Elsevier

Immunology Letters

Volume 70, Issue 3, 1 December 2000, Pages 143-149
Immunology Letters

Reactivity and assay restriction profiles of monoclonal and polyclonal antibodies to acid phosphatases: a preliminary study

https://doi.org/10.1016/S0165-2478(99)00154-6Get rights and content

Abstract

The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5C1 demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5C1 and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5C1 and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.

Introduction

Monoclonal reagents enable standardisation of both reagent and assay technique because they are highly specific and can be generated in unlimited quantities in vitro [1]. However, broad and rigorous characterisation is paramount prior to the application of MAbs for clinical diagnostic or therapeutic purposes. Key issues to be resolved include the potential problem of cross-reactivity and the phenomenon of assay restriction: an antibody being positive in one assay system but negative, or reacting poorly, to the same antigen in another [1], [2]. Thus there is a requirement to ascertain the reactivity profiles of novel antibodies in different assay systems.

Human acid phosphatases (APs) are a group of six isoenzymes previously identified by electrophoresis [3]. The type 5 isoenzyme differs from other APs in being insensitive to inhibition by l(+)-tartrate [4], hence the term tartrate-resistant acid phosphatase (TRAP). TRAP is a mannose-rich metallo-glycoprotein [5] synthesised by the differentiated cells of the monohistiocytic system. It is found in leukocytes, alveolar and monocyte-derived macrophages, placental cells, and in small amounts in erythrocytes, being localised either at the cell membrane or intracellular organelles [6], [7], [8], [9], [10]. The archetypal source of TRAP is the osteoclast, from whose ruffled plasmalemma border it is released in a hormone regulated manner [11] in relation to normal bone-remodelling and calcium homeostasis activity [12]. However, the identity and biological function of its natural substrate is unknown.

Abnormally high serum levels of TRAP are found in Gaucher’s Disease [13], hairy cell leukaemia [14], [15], [16], Paget’s disease, bone malignancy and osteoporosis. In the latter three, accelerated osteoclast activity, associated with increased breakdown of the bone matrix [17], [18], is thought to account for the high levels. Osteoclast-derived TRAP (OC-TRAP) has therefore been proposed as a marker for osteoclast-associated bone resorption, and for osteoclasts themselves, which could be utilised in diagnostic assays and in assays to monitor the effectiveness of therapy [19], [20].

Enzymological methods for detecting serum OC-TRAP are often unreliable due to interference from other serum enzymes and inhibitors, or lack of appropriate sensitivity. Immunoassays using MAbs, or polyclonal reagents, raised to TRAP are the obvious alternative approach [8], [21], [22], [23], [24]. However, to date the range and availability of suitable TRAP-specific reagents in the development of a secure diagnostic systems is limited. In this study, a panel of antibodies, raised to osteoclastoma and APs, were evaluated in enzyme-linked immunoassay (ELISA) against purified recombinant TRAP and native APs, and in immunocytochemistry using tissue samples and cell lines. Furthermore, their binding patterns were compared to the cytochemical localisation of APs. We conclude that the reagents investigated react to TRAP, or histological TRAP locations, although the binding varies in intensity and is governed by assay type. Cross-reactivity patterns suggest that a number of these reagents may bind to conformational epitopes shared by other APs.

Section snippets

Antigens

Recombinant TRAP (rTRAP) type 5 was supplied by SmithKline Beecham (Harlow, UK) [18], [25]. Human prostatic acid phosphatase type 2a (P-AP), bovine milk acid phosphatase type 4 (B-AP); potato acid phosphatase type 2 (Pt-AP), bovine serum albumin (BSA) were supplied by Sigma Chemicals (Poole, UK).

Antibodies

The production of MAbs G211D, G312G [24], 2H1, 4E6, 5C1 (anti-rTRAP) [18], and PAb 8023 (raised using a synthetic peptide of TRAP) [23] have previously been described. Hybridoma V35B was generated by

ELISA and immunocytochemistry

MAb G211D reacted weakly to rTRAP in ELISA (Table 1), but exhibited intense cytoplasmic staining of osteoclasts (Fig. 1) and cuboidal epithelial cells lining the collecting tubules of the GPK. In both cell types, notable staining occurred at the cytoplasm/membrane interface. MAb G211D bound intensely to cytoplasmic determinants of activated, and unactivated U937 cells, forming an apparent crescent-shape and granular pattern corresponding in location to Golgi apparatus and to vesicles,

Discussion

As preliminary work for the development of a standardised immunoassay for TRAP system, the present study characterised a panel of antibody reagents with putative specificity for APs. Evidently the availability of a standardised system would find useful applications in clinical diagnosis and in monitoring therapeutic regimens and exercise-induced bone resorption [26]. In considering the possibility of assay restriction, these antibodies were assessed for reactivity in ELISA, and against

References (43)

  • P.N. Nelson et al.

    Immunol. Methods

    (1991)
  • K.H.W. Lau et al.

    Int. J. Biochem.

    (1992)
  • K.H.W. Lau et al.

    Int J. Biochem.

    (1991)
  • A.R. Hayman et al.

    Biol. Chem.

    (1994)
  • P. Ling et al.

    J. Biol. Chem.

    (1993)
  • T. Klabunde et al.

    FEBS Lett.

    (1995)
  • C.M. Ketcham et al.

    J. Biol. Chem.

    (1989)
  • H.M. Blottiere et al.

    Biomaterials

    (1995)
  • D.P.C. Rice et al.

    Bone

    (1997)
  • S.M. Wilhelm et al.

    J. Biol. Chem.

    (1989)
  • R.C. Williams et al.

    Mol. Immunol.

    (1997)
  • P.N. Nelson et al.

    Immunol. Invest.

    (1994)
  • W.K.W. Lam et al.

    Clin. Chem.

    (1978)
  • P.T.K. Saunders et al.

    J. Biol. Chem.

    (1985)
  • D.W. Moss et al.

    Crit. Rev. Clin. Lab. Sci.

    (1995)
  • H.G. Drexler et al.

    Leukemia

    (1994)
  • M.E. Kraenzlin et al.

    Clin. Endocrinol. Metab.

    (1990)
  • O. Fukushima et al.

    Clin. Endocrinol. Med.

    (1991)
  • A.J. Janckila et al.

    Histochemistry

    (1996)
  • G. Vaes

    Clin. Orthop.

    (1998)
  • C. Minkin

    Calcif. Tissue Int.

    (1982)
  • Cited by (0)

    1

    Deceased

    View full text