Development of a new, more sensitive immunoassay for human tryptase: Use in systemic anaphylaxis

Abstract

Tryptase, a neutral protease, is selectively concentrated in the secretory granules of human mast cells, and its release into the circulation serves as a clinical marker of mast cell activation. The current study describes a new, more sensitive ELISA utilizing a newly developed, mouse monoclonal IgG1 antibody for capture called B12 and capable of detecting tryptase in normal plasma and serum. The greater sensitivity of the new immunoassay results in part from a greater portion of tryptase being detected. Mean levels of tryptase in serum from normal subjects from Richmond, Virginia (4.9 ng/ml; n = 56), Munich, Germany (3.8 ng/ml; n = 19), and Amersfoort, The Netherlands (1.9 ng/ml; n = 8) were as indicated. In 62 subjects with ongoing allergic rhinitis, tryptase levels were no different in serum than for 19 normal controls, indicating that local mast cell activation is not necessarily reflected in the circulation. In 61 subjects sensitive to honey bee or yellow jacket venom by history, the 17 destined to have a severe, hypotensive response to a sting challenge had higher levels of tryptase at baseline than mild reactors, nonreactors, and controls, suggesting that baseline levels of tryptase may predict the severity of the clinical response to allergen in sensitive subjects.

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“…The plasma tryptase levels in our patient, although elevated, are not as high as reported values in bee sting anaphylaxis [16]. The lower values in this case can be explained on two counts.…”

Tryptase Precursors Are Preferentially and Spontaneously Released, Whereas Mature Tryptase Is Retained by HMC-1 Cells, Mono-Mac-6 Cells, and Human Skin-Derived Mast Cells

“…Purified preparations of tryptase were stored at Ϫ70°C in 20% (v/v) glycerol, 0.01 M MES (pH 6.5), 0.8 M NaCl. IgG1 mAb B12, which recognizes a conformational epitope present on tryptase, was prepared as before (14,25). mAb B12 Affi-Gel was prepared by coupling B12 with activated Affi-Gel (Bio-Rad Laboratories, Hercules, CA).…”

“…In such cases, the magnitude of mast cell degranulation appears to be the primary determinant of clinical severity (11)(12)(13). In contrast, based on mAbs that recognize both ␣-protryptase and ␤-tryptase, a total tryptase immunoassay was developed that detected levels of tryptase in baseline serum from essentially all individuals (mean Ϯ SD, 4.9 Ϯ 2.3 ng/ml) (14). So-called total tryptase levels are elevated in subjects with systemic mastocytosis and reflect the total body burden of mast cells (10).…”

Utility of the World Heath Organization classification criteria for the diagnosis of systemic mastocytosis in bone marrow

“…Total serum tryptase was assessed at a reference laboratory using a fluorenzyme immunoassay. 6 This test measures both forms of tryptase, a and b, and our normal reference range is 2-10 ng/ml.…”