Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts

Lemons, Angela R.; Bledsoe, Toni A.; Siegel, Paul D.; Beezhold, Donald H.; Green, Brett J.

doi:10.1016/j.jim.2013.08.015

Cited by 7 publications

(6 citation statements)

References 9 publications

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“…However, Wisnewski and coworkers [19] have identified 12 MDI lysine peptide conjugation sites with IgG on human albumin which when utilized as a biomarker of exposure could increase specificity by not relying on the purity of the HSA fraction. Recent advances in the development of diisocyanate-specific monoclonal antibodies have provided a new approach to detecting diisocyanate adducts [20] , [21] , [22] . The advantages of measuring protein adduct biomarkers are specificity for at least one diisocyanate adduct, a slow turnover (i.e., half-life of HSA is approximately 20 days) and therefore represents a viable potential tool for monitoring long-term exposure [23] , [24] .…”

Section: Introductionmentioning

confidence: 99%

Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

et al. 2014

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4,4′-Methylene diphenyl diisocyanate (herein 4,4′-MDI) is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4′-MDI-Lys) adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) quantitation method via a signature peptide approach to enable biomonitoring of 4,4′-MDI adducted to human serum albumin (HSA) in plasma. A murine, anti-4,4′-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction) 4,4′-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF) system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM) mode was utilized for quantification of the adducted signature peptide biomarker both for in chemico and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4′-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4′-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4′-MDI-Lys adducts. Results indicated no positive results were obtained above the quantification limit by the signature peptide approach. If the 414 site of lysine adduction accounted for 100% of the 4,4′-MDI adductions in the signature peptide adduct approach, the three highest quantifiable samples by the 4,4′-MDI-Lys method should have at least been detectable by the signature peptide method. Results show that although the 4,4′-MDI signature peptide approach is more selective, it is 18 times less sensitive than the 4,4′-MDI-Lys method, thus limiting the ability to detect adduct levels relative to the 4,4′-MDI-Lys amino acid method.

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Section: Introductionmentioning

confidence: 99%

Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

et al. 2014

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“…These findings suggest that for clinical assay development, particularly for sandwich ELISA development, it is imperative to characterize the immunoreactivity of the standard antigens with the mAbs to ensure the highest level of sensitivity is achieved. (10–12, 22) …”

Section: Discussionmentioning

confidence: 99%

“…(1–7) Measuring the levels of the MDI byproduct, methylene dianiline (MDA), in hydrolyzed urine or blood is another method employed to monitor workers for exposure to MDI, however, this method does not reveal sensitization status, and is rather a reflection of exposure (8, 9) . Furthermore, sandwich ELISAs have been developed and proposed as an additional biomonitoring method (10) . These could be used to either measure MDI-conjugated proteins within blood or urine as an indicator of exposure, or to measure MDI-specific antibodies as an indicator of sensitization.…”

Section: Introductionmentioning

confidence: 99%

The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition

Hagerman

Law

Bledsoe

et al. 2016

Journal of Occupational and Environmental Hygiene

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Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences.

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“…A certain number of analytical techniques are nowadays applied in the proteic adduct analysis (26,50), as chemical and enzymatic digestions, electrophoresis or capillary liquid chromatography with different detection techniques, such as mass spectrometry and fluorescence, or immunochemical approaches, as enzymelinked immunosorbent assay (ELISA). The advancement in the instrumental performances allows high sensitivity levels, from 0.1 to 500 fmol of proteic adduct; nevertheless, there are no reports concerning the costs and time of the unavoidable purification procedure of the biological sample and the selective enrichment of the macromolecular adduct under study with respect to the unmodified proteic fraction.…”

Section: Discussionmentioning

confidence: 99%

New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I

Pieri¹

2014

PER

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Background: hemoglobin adducts are considered suitable biomarkers to evaluate human exposure to electrophile agents. One of the procedures used for the molecular dosimetry of hemoglobin adducts is the enzymatic digestion of the protein and the subsequent quantification of modified peptides by mass spectrometry. The aim of the study is to evaluate the ability of Calpain to discriminate between normal and alkylated globin chains. Methods: digestions of pure hemoglobin sample and of an alkylated one with Calpain I at 200:1 and 25:1 weight ratios, for 1, 5 and 18 hrs were carried out. Only the standard deviations (SD) of the ratios between alkylated globins with respect to the unmodified ones (αECH/α and bECH/b) were estimated because the aim of the study is to assess the ability of Calpain I to selectively digest the samples and, above all, to find the optimal digestion conditions and characterize obtained peptides. Results: by using a 200:1 weight ratio only peptides α(1-137), α(1-138) and b(1-142) were produced, pointing out that the first proteolytic sites are located at the C-terminus ends of both α-and b-chains. The identification of peptides obtained from the 25:1 digestions allowed the determination of internal secondary proteolytic sites, not reported in literature. In both cases, a non-negligible proteolysis was observed only when a reaction time of 18 hrs was used. Discussion: results showed a progressive enrichment of modified hemoglobin with respect to the unmodified one, particularly for the α-chain and represent a promising initial step in the development of a selective purification procedure to be applied to protein adducts analysis.

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Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts

Cited by 7 publications

References 9 publications

Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition

New perspectives in hemoglobin adducts analysis: selective digestion with Calpain I

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