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Characterization of New Polyclonal Antibodies Specific for 40 and 42 Amino Acid-Long Amyloid β Peptides: Their Use to Examine the Cell Biology of Presenilins and the Immunohistochemistry of Sporadic Alzheimer’s Disease and Cerebral Amyloid Angiopathy Cases

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Abstract

Background

In Alzheimer’s disease (AD), the main histological lesion is a proteinaceous deposit, the senile plaque, which is mainly composed of a peptide called Aβ. The aggregation process is thought to occur through enhanced concentration of Aβ40 or increased production of the more readily aggregating 42 amino acid-long Aβ42 species.

Materials and Methods

Specificity of the antibodies was assessed by dot blot, Western blot, ELISA, and immunoprecipitation procedures on synthetic and endogenous Aβ produced by secreted HK293 cells. Aβ and p3 production by wild-type and mutated presenilin 1-ex-pressing cells transiently transfected with βAPP751 was monitored after metabolic labeling and immunoprecipitation procedures. Immunohistochemical analysis was performed on brains of sporadic and typical cerebrovascular amyloid angiopathy (CAA) cases.

Results

Dot and Western blot analyses indicate that IgG-purified fractions of antisera recognize native and denaturated Aβs. FCA3340 and FCA3542 display full specificity for Aβ40 and Aβ42, respectively. Antibodies immunoprecipitate their respective synthetic Aβ species but also Aβs and their related p3 counterparts endog-enously secreted by transfected human kidney 293 cells. This allowed us to show that mutations on presenilin 1 triggered similar increased ratios of Aβ42 and its p342 counterpart over total Aβ and p3. ELISA assays allow detection of about 25–50 pg/ml of Aβs and remain linear up to 750 to 1500 pg/ml without any cross-reactivity. FCA18 and FCA3542 label diffuse and mature plaques of a sporadic AD case whereas FCA3340 only reveals the mature lesions and particularly labels their central dense core. In a CAA case, FCA18 and FCA3340 reveal leptomeningeal and cortical arterioles whereas FCA3542 only faintly labels such structures.

Conclusions

Polyclonal antibodies exclusively recognizing Aβ40 (FCA3340) or Aβ42 (FCA3542) were obtained. These demonstrated that FAD-linked presenilins similarly affect both p342 and Aβ42, suggesting that these mutations misroute the βAPP to a compartment where γ-secretase, but not α-secretase, cleavages are modified. Overall, these antibodies should prove useful for fundamental and diagnostic approaches, as suggested by their usefulness for biochemical, cell biological, and immunohistochemical techniques.

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Acknowledgments

We wish to acknowledge Drs. Thinakaran and Sisodia (John Hopkins Institute, Baltimore) for providing us with the ΔE9-PS1 cDNA. We sincerely thank Drs. A. Cupo and C. Cucumel for their advice on antigen coupling. We thank J. D. Bardes for animal care and blood sampling, F. Aguila for artwork, and J. Kervella for secretarial assistance. This work was supported by the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, and by a grant from the Fondation de France.

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Authors and Affiliations

  1. IPMC du CNRS, UPR411, 660 route des Lucioles, 06560, Valbonne, France

    Hélène Barelli, Anthony Lebeau, Nathalie Chevallier, Philippe Marambaud, Karine Ancolio &  Frédéric Checler

  2. URA CNRS 1845, Montpellier, France

    Jean Vizzavona & Jean Martinez

  3. Rhône-Poulenc Rorer, Vitry Alfortville, France

    Pia Delaere & Cyril Drouot

  4. The Rockefeller University, New York, New York, USA

    Joseph D. Buxbaum

  5. Hoechst-Marion-Roussel, Sommerville, New Jersey, USA

    Olga Khorkova, Jeff Heroux & Sudhir Sahasrabudhe

  6. Service des Maladies du Système Nerveux et du Muscle, Hopitaux Universitaires, Strasbourg, France

    Jean-Marie Warter

  7. Institut de Pathologie, Strasbourg, France

    Michel Mohr

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  1. Hélène Barelli
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Correspondence to Frédéric Checler.

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Barelli, H., Lebeau, A., Vizzavona, J. et al. Characterization of New Polyclonal Antibodies Specific for 40 and 42 Amino Acid-Long Amyloid β Peptides: Their Use to Examine the Cell Biology of Presenilins and the Immunohistochemistry of Sporadic Alzheimer’s Disease and Cerebral Amyloid Angiopathy Cases. Mol Med 3, 695–707 (1997). https://doi.org/10.1007/BF03401708

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