Monoclonal antibodies against tissue-nonspecific alkaline phosphatase. Report of the ISOBM TD9 workshop.
Nineteen monoclonal antibodies (MAbs) against tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (TNALP) were investigated in the ISOBM TD-9 Workshop. These MAbs were generated with antigens obtained from human bone tissue (n = 9), human osteosarcoma cell lines (SaOS-2 and TPX; n = 7) and human liver tissue (n = 3). The evaluation included the following antigen forms: (a) commercially available preparations of human bone ALP (BALP) and liver ALP (LALP); (b) human BALP isoforms, B/I, B1 and B2; and (c) soluble secreted epitope-tagged recombinant human TNALP (setTNALP) expressed in COS-1, osteosarcoma (SaOS-2) and hepatoma (Huh2) cell lines. In addition, 16 TNALP mutant cDNAs corresponding to a wide spectrum of reported hypophosphatasia mutations were used in an attempt to map specific immunoreactive epitopes on the surface of the TNALP molecule. The TD-9 MAbs were evaluated by immunoradiometric (IRMA) assays, cross-inhibition and different enzyme immunoassay designs. No indications of explicit tissue discriminatory immunoreactivities of the investigated MAbs against TNALP were found. However, certain IRMA combinations of MAbs increased the specificity of BALP measurements. All MAbs bound to the three BALP isoforms B/I, B1 and B2, but none of the investigated MAbs were specific for any of the isoforms. Significant differences were, however, found in immunoreactivity between these isoforms, with cross-reactivities ranging from 21 to 109% between the two major BALP isoforms B1 and B2. Desialylation with neuraminidase significantly increased the MAb affinity for the BALP isoforms B/I, B1 and B2, and also decreased the observed differences in cross-reactivity between these isoforms. We suggest, therefore, that the MAb affinity is dependent on the amount/number of terminal sialic acid residues located at the five putative N-glycosylation sites. Based on the overall results, we present a putative three-dimensional model of the TNALP molecule with positioning of the four major antigenic domains (designated A-D) of the investigated MAbs. The TNALP molecule is depicted as a homodimer, hence most, but not necessarily all, epitopes are displayed twice. The antigenic domains were positioned with the following assumptions: domain A was positioned close to the active site since most of these MAbs interfered with the catalytic activity. Interestingly, both MAbs included in the commercial BALP kits were grouped with domain A. Moreover, 4 of the 5 putative N-glycosylation sites (with terminal sialic acid residues) are located within, or with close proximity to, domain A. Domain B was localized at the top flexible loop (crown domain) of the TNALP molecule. Domain C was clearly defined by the IRMA assay combinations and by site-directed mutants of TNALP to be close to residue E281, which is located near the fourth metal binding site, likely to be occupied by a calcium ion. Domain D was positioned close to residues A115, A162 and E174, but this domain was also close to the GPI anchor site. In conclusion, none of the 19 investigated TD-9 MAbs were entirely specific for BALP or LALP, thus indicating that all MAbs bind mainly to epitopes on the common protein core of BALP and LALP and/or common glycosylated epitopes. However, some MAbs (either single or in combination with other MAbs) work sufficiently well to measure BALP when the assayed samples do not contain elevated levels of LALP.
Magnusson
P
P
Bone and Mineral Metabolic Unit, Division of Clinical Chemistry, Department of Biomedicine and Surgery, Linköping University Hospital, Linköping, Sweden. Per.Magnusson@lio.se
Arlestig
L
L
Paus
E
E
Di Mauro
S
S
Testa
M P
MP
Stigbrand
T
T
Farley
J R
JR
Nustad
K
K
Millán
J L
JL
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism; D001842:Bone and Bones / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004493:Education; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D007118:Immunoassay; D007074:Immunoglobulin G / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000276:immunology; D008958:Models, Molecular; D009439:Neuraminidase / Q000494:pharmacology; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000941:Antigens; D018076:DNA, Complementary; D000939:Epitopes; D007074:Immunoglobulin G; D020033:Protein Isoforms; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D009439:Neuraminidase