Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA)

Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA)

Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and...

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Bibliographic Details
Published in:Journal of immunological methods Vol. 406; pp. 34 - 42
Main Authors: Flanagan, J.J., Arjomandi, A., Delanoy, M.L., Du Paty, E., Galea, P., Laune, D., Rieunier, F., Walker, R.P., Binder, S.R.
Format: Journal Article
Language:English
Published: Netherlands Elsevier B.V 01-04-2014
Subjects:
Amino Acid Sequence
Animals
Antibodies, Monoclonal - immunology
Autoimmune
Enzyme-Linked Immunosorbent Assay
Epitope Mapping
Haptoglobins - analysis
Haptoglobins - immunology
Humans
Intestinal permeability
Mice
Molecular Sequence Data
Monoclonal antibodies
Pre-haptoglobin-2
Zonulin
Monoclonal antibodies
Zonulin
Autoimmune
Enzyme-linked immunosorbent assay
Pre-haptoglobin-2
Intestinal permeability
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Summary:Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1ng/mL to 200ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2ng/mL (95% CI: 106.5–335.9ng/mL) and a median value of 23.9ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum.
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ISSN:0022-1759
1872-7905
DOI:10.1016/j.jim.2014.02.009