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Immunoassay of serine-phosphorylated isoform of insulin-like growth factor (IGF) binding protein (IGFBP)-1. | LitMetric

AI Article Synopsis

  • The study aimed to develop a specific ELISA for detecting the phosphorylated form of IGFBP-1, a protein that plays a key role in regulating biological activity through serine phosphorylation.
  • The assay utilized a two-step method where an anti-IGFBP-1 antibody captured both phosphorylated and non-phosphorylated IGFBP-1, followed by detection with an anti-phosphoserine antibody, ensuring specificity in measuring the phosphorylated isoform.
  • Results showed that the new assay has strong analytical performance, with a low detection limit, good recovery rates, and significant correlations between phosphorylated and total IGFBP-1 levels in various serum and amniotic fluid samples, supporting its use in further research.

Article Abstract

Objectives: 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.

Design And 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.

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Source
http://dx.doi.org/10.1016/j.clinbiochem.2006.07.004DOI Listing

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