The validation of immunoblot SDS-PAGE as a qualitative and quantitative method for the determination of urinary Cystatin C in neonates.
Immunoblot SDS-PAGE
Method validation
Neonates
Qualitative and quantitative analysis
Reading of densitometry
Urinary cystatin C
Journal
Clinical biochemistry
ISSN: 1873-2933
Titre abrégé: Clin Biochem
Pays: United States
ID NLM: 0133660
Informations de publication
Date de publication:
Jan 2021
Jan 2021
Historique:
received:
26
06
2020
revised:
07
10
2020
accepted:
08
10
2020
pubmed:
18
10
2020
medline:
25
5
2021
entrez:
17
10
2020
Statut:
ppublish
Résumé
The quantitative determination of urinary Cystatin C (cyst-C) associated with the qualitative analysis of its polymorphisms is an excellent method for early identification of newborns predisposed to renal function impairment. PETIA, PENIA and EIA are the immunometric methods used for the quantitative determination of cyst-C in human biologic fluid but they have limitations and do not allow qualitative analysis. The present study is a validation of Immunoblot SDS-PAGE for the qualitative and quantitative analysis of urinary cyst-C. Urine was collected from neonates in the nursey at S. Maria della Misericordia Hospital. Urinary cyst-C was investigated by the immunoblot SDS-PAGE and by reading of optical density. The qualitative analysis showed two different molecular forms: a reactivity at about 70 KDa in all samples and a reactivity at 13 KDa in a limited number of samples. This analysis allows the correlation of the polymorphisms of cyst-C with specific alterations of renal function in newborns. The quantitative analysis is specific, sensitive and accurate. In fact the coefficient of variation for assay precision was 10% and for assay accuracy was ±10%, the detection limit was 0.009 ng/ µL and the calibration line has satisfactory linearity (range 0.02-0.3 ng/ µL). The stability of urinary cyst-C was acceptable, even without the use of protease inhibitors, as long as the assay was performed on freshly recruited urine or immediately after thawing the samples, which had been stored for up to six months. Immunoblot SDS-PAGE analysis is a valid method of obtaining a qualitative and quantitative analysis of urinary cyst-C. This method presents unique information about a previously unknown 70 KDa cyst-C form. The assay may offer potential diagnostic information not available with immunometric method.
Identifiants
pubmed: 33068572
pii: S0009-9120(20)30865-1
doi: 10.1016/j.clinbiochem.2020.10.005
pii:
doi:
Substances chimiques
Biomarkers
0
CST3 protein, human
0
Cystatin C
0
Types de publication
Journal Article
Validation Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
52-59Informations de copyright
Copyright © 2020 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.