Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays

Richard Bryant, B. N. Chamovitz, S. A. Morse, M. A. Apicella, V. H. Morthland

Research output: Contribution to journalArticle

Abstract

The specificity of the enzyme-linked immunosorbent assay(s) is thought to depend on the specificity of the antibody used in the assay system. Therefore, the association of broadly reactive antigens like endotoxin with enzyme conjugates or other enzyme-linked immunosorbent assay reagents has the potential of altering the specificity of reactions in the enzyme-linked immunosorbent assay. Using the Limulus amoebocyte lysate assay, we demonstrated that commercially prepared conjugates of goat anti-human immunoglobulin G peroxidase, goat anti-rabbit immunoglobulin G alkaline phosphatase, rabbit anti-human immunoglobulin G, and other enzyme conjugates contained endotoxin. Furthermore, the staphylococcal protein A, horseradish peroxidase, and bovine alkaline phosphatase used to prepare enzyme conjugates also contained endotoxin. Commercially obtained bovine alkaline phosphatase contained as much as 1.0 μg of endotoxin per ml of enzyme solution. Both commercially prepared enzyme conjugates and those prepared by us contained endotoxin as determined by their absorption to immobilized monoclonal antibody to lipid A or to immobilized Limulus amoebocyte lysate. The results of this study further suggest that the endotoxin was associated with the enzyme component of the conjugate. In a competitive inhibition enzyme immunoassay, 10 μg of lipid A per ml inhibited binding of the enzyme conjugate to absorbed Limulus amoebocyte lysate, thereby confirming that endotoxin mediated the binding of the conjugate in that system. The potential significance of endotoxin bound to enzyme conjugates may be far reaching because of the ubiquity of endotoxin in conjugates and the prevalence of antibodies to endotoxin in mammalian serum.

Original languageEnglish (US)
Pages (from-to)1050-1053
Number of pages4
JournalJournal of Clinical Microbiology
Volume17
Issue number6
StatePublished - 1983

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Endotoxins
Enzyme-Linked Immunosorbent Assay
Enzymes
Horseshoe Crabs
Alkaline Phosphatase
Lipid A
Immunoglobulin G
Goats
Immobilized Antibodies
Rabbits
Antibody Specificity
Staphylococcal Protein A
Horseradish Peroxidase
Immunoenzyme Techniques
Peroxidase
Monoclonal Antibodies
Antigens
Antibodies
Serum

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

Bryant, R., Chamovitz, B. N., Morse, S. A., Apicella, M. A., & Morthland, V. H. (1983). Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays. Journal of Clinical Microbiology, 17(6), 1050-1053.

Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays. / Bryant, Richard; Chamovitz, B. N.; Morse, S. A.; Apicella, M. A.; Morthland, V. H.

In: Journal of Clinical Microbiology, Vol. 17, No. 6, 1983, p. 1050-1053.

Research output: Contribution to journalArticle

Bryant, R, Chamovitz, BN, Morse, SA, Apicella, MA & Morthland, VH 1983, 'Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays', Journal of Clinical Microbiology, vol. 17, no. 6, pp. 1050-1053.
Bryant R, Chamovitz BN, Morse SA, Apicella MA, Morthland VH. Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays. Journal of Clinical Microbiology. 1983;17(6):1050-1053.
Bryant, Richard ; Chamovitz, B. N. ; Morse, S. A. ; Apicella, M. A. ; Morthland, V. H. / Endotoxin contamination of enzyme conjugates used in enzyme-linked immunosorbent assays. In: Journal of Clinical Microbiology. 1983 ; Vol. 17, No. 6. pp. 1050-1053.
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