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Development of a microcolumn one-site immunometric assay for a protein biomarker: Analysis of alpha₁-acid glycoprotein.

ABSTRACT: A one-site immunometric assay based on affinity microcolumns was developed for the analysis of alpha₁-acid glycoprotein (AGP) as a model protein biomarker. In this assay, a sample containing AGP was incubated with an excess amount of a labeled binding agent, such as fluorescein-labeled anti-AGP antibodies or F_ab fragments. The excess binding agent was removed by passing this mixture through a microcolumn that contained an immobilized form of AGP, while the signal was measured for the binding agent-AGP complex in the non-retained fraction. Theoretical and practical factors were both considered in selecting the concentration of labeled binding agent, the incubation time of this agent with the sample, and the application conditions for this mixture onto the microcolumn. The effects of using various labeling methods and intact antibodies vs F_ab fragments were also considered. The final assay was performed with fluorescein-labeled anti-AGP antibodies and a 2.1?mm i.d.?×?1.0?cm AGP microcolumn operated at 0.30?mL min^-1. This method required only 1 µL of serum or plasma, had a detection limit of 0.63?nM AGP, and gave a potential throughput of 2?min per sample. This assay was used to measure AGP in normal serum and plasma from patients with systemic lupus erythematosus, giving good agreement with the literature and a reference method. The same approach and guidelines can be used to create assays for other protein biomarkers by changing the labeled binding agent and immobilized protein within the microcolumn.

SUBMITTER: Zhang C

PROVIDER: S-EPMC6980775 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Development of a microcolumn one-site immunometric assay for a protein biomarker: Analysis of alpha1-acid glycoprotein.

Zhang Chenhua C Lott Shae S Clarke William W Hage David S DS

Journal of chromatography. A 20190920

A one-site immunometric assay based on affinity microcolumns was developed for the analysis of alpha1-acid glycoprotein (AGP) as a model protein biomarker. In this assay, a sample containing AGP was incubated with an excess amount of a labeled binding agent, such as fluorescein-labeled anti-AGP antibodies or Fab fragments. The excess binding agent was removed by passing this mixture through a microcolumn that contained an immobilized form of AGP, while the signal was measur ...[more]

PMID: 31564560

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Project description:Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases, and some changes appear to be disease-specific; thus, it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers, and enable better understanding of AGP's role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96-well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography-based solid-phase extraction and analyzed by reversed-phase-liquid chromatography-electrospray ionization-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in three time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP's second glycosylation site and lower sialylation of N-glycans on AGP's third and AGP1's fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.

Project description:Background:Inflammation is a key component of immune response to infections and pathogenesis of metabolic and cardiovascular diseases. Inflammatory biomarkers, including alpha-1-acid glycoprotein (AGP), are considered prognostic tools for predicting risk, monitoring response to therapy, and adjusting nutritional biomarkers for accurate interpretation. Serum is considered a primary source of biomarkers; urine and saliva are increasingly being explored and utilized as rapidly accessible, noninvasive biofluids requiring minimal sample processing and posing fewer biohazard risks. Methods:A lateral flow immunoassay was developed for an established mobile-based platform to quantify AGP in human serum, urine, and saliva. Assay performance was assessed with purified AGP in buffer, diluted human serum samples (n = 16) banked from a trial in people living with HIV, and saliva and urine (n = 15 each) from healthy participants. Reference methods were conventional clinical chemistry analyzer or commercial ELISA. Bootstrap analysis was used to train and validate sample calibration. Findings:The correlation between the assay and reference method for serum was 0.97 (P < 0.001). Mean (95% CI) best fit line slope was 1.0 (0.88, 1.15) and intercept was -0.003 (-0.08, 0.09). The correlation for urine was 0.93, and for saliva was 0.97 (both P < 0.001). The median CV for the LFIA for AGP in buffer was 13.2% and for all samples was 28.7%. Interpretation:The performance of the assay indicated potential use as a rapid, low sample volume input, and easy method to quantify AGP that can be licensed and adopted by commercial manufacturers for regulatory approvals and production. This has future applications for determining inflammatory status either alone or in conjunction with other inflammatory proteins such as C-reactive protein for prognostic, monitoring, or nutritional status applications, including large-scale country level surveys conducted by the DHS and those recommended by the WHO.

Project description:This study aimed to determine the association between umbilical cord leucine-rich alpha-2 glycoprotein (LRG) and fetal infection and investigate the underlying mechanism of LRG elevation in fetuses. We retrospectively reviewed the medical records of patients who delivered at Osaka University Hospital between 2012 and 2017 and selected those with histologically confirmed chorioamnionitis (CAM), which is a common pregnancy complication that may cause neonatal infection. The participants were divided into two groups: CAM with fetal infection (CAM-f[+] group, n = 14) and CAM without fetal infection (CAM-f[-] group, n = 31). Fetal infection was defined by the histological evidence of funisitis. We also selected 50 cases without clinical signs of CAM to serve as the control. LRG concentrations in sera obtained from the umbilical cord were unaffected by gestational age at delivery, neonatal birth weight, nor the presence of noninfectious obstetric complications (all, p > 0.05). Meanwhile, the LRG levels (median, Interquartile range [IQR]) were significantly higher in the CAM-f(+) group (10.37 [5.21-13.7] μg/ml) than in the CAM-f(-) (3.61 [2.71-4.65] μg/ml) or control group (3.39 [2.81-3.93] μg/ml; p < 0.01). The area under the receiver operating characteristic (ROC) curve of LRG for recognizing fetal infection was 0.92 (optimal cutoff, 5.08 μg/ml; sensitivity, 86%; specificity, 88%). In a mouse CAM model established by lipopolysaccharide administration, the fetal LRG protein in sera and LRG mRNA in the liver were significantly higher than those in phosphate-buffered saline (PBS)-administered control mice (p < 0.01). In vitro experiments using a fetal liver-derived cell line (WRL68) showed that the expression of LRG mRNA was significantly increased after interleukin (IL)-6, IL-1β, and tumor necrosis factor- alpha (TNF-α) stimulation (p < 0.01); the induction was considerably stronger following IL-6 and TNF-α stimulation (p < 0.01). In conclusion, LRG is an effective biomarker of fetal infection, and fetal hepatocytes stimulated with inflammatory cytokines may be the primary source of LRG production in utero.

Dataset Information

Development of a microcolumn one-site immunometric assay for a protein biomarker: Analysis of alpha₁-acid glycoprotein.

Publications

Development of a microcolumn one-site immunometric assay for a protein biomarker: Analysis of alpha<sub>1</sub>-acid glycoprotein.

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