Monoclonal Antibodies Against Toluene Diisocyanate Haptenated Proteins from Vapor-Exposed Mice

Ruwona, Tinashe B.; Johnson, Victor J.; Schmechel, Detlef; Simoyi, Reuben H.; Beezhold, Donald H.; Siegel, Paul D.

doi:10.1089/hyb.2009.0110

Cited by 13 publications

(8 citation statements)

References 24 publications

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“…For Western blot analysis, samples were electrophoresed under reducing conditions on precast 4–15% gradient gels and a nitrocellulose trans-blot system from BioRad (Hercules, CA). Nitrocellulose membranes were blocked with 3% dry milk in PBS, probed with 1 μg/mL of the anti-TDI mAb 79G7 (33), followed by anti-mouse IgG1 from Pharmingen (San Diego, CA), and developed with electrochemical luminescence reagent from Thermo Fisher Scientific (Rochester, NY).…”

Section: Methodsmentioning

confidence: 99%

Toluene Diisocyanate Reactivity with Glutathione Across a Vapor/Liquid Interface and Subsequent Transcarbamoylation of Human Albumin

Wisnewski

Hettick

Siegel

2011

Chem. Res. Toxicol.

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Glutathione has previously been identified as a reaction target for toluene diisocyanate (TDI) in vitro and in vivo, and has been suggested to contribute to toxic and allergic reactions to exposure. In this study, the reactivity of reduced glutathione (GSH) with TDI in vitro was further investigated using a mixed phase (vapor/liquid) exposure system to model the in vivo biophysics of exposure in the lower respiratory tract. HPLC/MS/MS was used to characterize the observed reaction products. Under the conditions tested, the major reaction products between TDI vapor and GSH were S-linked bis(GSH)-TDI and to a lesser extent mono(GSH)-TDI conjugates (with one N=C=O hydrolyzed). The vapor phase generated GSH-TDI conjugates were capable of transcarbamoylating human albumin in a pH-dependent manner, resulting in changes in the self-protein’s conformation/charge, based on electrophoretic mobility under native conditions. Specific sites of human albumin-TDI conjugation, mediated by GSH-TDI, were identified (Lys73, Lys159, Lys190, Lys199, Lys212, Lys351, Lys136/137, Lys413/414, Lys524/525) and overlap with those susceptible to direct conjugation by TDI. Together, the data extend proof-of-principle for GSH to act as a “shuttle” for a reactive form of TDI, which could contribute to clinical responses to exposure.

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Section: Methodsmentioning

confidence: 99%

Toluene Diisocyanate Reactivity with Glutathione Across a Vapor/Liquid Interface and Subsequent Transcarbamoylation of Human Albumin

Wisnewski

Hettick

Siegel

2011

Chem. Res. Toxicol.

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“…The ELISA format employed in the current study used an anti TDI–protein IgG that was produced in our lab against TDI-KLH 17 as the primary antibody and an alkaline phosphatase-labeled anti-IgG as the detection antibody. Both TDI-HSA and TDI-Hb reacted to the anti-TDI-protein monoclonal antibody, indicating that TDI conjugated Hb can be antigenic.…”

Section: Discussionmentioning

confidence: 99%

“…Although most reported cases of isocyanate sensitization occur at workplaces 13,14 , it has been suggested that non-occupational exposure to the general public may also occur through use of “do-it-yourself” free diisocyanate containing commercial products such as polyurethane foams and sprays 15,16 . Once allergic sensitization to isocyanates occurs, asthmatic reactions may be triggered by exceedingly minute concentrations of isocyanates 5,17,18 .…”

Section: Introductionmentioning

confidence: 99%

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin

Mhike

Hettick

Chipinda

et al. 2016

Journal of Immunological Methods

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Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25 % of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30 % that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity.

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“…We have reported 29 TDI-HSA specific IgG isotype mAbs (17) as well as 10 TDI-protein adduct specific IgM mAbs. (16) Recently, Wisnewski and Liu have produced 6 MDI-protein adduct specific IgG 1 isotype mAbs. (18) pAbs have also been produced for 1,6-HDI (15,30) and TDI.…”

Section: Discussionmentioning

confidence: 99%

“…(15) Ruwona et al have developed murine IgM and IgG mAbs with unique specificity for TDI-HSA and other protein adducts. (16,17) More recently, six IgG 1 mAbs with specific reactivity for MDI-protein adducts have been reported by Wisnewski and Liu. (18) Although these antibodies have provided potential new tools for the isolation and identification of TDI and MDI target proteins, to our knowledge there are no mAbs that react with either HDI or a combination of other occupationally relevant dNCOs.…”

Section: Introductionmentioning

confidence: 98%

A Murine Monoclonal Antibody with Broad Specificity for Occupationally Relevant Diisocyanates

Lemons¹,

Siegel²,

Mhike³

et al. 2013

Journal of Occupational and Environmental Hygiene

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Diisocyanates (dNCOs) used in industrial applications are well known low molecular weight allergens. Occupational exposure is associated with adverse health outcomes including allergic sensitization and occupational asthma. In this study, we report the production and initial characterization of a dNCO-hapten specific murine IgM monoclonal antibody (mAb). Female BALB/c mice were immunized intraperitoneally with 25 µg of 4,4′-methylene diphenyl diisocyanate (MDI)-keyhole limpet hemocyanin. Following six biweekly booster immunizations, splenocytes were recovered and fused to Sp2/0-Ag14 murine myeloma cell line for hybridoma production. Hybridomas were then screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against 40:1 4,4′-MDI– human serum albumin (HSA). mAb reactivity to dNCO-HSA conjugates and dNCO-HSA spiked human serum were characterized using a sandwich ELISA. One hybridoma produced a multimeric IgM mAb (15D4) that reacted with 4,4′-MDI-HSA. Sandwich ELISA analysis demonstrated comparable reactivity with other occupationally relevant dNCO-HSA adducts, including 2,4-toluene diisocyanate (TDI)-HSA, 2,6-TDI-HSA, and 1,6-hexamethylene diisocyanate (HDI)-HSA, but not other electrophilic chemical HSA conjugates. The limit of quantification (LOQ) of 4,4′-MDI-HSA, 2,4-TDI-HSA, 2,6-TDI-HSA, and 1,6-HDI-HSA sandwich ELISAs were 567.2, 172.7, 184.2, and 403.5 ng/mL (8.67, 2.60, 2.77, and 6.07 pmol/mL), respectively. In contrast, experiments using dNCO-supplemented human sera showed an increase in the detectable limit of the assay. A mAb has been produced that has potential utility for detecting mixed diisocyanate exposures in occupational environments. The mAb may have additional utility in the standardization of specific IgE detection immunoassays as well as chromatographic-mass spectrometric methods to enrich dNCO adducted HSA in the plasma of occupationally exposed workers.

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Monoclonal Antibodies Against Toluene Diisocyanate Haptenated Proteins from Vapor-Exposed Mice

Cited by 13 publications

References 24 publications

Toluene Diisocyanate Reactivity with Glutathione Across a Vapor/Liquid Interface and Subsequent Transcarbamoylation of Human Albumin

Toluene Diisocyanate Reactivity with Glutathione Across a Vapor/Liquid Interface and Subsequent Transcarbamoylation of Human Albumin

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin

A Murine Monoclonal Antibody with Broad Specificity for Occupationally Relevant Diisocyanates

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