Serum Alpha 1-acid Glycoprotein Research Articles

Alpha-1-Acid Glycoprotein Quantification via Spatial Proximity Analyte Reagent Capture Luminescence Assay: Application as Diagnostic and Prognostic Marker in Serum and Effusions of Cats with Feline Infectious Peritonitis Undergoing GS-441524 Therapy

Until recently, the diagnosis of feline infectious peritonitis (FIP) in cats usually led to euthanasia, but recent research has revealed that antiviral drugs, including the nucleoside analog GS-441524, have the potential to effectively cure FIP. Alpha-1-acid glycoprotein (AGP) has been suggested as a diagnostic marker for FIP. However, AGP quantification methods are not easily accessible. This study aimed to establish a Spatial Proximity Analyte Reagent Capture Luminescence (SPARCLTM) assay on the VetBio-1 analyzer to determine the AGP concentrations in feline serum and effusion samples. Linearity was found in serial dilutions between 1:2000 and 1:32,000; the intra-run and inter-run precision was <5% and <15%, respectively; and AGP was stable in serum stored for at least 8 days at room temperature, at 4 °C and at −20 °C. Cats with confirmed FIP had significantly higher serum AGP concentrations (median: 2954 µg/mL (range: 200–5861 µg/mL)) than those with other inflammatory diseases (median: 1734 µg/mL (305–3449 µg/mL)) and clinically healthy cats (median 235 µg/mL (range: 78–616 µg/mL); pKW < 0.0001). The AGP concentrations were significantly higher in the effusions from cats with FIP than in those from diseased cats without FIP (pMWU < 0.0001). The AGP concentrations in the serum of cats with FIP undergoing GS-441524 treatment showed a significant drop within the first seven days of treatment and reached normal levels after ~14 days. In conclusion, the VetBio-1 SPARCLTM assay offers a precise, fast and cost-effective method to measure the AGP concentrations in serum and effusion samples of feline patients. The monitoring of the AGP concentration throughout FIP treatment provides a valuable marker to evaluate the treatment’s effectiveness and identify potential relapses at an early stage.

Measurement of Feline Alpha-1 Acid Glycoprotein in Serum and Effusion Using an ELISA Method: Analytical Validation and Diagnostic Role for Feline Infectious Peritonitis.

Alpha-1 acid glycoprotein (AGP) may support a clinical diagnosis of feline infectious peritonitis (FIP). In this study, we assessed the analytical and diagnostic performances of a novel ELISA method to measure feline AGP. AGP was measured in sera and effusions from cats with FIP (n = 20) or with other diseases (n = 15). Precision was calculated based on the coefficient of variation (CV) of repeated testing, and accuracy was calculated by linearity under dilution (LUD). The test is precise (intra-assay CVs: <6.0% in individual samples, <15.0% in pooled samples; inter-assay CVs <11.0% and <15.0%) and accurate (serum LUD r2: 0.995; effusion LUD r2: 0.950) in serum and in effusions. AGP is higher in cats with FIP than in other cats in both serum (median: 1968, I-III interquartile range: 1216-3371 μg/mL and 296, 246-1963 μg/mL; p = 0.009) and effusion (1717, 1011-2379 μg/mL and 233, 165-566 μg/mL; p < 0.001). AGP discriminates FIP from other diseases (area under the receiver operating characteristic curve: serum, 0.760; effusion, 0.877), and its likelihood ratio is high (serum: 8.50 if AGP > 1590 μg/mL; effusion: 3.75 if AGP > 3780 μg/mL). This ELISA method is precise and accurate. AGP in serum and in effusions is a useful diagnostic marker for FIP.

Serum alpha 1-acid glycoprotein and interleukin-8 elevations in felines with localized and metastatic tumors.

This study aimed to investigate the expression of acute phase proteins and plasma cytokines in cats with various tumor types and varying metastatic statuses. 5 clinically healthy cats and 22 cats with neoplastic disease that underwent CT imaging before treatment were enrolled. Patients were grouped based on their tumor types and metastatic status. Blood samples were collected from all cats for general blood analyses before they underwent CT imaging. The remaining plasma sample was frozen for subsequent alpha 1-acid glycoprotein (AGP), serum amyloid A (SAA), and feline cytokine panel measurements. These results were compared with those of healthy cats as well as between metastatic status and tumor types. Only 4 cats (18%) exhibited elevated SAA levels, whereas 16 (73%) showed elevated AGP levels. AGP was significantly increased in cats with tumors (P = .016), while SAA was not. Only IL-8 showed a significant difference (P = .002) between cats with primary tumors and those with radiologically suspected tumor metastasis. While AGP is a more prominent biomarker than SAA in cats with tumors, a significant elevation of AGP and SAA levels in association with metastasis and specific tumor types could not be identified. Alternatively, further investigation is warranted to evaluate the potential significance of IL-8 in tumor progression and metastasis.

In ovo administration of the Marek's disease vaccine in conjunction with 25-hydroxyvitamin D3 and its subsequent effects on the performance and immunity-related characteristics of Ross 708 broiler hatchlings1,2,3

The combined effects of the in ovo injection of commercial Marek's disease vaccine (MDV) and various levels of 25-hydroxyvitamin D3 (25OHD3) on the hatch variables, immunological measurements, and gene expression of Ross 708 hatchling broilers were investigated. A total of 5 in ovo injection treatments that were applied at 18 d of incubation (doi) included: 1) noninjected (control); or a 50 μL solution volume of 2) MDV alone; or MDV combined with 3) 0.6 μg of 25OHD3; 4) 1.2 μg of 25OHD3; or 5) 2.4 μg of 25OHD3. At hatch, hatchability of set and live embryonated eggs, hatchling body weight, hatch residue analysis, serum IgY and alpha-1 acid glycoprotein (AGP) concentrations, and the expression of genes related to immunity (INFα, INFβ, INFγ, TLR-3, and TLR-21) and vitamin D3 activity (1 α-hydroxylase, 24 hydroxylase, and vitamin D receptor) were determined. No significant treatment differences were observed for hatchability of set and live embryonated eggs, or for serum IgY and AGP concentrations. However, hatchling body weight was higher when MDV was combined with either 1.2 or 2.4 μg of 25OHD3 than when MDV was provided alone or in combination with 0.6 μg of 25OHD3. Also, in comparison to the noninjected treatment group, the expression of the genes for 1 α-hydroxylase and 24 hydroxylase was improved when MDV was combined with either 1.2 or 2.4 μg of 25OHD3. Lastly, expression of the genes linked to viral detection (TLR-3) and antibody production (INF-β) was increased in those treatments that contained any level of 25OHD3. These results indicate that in comparison to controls, the effects of MDV were observed to be greater on hatchling BW and splenic gene expression when it was administered in combination with the 1.2 or 2.4 μg doses of 25OHD3. Further research is needed to determine the posthatch effects of the administration of various levels of 25OHD3 in combination with MDV.

AB1519 NEUTROPHIL-TO-LYMPHOCYTE RATIO AND PLATELET-TO-LYMPHOCYTE RATIO AS POTENTIAL MARKERS FOR DISEASE ACTIVITY EVALUATION IN BEHCET’S DISEASE

BackgroundBehcet’s disease (BD) is an auto-inflammatory disorder, characterized by recurrent aphthous stomatitis, genital ulcers, recurrent uveitis, erythema nodosum and other numerous mucocutaneous, gastrointestinal, neurological, vascular manifestations. Despite many biomarkers (anti-lysozyme, serum endocan, serum alpha 1-acid glycoprotein (AGP)) have been investigated for evaluating disease activity and predicting disease severity, none of mentioned above have accessibility to be used in clinical routine. During recent years neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been proposed as easy countable markers for severe inflammation and endothelial injury and therefor can be used in the assessment of Behcet’s disease activity.ObjectivesThe aim of this study is to analyze the potential role of NLR and PLR as markers of the Behcet’s disease activity.Methods62 patients with Behcet’s disease (76,4% male and 23,6 % female) and 56 healthy controls were included in this study. Patients with Behcet disease fulfilled The International Criteria for Behcet’s disease (ICBD 2006). Disease activity was assessed with Behcet’s Disease Clinical Activity Form (BDCAF). All patients at the moment of the study had active Behcet’s disease. Participants from the control group didn’t present any history of cardiac or endocrine disease, because mentioned above conditions can also cause high levels of NLR and PLR.ResultsThe mean age of patient group was 36±11,2 years. All patients had oral ulcers at any episode of the disease, 82,26% of patients had erythema nodosum, 61,29% -genital ulcers. 40,38%- eye lesions, 0,05%-entero-Behcet, 0,032%- neuro-Behcet. According to our study, levels of NLR positively correlate with the BDCAF score (r=0.401, p&lt;0.01) and levels of CRP (r=0,571, p&lt;0,01), while there was no correlation found between PLR and disease activity. Levels of NLR and PLR were both significantly higher in the patient group compare to the controls (p&lt;0,001 and p&lt;0,05 accordingly).ConclusionTaking into consideration data from our study NLR could be implemented in clinical practice as a cheap and easy accessible marker of the Behcet’s disease activity. On the other hand, further studies with bigger samples are required to evaluate changes of PLR in BD. Assessment of NLR and PLR among BD patient in remission is required to find out if there are any changes compared to healthy controls.

Potential Clinically Relevant Effects of Sialylation on Human Serum AAG-Drug Interactions Assessed by Isothermal Titration Calorimetry: Insight into Pharmacoglycomics?

Human serum alpha-1 acid glycoprotein is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. It has been reported that the sialic acid groups that terminate the N-glycan chains of alpha-1 acid glycoprotein change in response to certain health conditions and may have a major impact on drug binding to alpha-1 acid glycoprotein. The interaction between native or desialylated alpha-1 acid glycoprotein and four representative drugs-clindamycin, diltiazem, lidocaine, and warfarin-was quantitatively evaluated using isothermal titration calorimetry. The calorimetry assay used here is a convenient and widely used approach to directly measure the amount of heat released or absorbed during the association processes of biomolecules in solution and to quantitatively estimate the thermodynamics of the interaction. The results showed that the binding of drugs with alpha-1 acid glycoprotein were enthalpy-driven exothermic interactions, and the binding affinity was in the range of 10-5-10-6 M. Desialylated alpha-1 acid glycoprotein showed significantly different binding with diltiazem, lidocaine, and warfarin compared with native alpha-1 acid glycoprotein, whereas clindamycin showed no significant difference. Therefore, a different degree of sialylation may result in different binding affinities, and the clinical significance of changes in sialylation or glycosylation of alpha-1 acid glycoprotein in general should not be neglected.

Effect of the sialic acid residues upon the binding of beta blocker propranolol to human serum alpha-1 acid glycoprotein

Effect of the sialic acid residues upon the binding of beta blocker propranolol to human serum alpha-1 acid glycoprotein

Potential use of Cypridina luciferin for quantifying alpha 1-acid glycoprotein in human serum.

Alpha 1-acid glycoprotein (AGP) is an acute phase protein in mammals, including humans. The amount of AGP in human serum varies in response to certain diseases; thus, many efforts have been made to develop methods for quantifying human AGP. We recently discovered that luminescence occurs merely by mixing Cypridina luciferin with human AGP under human serum-free neutral or basic buffer conditions. In this study, we tested an application of Cypridina luciferin for quantifying AGP contained in human serum. Our luminescence spectrum measurements of Cypridina luciferin with human serum samples showed that the maximum emission wavelength with human serum (480nm) differed from that with human AGP (464nm) due to the abundant presence of endogenous human serum albumin (HSA). Furthermore, the luminescence intensities of Cypridina luciferin with human AGP in HSA-depleted human serum were consistent with those in a human serum-free basic buffer, but those in human serum were not. These results indicated that depletion of HSA in human serum was required to use Cypridina luciferin for quantifying AGP in human serum. Additionally, we found that the luminescence intensity of Cypridina luciferin with bovine AGP was approximately tenfold lower than that with human AGP.

The Role of Uremic Retention Solutes in the MIA Syndrome in Hemodialysis Subjects

Introduction: In chronic kidney disease (CKD), the high morbidity and mortality risk for cardiovascular disease (CVD) are not easily explained only on the basis of traditional factors. Among nontraditional ones involved in CKD, malnutrition, inflammation, and atherosclerosis/calcification have been described as the “MIA syndrome.” Methods: In this pilot study, we evaluated the association between the variation in serum levels of 27 uremic retention solutes plus 6 indexes related to the MIA syndrome processes in a population of dialysis patients. Results: As expected, we found a direct correlation between serum albumin and both phosphate and total cholesterol (r = 0.54 and 0.37, respectively; p < 0.05). Moreover, total cholesterol and phosphate directly correlate (r = 0.40, p < 0.05). The relationship between malnutrition and inflammation is highlighted by the correlation of serum cholesterol levels with serum alpha-1 acid glycoprotein and IL-6 levels (r = −0.56, r = −0.39, respectively; p < 0.05). Moreover, the relation between inflammation and atherosclerosis/calcification is supported by the correlation of IL-6 with VEGF levels and vascular smooth muscle cell high-Pi in vitro calcification (r = 0.81, r = 0.66, respectively; p < 0.01). Conclusion: We found significant correlations between several uremic retention solutes and malnutrition, inflammation, and atherosclerosis/calcification. Our findings support the hypothesis of a central role of the uremic milieu in the MIA syndrome and ultimately in the pathogenesis of CKD-specific CVD risk factors.

MO736: The Role of Uremic Retention Solutes in the MIA Syndrome in Haemodialysis Subjects

Abstract BACKGROUND AND AIMS In end-stage renal disease (ESRD), the high morbidity and mortality risk for cardiovascular disease (CVD) is not easily explained only on the basis of traditional factors. Among non-traditional ones, malnutrition, inflammation and atherosclerosis/calcification have been described as the ‘MIA syndrome’. METHOD In this study, we evaluated the association between the variation in serum levels of 27 uremic retention solutes plus 6 indexes related to the MIA syndrome processes in dialysis patients. Uremic retention solute levels were detected by the ELISA or using an ultra-performance liquid chromatography—tandem mass spectrometry method. The pro-calcifying, pro-apoptotic and pro-necrotic effects of uremic serum were evaluated in an in vitro model of high-Pi induced calcification in vascular smooth muscle cells (VSMC). RESULTS As expected, we found reciprocal correlations between serum albumin, total cholesterol and phosphate, linking a better nutritional status with higher phosphate levels (r = 0.37, r = 0.40, r = 0.54, respectively; P &amp;lt; 0.05). The relationship between malnutrition and inflammation is highlighted by the negative correlation of serum cholesterol levels with serum alpha-1 acid glycoprotein and IL-6 levels (r = –0.56, r = –0.39, respectively; P &amp;lt; 0.05). These results suggest that the increase in inflammatory mediators is probably higher in malnourished patients, and a better nutritional state seems to correlate with lower inflammatory marker levels. Moreover, we found a significant direct correlation between alpha-1 acid glycoprotein and IL-6 with a correlation coefficient of 0.54 and P &amp;lt; 0.01, indicating that both these uremic toxins might participate in the maintenance of the inflammation. The relation between inflammation and atherosclerosis/calcification is supported by the correlations between IL-6 variations that significantly correlate with both PTX3 and VEGF (r = 0.36 and r = 0.81; P &amp;lt; 0.05) and with VSMC in vitro high-Pi induced calcification (r = 0.66; P &amp;lt; 0.01). Thus, IL-6, a regulator of acute phase protein synthesis, seems to be related to CVD markers (PTX3 and VEGF) and in vitro calcification, two robust indexes of increased cardiovascular risk. In addition, the Kyn/Try ratio directly correlates with serum FGF23 levels with a correlation coefficient r of 0.60 and P &amp;lt; 0.01. Regarding atherosclerosis/calcification, we found that PTX3, TMAO, IS, myoglobin and FGF23 correlate with each other in several combinations, reflecting the elevated cardiovascular risk present in ESRD. PTX3 shows a trend towards a direct correlation with TMAO (r = 0.37 and P &amp;lt; 0.05), and IS shows a trend towards a direct correlation both with TMAO, FGF23 and myoglobin (r = 0.46, r = 0.45 and r = 0.42; P &amp;lt; 0.05). Moreover, IS and TMAO correlate also with two processes strictly related to VSMC calcification, such as apoptosis and necrosis (IS: apoptosis r = 0.69 and necrosis r = 0.47; P &amp;lt; 0.05; TMAO: apoptosis r = 0.42 and necrosis r = 0.40 with P &amp;lt; 0.05). Finally, VEGF shows a robust correlation with in vitro calcification (r = 0.74; P &amp;lt; 0.01). CONCLUSION Among all the correlations found between uremic toxin variations in dialysis patients, IL-6 seems to have a central role correlating with malnutrition and several CV markers together with vascular calcification (VC). This might be explained by the characteristics of IL-6 being contemporary an inflammatory and a CVD marker in ESRD. On the other hand, IS has a potential role in VC, suggesting a central role of the uremic milieu in MIA syndrome development. Therefore, there is an urgent need to find a more efficient dialysis treatment to improve the clearance of uremic retention solutes in order to try to ameliorate CV outcomes in ESRD patients.

On the binding reaction of loratadine with human serum acute phase protein alpha 1-acid glycoprotein

Loratadine is an important anti-allergic drug. It is a second generation antihistamine drug used to treat allergic rhinitis, hay fever and urticaria. Human serum alpha 1-acid glycoprotein (AG) is an important acute phase protein and its serum concentration is found to increase in inflammation and acute response.The binding interaction between loratadine and AG is studied using spectroscopy and molecular docking techniques. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of AG is quenched by loratadine. Loratadine was found to bind AG with the binding constant of ≈104 at 298 K. The Gibb’s free energy change was found to be negative for the interaction of loratadine with AG indicating the binding process is spontaneous. Binding of loratadine with AG induced ordered structures in the protein. Hydrogen bonding and hydrophobic interactions were the main bonding forces between AG–loratadine as revealed by molecular docking results. This study suggests the importance of binding of anti-allergic drug to AG spatially in the diseases where the plasma concentration of AG increases many folds and interaction with this protein becomes significant. This study will help in design of drug dosage and adjustment accordingly to achieve optimal treatment outcome. Communicated by Ramaswamy H. Sarma

Studies of binding by 2-imidazolines to human serum albumin and alpha1-acid glycoprotein by high-performance affinity chromatography

2-Imidazoline drugs are used in a variety of applications, such as the treatment of hypertension and opioid withdrawal. It is known these drugs bind to serum proteins and have significant variations within this class of compounds in the overall level of this binding. However, little specific information is available on the interactions of these compounds with the two major transport proteins for many drugs, human serum albumin (HSA) and alpha1-acid glycoprotein (AGP). This study examined binding by 2-imidazolines to these proteins by using 25 mm × 2.1 mm i.d. high-performance affinity microcolumns that contained HSA or AGP. The drugs that were examined were antazoline, clonidine, dexmedetomidine, lofexidine, moxonidine, phentolamine, and tizanidine, which represented a wide range of structures and pharmaceutical applications. The major metabolite of lofexidine, N-(2-aminoethyl)-2-(2,6-dichlorophenoxy) propenamide (LADP), was also examined. All these 2-imidazolines were found to have weak-to-moderate binding to HSA, with global affinities that ranged from 1.62 × 102 to 1.07 × 104 M−1 at pH 7.4 and 37 °C. These compounds had stronger binding with AGP, with global affinities constants ranging from 3.80 × 102 to 1.85 × 104 M−1. No stereoselectivity was observed by HSA for the enantiomers of dexmedetomidine, lofexidine, or LADP. However, AGP did show some stereoselectivity for lofexidine and LADP but not for dexmedetomidine. These results provide a better understanding of interactions of 2-imidazoline with HSA vs AGP in the circulation and of how this binding can change between drugs within this class of compounds.

The Acute Host-Response of Turkeys Colonized With Campylobacter coli.

Consumption of contaminated poultry products is one of the main sources of human campylobacteriosis, of which Campylobacter jejuni subsp. jejuni (C. jejuni) and C. coli are responsible for ~98% of the cases. In turkeys, the ceca are an important anatomical site where Campylobacter asymptomatically colonizes. We previously demonstrated that commercial turkey poults colonized by C. jejuni showed acute changes in cytokine gene expression profiles, and histological intestinal lesions at 2 days post-inoculation (dpi). Cecal tonsils (CT) are an important part of the gastrointestinal-associated lymphoid tissue that surveil material passing in and out of the ceca, and generate immune responses against intestinal pathogens. The CT immune response toward Campylobacter remains unknown. In this study, we generated a kanamycin-resistant C. coli construct (CcK) to facilitate its enumeration from cecal contents after experimental challenge. In vitro analysis of CcK demonstrated no changes in motility when compared to the parent isolate. Poults were inoculated by oral gavage with CcK (5 × 107 colony forming units) or sterile-media (mock-colonized), and euthanized at 1 and 3 dpi. At both time points, CcK was recovered from cecal contents, but not from the mock-colonized group. As a marker of acute inflammation, serum alpha-1 acid glycoprotein was significantly elevated at 3 dpi in CcK inoculated poults compared to mock-infected samples. Significant histological lesions were detected in cecal and CT tissues of CcK colonized poults at 1 and 3 dpi, respectively. RNAseq analysis identified 250 differentially expressed genes (DEG) in CT from CcK colonized poults at 3 dpi, of which 194 were upregulated and 56 were downregulated. From the DEG, 9 significantly enriched biological pathways were identified, including platelet aggregation, response to oxidative stress and negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway. These data suggest that C. coli induced an acute inflammatory response in the intestinal tract of poults, and that platelet aggregation and oxidative stress in the CT may affect the turkey's ability to resist Campylobacter colonization. These findings will help to develop and test Campylobacter mitigation strategies to promote food safety in commercial turkeys.

A pilot study to evaluate the serum Alpha-1 acid glycoprotein response in cats suffering from feline chronic gingivostomatitis

BackgroundFeline chronic gingivostomatitis (FCGS) is a multifactorial immune-mediated disease that can lead to chronic pain, anorexia, and weight loss and has substantial health and welfare effects. Currently, the recommended treatment includes dental extractions to decrease the inflammatory stimulation associated with dental plaque. However, complete remission is observed in less than half of the cases, and the majority need comprehensive medical management. This study aimed to evaluate the serum levels of the acute phase protein alpha-1 acid glycoprotein (AGP) in cats with FCGS and to examine whether dental extractions contribute to a significant decrease in the systemic inflammatory response at two postoperative time points.ResultsAGP serum concentrations in the cats with FCGS were significantly higher at all time points than that in the control groups and were significantly correlated with the global caudal stomatitis score at day 0 but not at day 30 or 60. A significant improvement of some clinical scores, such as perceived comfort and global caudal stomatitis, was observed 60 days after the dental extraction. However, the levels of AGP did not significantly change over time.ConclusionsCats with FCGS were more likely to have a systemic inflammatory response compared with age- and dental disease-matched controls. Dental extractions, in most cases, did not contribute to a significant decrease of AGP both at 30 and 60 days. Therefore, this study reinforces the need to pursue comprehensive medical management after dental extractions to attenuate the systemic inflammatory response as a result of this disease.

Serum Levels and Glycosylation Changes of Alpha-1-Acid Glycoprotein According to Severity of Breast Cancer in Korean Women.

Elevated serum levels of alpha-1-acid glycoprotein (AGP) are known to be associated with several types of cancer. In addition, some reports have indicated that changes in glycosylation of AGP are associated with cancer progression. However, changes in AGP levels of serum and changes in glycosylation of AGPs in breast cancer have not been specifically studied. In the present study, serum AGP levels in benign (BN) cancer and breast cancer stage I (BC I), BC IIA, BC IIB, and BC III in Korean women were measured using an enzyme-linked immunosorbent assay (ELISA). AGP was purified from individual sera by hot phenol extraction and then subjected to AGP glycosylation analysis. Three types of AGP glycosylation (fucosylation, high-mannose-type and sialylation) were detected using enzyme-linked lectin assays (ELLAs). Serum AGP levels were higher in BC I, BC IIA, BC IIB, and BC III, than in the BN group, and the level in BC I and BC IIA was high enough to be distinguished from BN. Meanwhile, terminal fucosylation and high-mannose-type glycans appeared to be lowest in BC I. The glycosylation levels of BC I provide sensitivity and specificity that make BC I clearly distinguishable from BC IIA, BC IIB, and BC III as well as BN. Therefore, determination of serum AGP or AGP glycosylation level could be useful for detecting the early stages of breast cancer.

A point-of-care assay for alpha-1-acid glycoprotein as a diagnostic tool for rapid, mobile-based determination of inflammation.

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. 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. 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%. 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.

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

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 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 Fab 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.

Glycoform Analysis of Alpha1-Acid Glycoprotein by Capillary Electrophoresis Using Electrophoretic Injection.

Human alpha1-acid glycoprotein (AGP) is an acute phase glycoprotein that has a heterogeneous glycosylation pattern. This pattern can change in certain diseases, which has resulted in interest in using AGP glycoforms as potential biomarkers for these diseases. This report describes a method that uses capillary electrophoresis to characterize and analyze AGP glycoforms both in purified samples of AGP and in human serum. This method uses static and dynamic coatings of poly(ethylene oxide) that are applied to a silica capillary for separation of AGP glycoforms in the reversed-polarity mode of CE and in the presence of negligible electroosmotic flow. Electrophoretic injection is performed onto such capillaries by using a stationary stacking interface between the sample and running buffer. In addition, acidic precipitation and desalting are used to allow for the isolation and the analysis of AGP from only 65μL of serum. Up to eleven AGP glycoform bands can be reproducibly separated by this method, with the difference in migration time between neighboring bands being 12- to almost 60-fold larger than the standard deviation for the migration time of any given band. A limit of detection down to about 2nM per glycoform band can be obtained by this method for AGP in serum based on absorbance detection and without the need for further sample modification or labeling.

Evaluation of Serum Alpha 1-Acid Glycoprotein as a Diagnostic Marker in Early Neonatal Sepsis

Evaluation of Serum Alpha 1-Acid Glycoprotein as a Diagnostic Marker in Early Neonatal Sepsis

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.