title: Immunoassay of serine-phosphorylated isoform of insulin-like growth factor (IGF) binding protein (IGFBP)-1.
abstract: OBJECTIVE
  Development of an ELISA for phosphorylated isoform of IGFBP-1. Serine phosphorylation is an important regulator of IGFBP-1 bioactivity, but specific immunoassays for its measurement are currently lacking.
  
  
  METHODS
  Assay design was based on a novel approach of first capturing the phosphorylated and non-phosphorylated IGFBP-1 by an anti-IGFBP-1 antibody and then selectively detecting the phosphorylated form by an anti-phosphoserine antibody. Method development involved pair-wise evaluation of the candidate antibodies and determinations of analytical performance and specificity. Specificity was monitored by reactivity with dephosphorylated IGFBP-1, with antibodies against other phosphorylated residues that are not expressed, and by comparative analysis of sample containing different IGFBP-1 phosphorylation profile.
  
  
  RESULTS
  Analytical evaluation demonstrated acceptable performance; detection limit 0.3 microg/L, dynamic range 1.56-100 microg/L; intra- and inter-assay CVs 2.1-8.6%; mean recovery (+/-SD) 97.8+/-9.2%, and mean recovery of sample dilution 93.4+/-6.0%. The phosphorylated and total IGFBP-1 medians in non-pregnant adult serum, which mostly contain the highly phosphorylated isoform, were 11.9 and 18.6 microg/L, respectively, and the sample values were tightly correlated (r=0.99). As expected, the corresponding medians in 1st trimester (17.4 and 63.0 microg/L) and 2nd trimester (30.9 and 75.8) samples with altered IGFBP-1 phosphorylation were significantly different (p<0.001). Similarly, a fraction (1.29%) of total IGFBP-1 (13.3 mg/L) in amniotic fluids was found to be phosphorylated (0.172 mg/L). There was no reactivity with dephosphorylated IGFBP-1.
  
  
  CONCLUSIONS
  The present ELISA is highly specific for the phosphorylated isoform of IGFBP-1 and its availability should help expedite further investigations of IGFBP-1 phosphorylation.
authors.affiliation: Diagnostic Systems Laboratories (Canada) Inc., Toronto, Ontario, Canada M5G 1L7. jkhosravi@101college.net
authors.forename: Javad
authors.initials: J
authors.lastname: Khosravi
authors.forename: Radha G
authors.initials: RG
authors.lastname: Krishna
authors.forename: Umesh
authors.initials: U
authors.lastname: Bodani
authors.forename: Anastasia
authors.initials: A
authors.lastname: Diamandi
authors.forename: Najmuddin
authors.initials: N
authors.lastname: Khaja
authors.forename: Bhanu
authors.initials: B
authors.lastname: Kalra
authors.forename: Ajay
authors.initials: A
authors.lastname: Kumar
mesh_terms: D000293:Adolescent; D000328:Adult; D000653:Amniotic Fluid / Q000737:chemistry; D000906:Antibodies / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000097:blood*; D008875:Middle Aged; D010768:Phosphoserine / Q000097:blood*; D011247:Pregnancy; D020033:Protein Isoforms / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D018970:Insulin-Like Growth Factor Binding Protein 1; D020033:Protein Isoforms; D010768:Phosphoserine