Comparative analyses of methods used to prepare diisocyanate conjugates; implications in clinical assay development

Details

• Personal Author:
  Bledsoe TA ; Hettick JM ; Kashon ML ; Law, Brandon F. ; Lemons, Angela R. ; Siegel, Paul D. ; Wagner LM ; Wisnewski AV
• Description:
  Exposure to diisocyanates (dNCOs), such as methylene diphenyldiisocyanate (MDI) can cause occupational asthma. Recently we observed differences in MDIspecific mAb reactivity with MDI-protein conjugates prepared using different methods. The consistent preparation of dNCO-protein adducts is crucial for dNCO- specific antibody screening. Therefore, the aim of this study was to identify the dNCO-protein conjugation method that resulted in the most extensive and consistent conjugation. Four methods were used: 1) MDI was slowly dripped into human serum albumin (HSA) while vortexing (drip), 2) MDI was quickly dispensed into HSA and vortexed (fast dispense), 3) MDI was pumped into HSA while vortexing (infusion), and 4) MDI was dissolved in an non-water miscible solvent forming 2-phases and was stirred overnight (2-phase). On average the infusion method resulted in approximately 1.5, 3.7, and 3.9 times more reactivity in a MDI specific sandwich ELISA than the drip, fast dispense, or 2-phase methods, respectively. Interestingly, the amount of crosslinking, as measured by the binding of free amines by trinitrobenzene sulfonic acid, was similar amongst samples prepared using all of the described methods. Intrapersonal variability was examined by instructing 3 individuals to prepare conjugates using either the drip or fast dispense methods. A high variability in protein conjugation was observed between users, particularly in the drip method. This may correlate to the drip speed of the MDI and vortex speed of the HSA. This work demonstrates intra- and interpersonnel variability in non-automated methods for conjugate preparation and suggests utility of infusion pump for removal of that variability. [Description provided by NIOSH]
• Subjects:
  Acids Amines Antibodies Chemistry Clinical Laboratory Techniques Isocyanates Laboratories Occupational Health Safety Solvents Toxicology

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• Keywords:
  Analytical-models Antibody-response Chemical-binding Chemical-reactions Clinical-techniques Laboratory-techniques Medical-screening Nanotechnology Performance-capability Proteins Sample-preparation Serological-techniques

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• ISSN:
  1096-6080
• Document Type:
  Text
• Genre:
  Abstract or Summary
• Place as Subject:
  California ; Connecticut ; OSHA Region 1 ; OSHA Region 3 ; OSHA Region 9 ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  HELD - Health Effects Laboratory Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  144
• Issue:
  1
• NIOSHTIC Number:
  nn:20045913
• Citation:
  Toxicologist 2015 Mar; 144(1):262
• CAS Registry Number:
  Diphenylmethane diisocyanate (CAS RN 101-68-8) ; Trinitrobenzenesulfonic acid (CAS RN 2508-19-2)
• Federal Fiscal Year:
  2015
• NORA Priority Area:
  Manufacturing
• Peer Reviewed:
  False
• Source Full Name:
  The Toxicologist. Society of Toxicology 54th Annual Meeting and ToxExpo, March 22-26, 2015, San Diego, California
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:d562ddeff5a06ecc6083cd214a1575c976330fd20f8d2afdee999864279e64379c2f5d3406bae4091c64bded017930108cc3ce3d67a9dd98be77fe55798c2937
• Download URL:
  https://stacks.cdc.gov/view/cdc/201152/cdc_201152_DS1.pdf
• File Type:
  [PDF - 229.07 KB ]