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Human fetal intestinal alkaline phosphatase: molecular heterogeneity and immunological detection in amniotic fluids
G F Verpooten1, M F Hoylaerts, E J Nouwen
1Department of Nephrology-Hypertension, University of Antwerp, Belgium.
Insights
Human fetal intestinal alkaline phosphatase (fIALP) exists in amniotic fluid in dimeric and tetrameric forms. Measuring fIALP antigen levels offers a more sensitive detection method than enzyme activity assays, particularly for cystic fibrosis.
Area of Science:
- Biochemistry
- Molecular Biology
- Clinical Chemistry
Background:
- Human fetal intestinal alkaline phosphatase (fIALP) is found in amniotic fluid.
- fIALP exists as free dimers or membrane-bound tetrameric forms.
- Both forms of fIALP are sialylated to varying degrees.
Purpose of the Study:
- To investigate the forms and levels of fIALP in amniotic fluid.
- To compare antigen detection versus enzyme activity measurements for fIALP.
- To assess the utility of fIALP measurements in prenatal diagnostics.
Main Methods:
- Extraction of particulate material with Triton X-100 to isolate tetrameric fIALP.
- Analysis of fIALP using double sandwich-ELISA for antigen levels.
- Comparison with enzyme antigen immunoassay for fIALP enzyme activity.
Main Results:
- Double sandwich-ELISA detected up to 10-fold higher fIALP antigen levels than enzyme activity assays.
- fIALP antigen levels showed a more symmetrical distribution and less overlap with bulk values.
- fIALP antigen measurements reduced false-negativity in cystic fibrosis cases.
Conclusions:
- fIALP antigen quantification is a more sensitive method than enzyme activity measurement.
- This improved sensitivity has implications for prenatal screening, especially for cystic fibrosis.
- Further research is needed to determine fIALP's predictive value in trisomy pregnancies.
Abstract:
Human fetal intestinal alkaline phosphatase (fIALP) is present in amniotic fluids as free dimers (Mr 140,000) or membrane-bound through phosphatidylinositol residues. Extraction of corresponding particulate material with Triton X-100, resulted in release of tetrameric high Mr fIALP forms (Mr 380,000). In individual amniotic fluids, as well as in meconeum, both dimeric and tetrameric fIALP are sialylated to various extents. When measured by a double sandwich-ELISA, up to 10-fold higher fIALP antigen levels were found in amniotic fluids than when determined by an enzyme antigen immunoassay, based upon fIALP enzyme activity measurements. Frequency analysis of fIALP antigen levels, showed a more symmetrical distribution than analysis of fIALP enzyme activities; likewise, the lower 95% confidence limit, calculated for the fIALP antigen distribution curve, overlapped less with the bulk of values. In cystic fibrosis amniotic fluids, measurements of fIALP antigen levels resulted in a lower false-negativity outcome than fIALP enzyme activity measurements, whereas in amniotic fluids of trisomy pregnancies fIALP enzyme activities and fIALP antigen levels were equally unpredictive.