen." Clinical Chemistry 35, no. 1 (January 1, 1989): 146–51. http://dx.doi.org/10.1093/clinchem/35.1.146.

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Abstract We evaluated the effect of human anti-murine antibodies (HAMA) on apparent concentrations of carcinoembryonic antigen (CEA) as measured in serum with commercial enzyme immunoassay (EIA) kits manufactured by Abbot ("two-step" double monoclonal antibody assay), Roche, and Hybritech (room-temperature protocol). In sera from patients given parenteral murine monoclonal antibody for experimental diagnostic and immunotherapy studies, HAMA titers were determined with Immunomedics' "ImmuSTRIP HAMA-EIA" kit reagents. "True" CEA titers were established by using the ImmuCEA/MA-EIA and heat-extraction to destroy HAMA before assay for CEA. The concordance of the ImmuCEA/MA assay with the Abbott and Roche CEA EIAs was established with sera from normal individuals and from patients who had not received parenteral injections of murine monoclonal antibody. At high (100 mg/L) concentrations of HAMA, false-positive results were observed with all three kits. The Hybritech and Roche assays were more sensitive to interference by HAMA than was the Abbott CEA-EIA, false-positive results being observed at HAMA concentrations between 1 and 10 mg/L. Similar sensitivity of the three kits to interference by primate anti-MAb sera was demonstrated. Use of primate anti-MAb sera to create controls with HAMA activity and of analyte is recommended to evaluate MAb assays for potential HAMA interference and for use to devise methods to eliminate HAMA interference.

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Zhang, Zhigang, Tianying Zhang, Lu Cao, Xin Wang, Jiali Cao, Xiaofen Huang, Yashuang Cai, et al. "Simultaneous in situ visualization and quantitation of dual antigens adsorbed on adjuvants using high content analysis." Nanomedicine 14, no. 19 (October 2019): 2535–48. http://dx.doi.org/10.2217/nnm-2019-0016.

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Aim: Traditional antigenicity assay requires antigen recovery from the particulate adjuvants prior to analysis. An in situ method was developed for interrogating vaccine antigens with monoclonal antibodies while being adsorbed on adjuvants. Materials & methods: The fluorescence imaging-based high content analysis was used to visualize the antigen distribution on adjuvant agglomerates and to analyze the antigenicity for adsorbed antigens. Results: Simultaneous visualization and quantitation were achieved for dual antigens in a bivalent human papillomavirus vaccine with uniquely labeled antibodies. Good agreement was observed between the in situ multiplexed assays with well-established sandwich enzyme-linked immunosorbent assays. Conclusion: The streamlined procedures and the amenability for multiplexing make the in situ antigenicity analysis a favorable choice for in vitro functional assessment of bionanoparticles as vaccine antigens.

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Nicholson, S., M. Fox, A. Epenetos, and G. Rustin. "Immunoglobulin Ihhibiting Reagent®: Evaluation of a New Method for Eliminating Spurious Elevations in CA125 Caused by HAMA." International Journal of Biological Markers 11, no. 1 (January 1996): 46–49. http://dx.doi.org/10.1177/172460089601100109.

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Cancer therapy utilising radiolabelled murine monoclonal antibodies frequently leads to the production of Human Anti-Mouse Antibodies (HAMA) in the recipient. HAMA interferes with “sandwich” immunoassays, such as those for tumour markers, rendering results unreliable. Published methods for eliminating HAMA from serum are not suitable for use in a laboratory which is processing a large number of assays using an automated system. We report on the use of Immunoglobulin Inhibiting Reagent (IIR) in CA125 assays from recipients of intraperitoneal radioimmunotherapy who had spuriously elevated results due to HAMA. IIR was found to be comparable to the admixture of mouse serum as a way of eliminating the effect of HAMA. IIR is ideally suited to use with an automated assay process.

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Tolnai, Zoltán János, Judit András, Zsuzsanna Szeitner, Krisztina Percze, László Ferenc Simon, Róbert E. Gyurcsányi, and Tamás Mészáros. "Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection." International Journal of Molecular Sciences 21, no. 14 (July 14, 2020): 4963. http://dx.doi.org/10.3390/ijms21144963.

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Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

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Speirs, Joan I., and Mumtaz Akhtar. "Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays." Canadian Journal of Microbiology 37, no. 8 (August 1, 1991): 650–53. http://dx.doi.org/10.1139/m91-110.

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Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin. Key words: Escherichia coli, cytotoxin, ELISA.

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STALS, AMBROOS, MIEKE UYTTENDAELE, LEEN BAERT, and ELS VAN COILLIE. "Norovirus Transfer between Foods and Food Contact Materials." Journal of Food Protection 76, no. 7 (July 1, 2013): 1202–9. http://dx.doi.org/10.4315/0362-028x.jfp-12-392.

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Human infective noroviruses (NoVs) are a worldwide leading cause of foodborne illness and are frequently spread via infected food handlers preparing and manipulating food products such as deli sandwiches. The objective of the current study was to determine the efficiencies whereby NoV could be transferred between surfaces associated with the preparation of manually prepared foods such as deli sandwiches. Nonfood surfaces included gloves and stainless steel discs, and boiled ham, lettuce, and a sandwich bun were the ingredients of the deli sandwich. Both NoV GII.4 and the murine NoV 1 (MNV-1, a cultivable human NoV surrogate) were included in the presented study. Transfer of NoV GII.4 and MNV-1 between surfaces was performed by pressing an inoculated donor surface against an acceptor surface. To evaluate the effect of subsequent contact, donor surfaces were pressed a second time to an identical acceptor surface. Subsequently, NoV GII.4 and MNV-1 were detected using real-time reverse transcription PCR assays and plaque assays, respectively. Transfer of both viruses from gloves to stainless steel was inefficient, and virus transfer from food products to stainless steel occurred with more variability for NoV GII.4 than for MNV-1. Virus transfer from the stainless steel discs to the gloves was substantially more efficient than from the gloves to the stainless steel. NoV GII.4 and MNV-1 transfer from food products to the gloves occurred with varying efficiencies, although this variation was more evident for NoV GII.4. The MNV-1 inoculum was significantly less efficiently transferred to the acceptor surface at the second contact, which was not the case for NoV GII.4. The obtained transfer efficiency data may provide insights into the transfer of NoV during preparation of foods and can be included in risk assessment models describing the transmission of NoVs in this context.

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Cheng, Xiao-Dong, Liu-Wei Song, Lin-Lin Fang, Lin Yang, Yong Wu, Sheng-Xiang Ge, Quan Yuan, Jun Zhang, Ning-Shao Xia, and Xiao-Ke Hao. "Comparison of Three Luminescent Immunoassays for Hepatitis B Virus Surface Antigen Quantification during the Natural History of Chronic Hepatitis B Virus Infection." Clinical and Vaccine Immunology 21, no. 11 (September 10, 2014): 1521–27. http://dx.doi.org/10.1128/cvi.00529-14.

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Abstract:

ABSTRACTHepatitis B surface antigen (HBsAg) quantification has garnered attention because of its high predictive value in determining treatment responses. The HBsAg quantification assays, such as Architect and Elecsys, are commercially available, and more assays are in development. We aimed to compare the results of the Architect and Elecsys assays with those of a new assay, WTultra. The WTultra HBsAg assay is a sandwich chemiluminescent microplate enzyme immunoassay and provides an alternative choice which is more cost-effective and potentially applicable in developing or resource-constrained countries and areas. A total of 411 serum samples were collected from patients during various phases of chronic hepatitis B (CHB) infection. The samples were assessed using the three assays, and the results were compared and analyzed. The results for the Architect, Elecsys, and WTultra assays were well correlated according to the overall results for the samples (correlation coefficients,rArchitect versus WTultra= 0.936,rArchitect versus Elecsys= 0.952, andrWTultra versus Elecsys= 0.981) and the various infection phases (rArchitect versus WTultraranging from 0.67 to 0.975,rArchitect versus Elecsysranging from 0.695 to 0.982, andrWTultra versus Elecsysranging from 0.877 to 0.99). Additionally, consistent results were observed according to genotype (genotype B:rArchitect versus WTultra= 0.976,rArchitect versus Elecsys= 0.978, andrWTultra versus Elecsys= 0.979; genotype C:rArchitect versus WTultra= 0.950,rArchitect versus Elecsys= 0.963, andrWTultra versus Elecsys= 0.981) and hepatitis B virus (HBV) DNA levels (rArchitect= 0.540,rWTultra= 0.553, andrElecsys= 0.580). In conclusion, the Elecsys and WTultra assays were well correlated with the Architect assay, irrespective of the CHB infection phase or genotype. All of these assays are reliable for HBsAg quantification.

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