Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme
- Anthony DelleDonne†1,
- Naomi Kouri†1,
- Lael Reinstatler1,
- Tomoko Sahara1,
- Lilin Li2,
- Ji Zhao2,
- Dennis W Dickson1,
- Nilufer Ertekin-Taner1, 2, 3 and
- Malcolm A Leissring1, 2Email author
© DelleDonne et al; licensee BioMed Central Ltd. 2009
Received: 2 September 2009
Accepted: 16 October 2009
Published: 16 October 2009
Insulin-degrading enzyme (IDE) is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD) and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs) targeting natively folded human and rodent IDE.
Eight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular—designated 6A1 and 6H9—proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts.
We succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.
Insulin-degrading enzyme (IDE; EC 22.214.171.124; a.k.a. insulysin, insulinase, insulin protease) is an atypical zinc-metalloprotease that hydrolyzes several biomedically important intermediate-sized peptide substrates, including insulin, insulin-like growth factor-2, glucagon, amylin, and the amyloid β-protein [1–3]. IDE is implicated in the pathogenesis of Alzheimer disease (AD) [4, 5] and type-2 diabetes mellitus [6–8], and has also been identified as the cellular receptor for varicella zoster virus infection and cell-to-cell spread .
Despite the clear biomedical significance of this protease, many fundamental questions about the basic biology of IDE remain unresolved, due in part to a lack of sufficiently selective reagents targeting this ubiquitous protease. In particular, the precise subcellular localization of IDE remains poorly defined. Although IDE is well-established to reside in cytosol  and mitochondria , reports of IDE's localization to other pathophysiologically important subcellular compartments—such as endosomes —have not been confirmed by microscopic analysis of intact cells with well-characterized anti-IDE antibodies. Moreover, the mechanisms underlying the export of IDE from the cell are completely unknown, though it has recently been demonstrated that they involve an unconventional, non-classical secretion pathway . Methods capable of detecting and quantifying secreted forms of IDE would greatly facilitate the elucidation of this important pathway. Finally, it will be important to detect genetically or environmentally induced variations in IDE protein levels, which will require the development of assays permitting accurate quantification of IDE levels in relevant tissues.
To help close these gaps in our understanding of the biology of IDE, we developed eight novel mouse mAbs that detect rodent and/or human IDE in diverse applications in a highly selective and species-specific manner. Notably, a subset of these mAbs were particularly well suited for detecting endogenous IDE by immunocytochemistry and immunohistochemistry. We also describe the development of sensitive and quantitative sandwich ELISAs capable of detecting variations in IDE levels in human brain extracts. Collectively, these novel anti-IDE mAbs, and the ELISA incorporating them, constitute important new tools for investigating both the basic biology of IDE and its potential derangement in disease.
Detailed methods for all experimental procedures are provided in the Additional File 1.
Generation of Monoclonal Hybridomas Expressing Anti-IDE mAbs
Properties of anti-IDE monoclonal antibodies
ELISA half-titer (ng/mL):
Western Blotting and Immunoprecipitation
With the exception of 4H5, human IDE was successfully immunoprecipitated by all anti-IDE mAbs, albeit the efficiency of 4C5 was less than the other mAbs (Fig. 1C). By contrast, rodent IDE was successfully immunoprecipitated by only a single mAb, 6A1 (Fig. 1C).
To evaluate the specificity of our mAbs for detecting human but not rodent IDE by immunocytochemistry, we analyzed Chinese hamster ovary (CHO) cells transiently transfected with a vector encoding human IDE. Among the antibodies tested, superior results were obtained with 6H9, which was found to intensely stain cells expressing human IDE, while showing no background staining in neighboring, nontransfected cells expressing rodent IDE (Fig. 1C). These results are consistent with the western blotting and immunoprecipitation results obtained for this mAb (Fig 1). 6H9 also readily detected endogenous levels of human IDE, as revealed by prominent staining present in unmodified HeLa cells (Fig. 1D).
Immunofluorescence and Immunohistochemistry
IDE Sandwich ELISAs
To validate the ability of this ELISA to detect variations in IDE levels from human brain samples, we quantified IDE levels in cerebellar extracts from a large set of autopsied AD brains both by western blot analysis with 2A1 (see Fig. 4B) and by 6H9/6A1 sandwich ELISA. For both methods, absolute IDE levels were determined by calibration to internal recombinant human IDE standards. After appropriate quality control measures and normalization to internal control samples (see Additional File 1), IDE levels within a total of 49 human cerebellar samples were successfully measured by both methodologies (Fig. 4C). Despite the different set of antibodies employed and potential batch effects, we observed a highly significant correlation between results obtained with the two methodologies (p < 0.0001, r2 = 0.3). Overall, the 6H9/6A1 ELISA consistently detected less IDE protein (7 ± 1%, mean ± SD) relative to that detected by western blotting (see Discussion). Using a subset of brain samples as a reference, qualitatively and quantitatively similar results were also obtained using ELISAs configured with multiple different anti-IDE mAbs combinations (not shown).
In the present study, we succeeded in developing 8 monoclonal hybridoma lines in house that express a versatile set of anti-IDE mAbs. Two mAbs in particular—6A1 and 6H9—were found to be useful in a wide array of applications, detecting human and rodent IDE in a highly selective and species-specific manner. 6A1 was found to detect both human and rodent IDE by western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. 6H9, by contrast, detected human but not rodent IDE, as determined by the same methods.
We also developed sandwich ELISAs capable of detecting human IDE in brain extracts and validated the ELISA results with quantitative western blot analysis. There was a significant correlation between IDE levels detected by western blotting with 2A1 and with the 6H9/6A1 sandwich ELISA. Despite the strong correlation between the two methods, the absolute amounts of IDE detected by ELISA were consistently lower than those detected by western blotting. This disparity may be attributable technical considerations, such as the particular protein extraction conditions used in this study (see Additional File 1), which could have denatured IDE sufficiently to affect its detection by ELISA. Future studies comparing the outcome obtained under different extraction conditions should resolve this question. On the other hand, several intriguing biological explanations also exist. For example, endogenous IDE might normally be complexed to other proteins, or may contain post-translational modifications, either or both of which could sterically block or remove the epitopes recognized by the antibodies used for ELISAs. Alternatively, or in addition, it may be that a substantial portion of IDE present in post-mortem extracts is itself not natively folded or is modified in other ways.
Although outside the scope of this methodology paper, it is notable that there was substantial variation in absolute IDE levels (~10-fold) detected by both ELISA and Western blotting in the large set of AD cerebellar samples we examined. In the future, it will be important to evaluate whether these changes correlate with genetic or other risk factors for AD. Further insight into these and many other important questions will be facilitated by the development of this well-characterized and versatile set of anti-IDE mAbs.
enzyme-linked immunosorbent assay
hypoxanthine, aminopterin, and thymidine
horse radish peroxidase
insulin-degrading enzyme knockout
The authors thank Dennis Selkoe (Harvard Medical School) for the αIDE-1 antibody and Mses. Qun Lu and Christelle Cabrol for superb technical assistance. Supported by National Institutes of Health Grants AG025070 and DA024888 to MAL and CTSA KL2 RR024151 award to NET. We thank the patients and their families for their participation in our research studies. We gratefully acknowledge the Mayo Clinic Alzheimer's Disease Research Center for its support.
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