Specific Sandwich Enzyme-linked Immunosorbent Assay Research Articles

Development and validation of bioanalytical assays for the quantification of 9MW2821, a nectin-4-targeting antibody–drug conjugate

We designed and developed 9MW2821, an anti-Nectin-4 antibody–drug conjugate (ADC) with an enzymatically cleavable valine–citrulline linker and monomethyl auristatin E (MMAE) as the payload. Four bioanalytical assays for total antibodies, conjugated antibodies, conjugated payload, and free payload were then developed and validated for the comprehensive evaluation of the multiple drug forms of 9MW2821. Specific sandwich enzyme-linked immunosorbent assays were used to quantify total antibodies and conjugated antibody, showing good drug-to-antibody ratio (DAR) tolerance. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine free MMAE, and conjugated MMAE was quantified using a combination of ligand-binding assay (LBA) and LC-MS/MS. Based on these four assays, we studied the serum stability and monkey pharmacokinetic profiles of 9MW2821, and the in vivo DAR of 9MW2821 was calculated and dynamically monitored. In conclusion, we developed and validated series of bioanalytical assays to quantify multiple forms of 9MW2821, a new ADC, and used the assays to evaluate the serum stability and monkey pharmacokinetic characteristics. The results indicate good linker stability and suggest that the developed assays can be further used in clinical settings.

Novel monoclonal antibodies against house dust mite allergen Der p 21 and their application to analyze allergen extracts.

Allergen extracts and recombinant allergens are used in allergy diagnostics and immunotherapy. Since allergen extracts from different manufacturers lack proper standardization regarding their composition, monoclonal antibodies (MAbs) against specific allergen components can be used for their identification and quantification in allergen extracts. This study aimed to generate MAbs against allergen Der p 21 of Dermatophagoides pteronyssinus for the analysis of allergen extracts. Recombinant Der p 21 was expressed in E. coli and purified using affinity chromatography. MAbs against Der p 21 were generated using hybridoma technology. House dust mite (HDM) allergen extracts were analyzed using the newly developed sandwich enzyme-linked immunosorbent assay, Western blotting and microarray immunoassay. MAbs raised against recombinant Der p 21 were characterized in detail and proven to be reactive with natural Der p 21. Highly specific sandwich enzyme-linked immunosorbent assay for the quantification of Der p 21 was developed and optimized. The allergen was detected and its concentration was determined in only three of six analyzed HDM allergen extracts from different manufacturers. HDM analysis by MAb-based immunoassays shows their differences in allergen composition. The results demonstrate the importance of allergen-specific MAbs as a tool for the characterization of allergen extracts and the need for their appropriate standardization before their use for allergy diagnostics or immunotherapy.

Development of a specific and sensitive sandwich enzyme-linked immunosorbent assay for the quantification of dasatinib

This study sought to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of dasatinib, a tyrosine kinase inhibitor. Anti-dasatinib antibodies were obtained from mice or rabbits by using two partial structures of dasatinib as haptens: 2-amino-N-(2-chloro-6-methylphenyl)-thiazole-5-carboxamide and 2-{4-(2-hydroxyethyl)-1-piperazinyl}-isonicotinic acid. The best combination of two antibodies for sandwich ELISA of dasatinib was determined using four anti-dasatinib antibodies derived from mice and rabbits. Using two dasatinib-specific rabbit antibodies, we successfully developed an ultra-specific and highly sensitive sandwich ELISA that is hardly affected by the main metabolite of dasatinib. The sandwich ELISA showed a linear detection range from 320 pg/mL to 1000 ng/mL. Serum dasatinib concentrations lower than 320 pg/mL were reproducibly measurable using the sandwich ELISA. The ELISA was specific to dasatinib and there were no cross-reactivities with the major metabolites 4′-hydroxy dasatinib and dasatinib carboxylic acid. The developed sandwich ELISA will be a valuable tool for pharmacokinetic studies of dasatinib. Furthermore, this study revealed that rabbit antibodies can sandwich drug molecules of a smaller size than mouse antibodies in sandwich ELISA.

Development and validation of a specific sandwich ELISA for determination of soybean allergens and its application in processed foods

Soybean allergens are commonly quantified using enzyme-linked immunosorbent assays (ELISAs) with antibodies produced by the single allergen. To enhance the specificity and avoid the false-negative results, the sandwich ELISA method was developed using a rabbit anti-bulk soybean proteins polyclonal antibody and a goat anti- bulk soybean proteins polyclonal antibody as the detection and capture antibody, respectively. The proposed method has been optimized and validated according to the requirements of the AOAC Allergen Community. The method has a wide quantification range from 0.0625 μg/mL to 6.0 μg/mL and can quantify soybean residues at a low level down to 0.25 mg/kg with satisfactory accuracy (recovery range from 89.5% to 118%) and precision (RSDs < 16.67%). The new approach performs high specificity to soybean residues without obvious cross-reactivity to common allergic foods and legumes, and has been successfully applied to detect soybean proteins in commercial foods. Our data demonstrated that the established sandwich ELISA method has a higher specificity, accuracy, and robustness that could meet the quantification requirements for soybean allergens.

Evidence for Glucagon Secretion and Function Within the Human Gut.

Glucagon is secreted by pancreatic α cells in response to hypoglycemia and increases hepatic glucose output through hepatic glucagon receptors (GCGRs). There is evidence supporting the notion of extrapancreatic glucagon but its source and physiological functions remain elusive. Intestinal tissue samples were obtained from patients undergoing surgical resection of cancer. Mass spectrometry analysis was used to detect glucagon from mucosal lysate. Static incubations of mucosal tissue were performed to assess glucagon secretory response. Glucagon concentration was quantitated using a highly specific sandwich enzyme-linked immunosorbent assay. A cholesterol uptake assay and an isolated murine colonic motility assay were used to assess the physiological functions of intestinal GCGRs. Fully processed glucagon was detected by mass spectrometry in human intestinal mucosal lysate. High glucose evoked significant glucagon secretion from human ileal tissue independent of sodium glucose cotransporter and KATP channels, contrasting glucose-induced glucagon-like peptide 1 (GLP-1) secretion. The GLP-1 receptor agonist Exendin-4 attenuated glucose-induced glucagon secretion from the human ileum. GCGR blockade significantly increased cholesterol uptake in human ileal crypt culture and markedly slowed ex vivo colonic motility. Our findings describe the human gut as a potential source of extrapancreatic glucagon and demonstrate a novel enteric glucagon/GCGR circuit with important physiological functions beyond glycemic regulation.

Associating Insulin Instillation with Negative Pressure Wound Therapy Reduces Insulin Resistance in Diabetic Patients with Infected Foot Wounds

In this work we investigated whether a joint treatment with insulin instillation and negative pressure wound therapy (NPWT) would reduce insulin resistance (IR) in diabetic patients with infected diabetic foot ulcers (DFUs) and improve their healing. We also studied the effects of insulin+NPWT treatment on the inflammatory response coupled with wound healing. Seventy-five diabetic patients with infected DFUs were recruited and randomly divided into equal (n=25) groups treated respectively with NPWT alone, NPWT+insulin, and conventional dressings (controls). Thereafter, the ulcers’ healing progress was assessed. The serum levels of Tumor Necrosis Factor-α (TNF-α), Interleukin-2 (IL-2), and Interleukin-6 (IL-6) were measured at 1 day before and at 7, 14, and 21 days after each treatment using specific double-antibody sandwich enzyme-linked immunosorbent assays (ELISA). Serum glucose was measured via a glucose-oxidase method, and serum insulin via radioimmunoassay. In each patient, IR was appraised via the Homeostasis Model Assessment (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI). Our results showed that at 7, 14 and 21 days post-treatment, with respect to the control group the two NPWT-treated groups had (i) significantly (p &lt; 0.05) lower serum levels of TNF-α, IL-2 and IL-6; (ii) a significantly (p &lt; 0.01) greater reduction in IR; and (iii) significantly (p &lt; 0.05) higher wound healing rates. Moreover, at 14- and 21-days post-treatment, the NPWT+insulin-treated group had a significantly (p &lt; 0.05) higher wound healing rate than the NPWT alone-treated group. In conclusion, the combined NPWT+insulin instillation treatment gave superior results by improving wound healing, decreasing inflammation at the wounds surface, and reducing IR in diabetic patients with infected DFUs.

Combining Negative Pressure Wound Therapy with Insulin Instillation Reduces Insulin Resistance and Accelerates Healing in Patients with Chronically Infected Wounds

Aim: This study aimed at evaluating the effects of negative pressure wound therapy (NPWT) combined with insulin instillation on wound healing and insulin resistance (IR) in patients with chronically infected wounds. Methods: Sixty-three patients were recruited and randomly divided into three groups (21 cases each) according to treatments: NPWT only; NPWT+insulin; and controls (conventional dressings). Both NPWT treatments lasted for three weeks. Thereafter, wounds’ healing conditions were assessed. At 1 day before and 7, 14, and 21 days posttreatment Interleukin- 6 (IL-6), Interleukin- 2 (IL-2), and Tumor Necrosis Factor-α (TNF-α) serum levels were assessed via specific double-antibody sandwich enzyme-linked immunosorbent assays; and plasma glucose and insulin were quantified via glucose-oxidase and radioimmunoassay, respectively. IR was appraised via homeostasis model assessment (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI). Results: At 7, 14, and 21 days after treatments onset the two NPWT groups had significantly (P = 0.0001) lower IL-6, IL-2, and TNF-α serum levels and HOMA-IR values than controls while wound healing rates were higher (P &lt; 0.001) than controls. Notably, the NPWT+insulin treatment results were better (P &lt; 0.01) than those of NPWT alone. Conclusion: NPWT+insulin instillation improved wound healing, mitigated inflammation, and reduced IR in patients with chronically infected wounds.

Measurement of the concentration of serum soluble interleukin-2 receptor alpha chain in dogs with lymphoma

Soluble interleukin-2 receptor (sIL-2r) is released directly from the surface of lymphocytes expressing interleukin-2 receptor alpha chain (CD25), and its serum concentration has been found to reflect the prognosis of human lymphoproliferative malignancies. In this study, we demonstrated the presence of sIL-2r in canine serum and developed a specific sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the concentration of canine serum sIL-2r. In the immunoprecipitation (IP) assay, CD25 protein weighing approximately 45 kDa was detected in canine serum, smaller than the membrane-bound CD25 (approximately 55 kDa). To measure the concentration of serum sIL-2r in dogs, an ELISA system was developed. Serum sIL-2r levels were significantly higher in dogs with multicentric high-grade B-cell lymphoma before therapy than that in healthy dogs. Serum sIL-2r concentration was also found to be elevated in a proportion of dogs with other types of lymphoma. Changes in serum sIL-2r levels generally paralleled the changes in mass and lymph node size in dogs with high-grade B-cell lymphoma. This study demonstrated that serum sIL-2r level would be a marker to monitor tumour growth and regression in canine lymphoma.

Plasma CD5L and non-invasive diagnosis of acute heart rejection

Acute rejection is one of the most important direct contributors to mortality after heart transplantation. Advances in the development of novel non-invasive approaches for the early identification of allograft rejection are necessary. We conducted a non-targeted proteome characterization focused on identifying multiple plasmatic protein differences to evaluate their diagnostic accuracy for rejection episodes. We included consecutive plasma samples from transplant recipients undergoing routine endomyocardial biopsies. A liquid chromatography-tandem mass spectrometry analysis using isobaric tags (tandem mass tag 10-plex) was performed and concentrations of CD5L were validated using a specific sandwich enzyme-linked immunosorbent assay. A total of 17 altered proteins were identified as potential markers for detecting heart transplant rejection, most involved in inflammation and immunity. CD5L, an apoptosis inhibitor expressed by macrophages, showed the best results in the proteomic analysis (n = 30). We confirm this finding in a larger patient cohort (n = 218), obtaining a great diagnostic capacity for clinically relevant rejection (≥Grade 2R: area under the curve = 0.892, p < 0.0001) and preserving the accuracy at mild rejection (Grade 1R: area under the curve = 0.774, p < 0.0001). CD5L was a strong independent predictor, with an odds ratio of 14.74 (p < 0.0001), for the presence of rejection. Episodes of acute cardiac allograft rejection are related to significant changes in a key inhibitor of apoptosis in macrophages, CD5L. Because of its precision to detect acute cellular rejection, even at mild grade, we propose CD5L as a potential candidate to be included in the studies of molecule combination panel assays. This finding could contribute to improving the diagnostic and preventive methods for the surveillance of cardiac transplanted patients.

Validation of antibody-based tools for galanin research

Antibodies are an integral biomedical tool, not only for research but also as therapeutic agents. However, progress can only be made with sensitive and specific antibodies. The regulatory (neuro)peptide galanin and its three endogenous receptors (GAL1-3-R) are widely distributed in the central and peripheral nervous systems, and in peripheral non-neuronal tissues. The galanin system has multiple biological functions, including feeding behavior, pain processing, nerve regeneration and inflammation, to name only a few. Galanin could serve as biomarker in these processes, and therefore its receptors are potential drug targets for various diseases. For that reason, it is of paramount interest to precisely measure galanin peptide levels in tissues and to determine the cellular and subcellular localization of galanin receptors. A plethora of antibodies and antibody-based tools, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) kits, are commercially available to detect galanin and its receptors. However, many of them lack rigorous validation which casts doubt on their specificity. A goal of the present study was to raise awareness of the importance of validation of antibodies and antibody-based tools, with a specific focus on the galanin system. To that end, we tested and report here about commercially available antibodies against galanin and galanin receptors that appear specific to us. Furthermore, we investigated the validity of commercially available galanin ELISA kits. As the tested ELISAs failed to meet the validation requirements, we developed and validated a specific sandwich ELISA which can be used to detect full-length galanin in human plasma.

Development of a double-antibody sandwich ELISA for rapid detection to C-peptide in human urine

C-peptide level is recognized as an important indicator of diabetes diagnosis. A sensitive and specific double-antibody sandwich enzyme-linked immunosorbent assay for the detection of C-peptide based on double antibody sandwich method was studied in this paper. The rabbit and hen were innunized with PLL-C-peptide and BSA-C-peptide respectively to obtain specific Yolk antibody (IgY) and polyclonal antibody used to construct the sandwich ELISA for the measurement of C-peptide. The limit of detection was 0.51 μg/mL and the half maximal inhibitory concentration (IC50) was 3.26 μg/mL. The method developed in the study showed no evident cross-reactivity with other similar analogs. The detection standard curve of C-peptide exhibited a good linearity (R2 = 0.9896, n = 15). 17 types of the urine of diabetes patients on c-peptide levels compared with the hospital type of diabetes information, with a conclusion of a high consistent rate. Therefore, the methods could be selectively used for rapid screening of C-peptide in human urine, and the type of diabetes has some referential significance.

Circulating soluble RAGE and cell surface RAGE on peripheral blood mononuclear cells in healthy children.

The receptor for advanced glycation end products (RAGE) has a critical role in the pathogenesis of inflammation. In healthy children, its basal expression on the peripheral blood mononuclear cell (PBMC) and the basal circulating soluble RAGE (sRAGE) levels are unknown. The aim of this study was to describe both. This is a monocentric, observational and descriptive study of samples obtained from healthy children. The RAGE expression on PBMC was analyzed using flow cytometry. The sRAGE values were determined with a specific sandwich enzyme-linked immunosorbent assay (ELISA) kit, later the relation between cellular RAGE and sRAGE was described. Forty-three children were included. The median sRAGE level was 849.0±579.0 pg/mL. The RAGE mean fluorescence intensity (MFI) was 1382±506 in monocytes and 792±506 in lymphocytes. There were no differences between genders. A negative correlation was found between sRAGE and RAGE MFI in lymphocytes (r=-0.3; p=0.04). We describe for the first time the RAGE surface levels on PBMC in children. It showed a negative correlation with sRAGE. The sRAGE circulating level is lower than the sRAGE level described in adult population or non-healthy children. Our findings should be confirmed in order to apply them as reference values for future investigations.

ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts

The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted.In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4×108molecules/mL particles.The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease.

Development of a sandwich enzyme-linked immunosorbent assay for dTMP-GH fusion protein by rational immunogen selection

dTMP-GH is a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide (dTMP) fused to human growth hormone (hGH) prepared previously by our team. It shows significant bioactivity in promoting thrombocytopoiesis, but detection of intact dTMP-GH in plasma is still a challenge due to the presence of endogenous hGH. In this study, a rabbit polyclonal antibody with high affinity to dTMP was obtained with a BSA-conjugated immunogen composed of 20 amino acids sequence spanning two TMP and the linker. A monoclonal antibody termed as 3B2 was screened out by using immunizing mice with whole dTMP-GH, which was proved to simultaneously interact with rhGH, TMP-GH, and dTMP-GH, respectively. In this study, we developed a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies (one polyclonal and one HRP-conjugated monoclonal) to quantify dTMP-GH. The polyclonal antibody and HRP-conjugated monoclonal antibody 3B2 were applied as the capture antibody and detection antibody, respectively. A good correlation between ELISA and bicinchoninic acid (BCA) assay in the quantification of diluted dTMP-GH was observed (r2 = 0.996). Meanwhile, the standard curve of this ELISA method was found in a linear relationship between 0.2 and 10 ng/mL in the presence of rabbit plasma. In vivo experiments demonstrate that the newly developed method is effective to detect dTMP-GH in rabbits, which paves the way for further pharmacokinetic evaluation.

SERCA2a: A potential non-invasive biomarker of cardiac allograft rejection

The detection of heart transplant rejection by non-invasive methods remains a major challenge. Despite the well-known importance of the study of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) in the heart, its role as a rejection marker has never been analyzed. Our objective in this study was to determine whether circulating SERCA2a could be a good marker of cardiac rejection. We collected 127 consecutive endomyocardial biopsies (EMBs) and serum samples from adult heart transplant recipients (49 without allograft rejection and 78 with the diagnosis of biopsy allograft rejection, including 48 Grade 1R, 21 Grade 2R and 9 Grade 3R). Serum concentrations of SERCA2a were determined using a specific sandwich enzyme-linked immunosorbent assay. We also analyzed SERCA2a expression changes on EMBs using immunofluorescence. SERCA2a cardiac tissue and serum levels were decreased in patients with cardiac rejection (p < 0.0001). A receiver-operating characteristic analysis showed that SERCA2a strongly discriminated between patients with and without allograft rejection: normal grafts vs all rejecting grafts (AUC = 0.804); normal grafts vs Grade 1R (AUC = 0.751); normal grafts vs Grade 2R (AUC = 0.875); normal grafts vs Grade 3R (AUC = 0.922); normal grafts vs Grade 2R and 3R (AUC = 0.889), with p < 0.0001 for all comparisons. We demonstrated that changes in SERCA2a cardiac tissue and serum levels occur in cardiac allograft rejection. Our findings suggest that SERCA2a concentration assessment may be a relatively simple, non-invasive test for heart transplant rejection, showing a strong capability for detection that improves progressively as rejection grades increase.

Identification and measurement of staphylococcal enterotoxin M from Staphylococcus aureus isolate associated with staphylococcal food poisoning.

Staphylococcal enterotoxins (SE) are the main causative agents of staphylococcalfood poisoning. Unlike classical SEs (SEA to SEE), the relationship between newly identified SEs (SEG to SEX) and staphylococcalfood poisoning has not been clearly elucidated. Recently, mild emetic potential of staphylococcal enterotoxin M (SEM) has been demonstrated, which indicated that SEM might be associated with food poisoning. However, there is currently no commercial enzyme-linked immunosorbent assay (ELISA) kit available for immunological detection of it. Therefore, we developed a highly sensitive, specific sandwich ELISA system and assayed SEM in food matrixes. This assay facilitates the study of SEM expression invitro and invivo. In addition, the investigations would be helpful in addressing the relative incidence of SEM+ strains in near future.

Amniotic Fluid-Soluble Form of Triggering Receptors Expressed on Myeloid Cells: A Physiologic Constituent at Term

Background: Triggering receptors expressed on myeloid cells-1 (TREM-1) are innate immune receptors that mediate inflammatory response. In recent times, the role of these receptors has become the focus of several studies in the gestational context. Evaluation of soluble TREM-1 concentration in normal pregnancy is scarce, and no data are available on the levels of this receptor in the last weeks of normal pregnancy. Method: Amniotic fluid (AF) samples were obtained from 77 patients in the last weeks of normal pregnancy during cesarean section, and the soluble form of TREM-1 (sTREM-1) levels determined using specific sandwich enzyme-linked immunosorbent assay kit. Results: sTREM-1 was detectable in all AF samples, and its levels did not vary in the last weeks of third trimester pregnancy. There was no correlation between sTREM-1 levels in AF and advancing gestational age. Conclusion: sTREM-1 is a physiologic constituent of AF and its levels are not temporally regulated in pregnancies at term.

L(59) TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice.

BackgroundHepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-β, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-β is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-β must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-β by cleavage between R58 and L59 residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R58 cleavage edges and functions as a footprint of PLK-dependent TGF-β activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L59 (L59 LAP-DPs). We demonstrated that the L59 LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis.ResultsWe established a specific sandwich ELISA to quantify L59 LAP-DPs as low as 2 pM and measured L59 LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L59 LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-β receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L59 LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) α1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L59 LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with α-smooth muscle actin (αSMA) expression in liver tissues. At this time, αSMA-positive cells as well as R58 LAP-DPs were seen in their liver tissues.ConclusionsL59 LAP-DPs reflect PLK-dependent TGF-β activation and the increase in αSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-015-0034-9) contains supplementary material, which is available to authorized users.

Evaluation of circulating markers of hepatic apoptosis and inflammation in obese children with and without obstructive sleep apnea

ObjectiveHepatocyte apoptosis and macrophage activation contribute to the disease progression of nonalcoholic fatty liver disease (NAFLD). Obstructive sleep apnea (OSA) in obese children is associated with the severity of NAFLD. The aim of this study was to evaluate plasma levels of soluble Fas (sFas), soluble Fas ligand (sFasL), cytokeratin 18 (CK18) (markers of apoptosis), and soluble CD163 (sCD163) (marker of macrophage activation) in obese children with and without OSA. MethodsConsecutive obese children who were evaluated for OSA were recruited. The diagnosis of OSA was made using overnight polysomnography (PSG). Fasting blood samples were used to determine plasma CK18, sFas, sFasL, and sCD163 levels using specific sandwich enzyme-linked immunosorbent assay (ELISA). ResultsFifty-eight subjects were included in the analysis with a mean age of 8.9 ± 3.2 years and a mean body mass index (BMI) z-score of 2.4 ± 0.49. Circulating sFas and sFasL levels were significantly lower in subjects with mild and severe OSA compared with those without OSA (p < 0.005 for both). In addition, sCD163 levels increased with an increasing severity of OSA (no OSA = 1.6 ± 0.25 mg/L, mild OSA = 2.3 ± 0.45, and severe OSA = 3.0 ± 0.52; p < 0.001), and they correlated with the apnea–hypopnea index (AHI) [rho (95% confidence interval, CI) of 0.71 (0.41, 1.00), p-value <0.001]. In six patients with severe OSA from whom samples were taken before and after tonsillectomy, the sCD163 level decreased significantly after treatment, and there was a trend toward an increase in sFasL. ConclusionMarkers of apoptosis and macrophage activation are altered in obese children with OSA, indicating increased apoptotic and inflammatory pressures.

A monoclonal antibody-based VZV glycoprotein E quantitative assay and its application on antigen quantitation in VZV vaccine.

Varicella-zoster virus (VZV) is a highly infectious agent that causes varicella and herpes zoster (HZ), which may be associated with severe neuralgia. Vaccination is the most effective way to reduce the burden of the diseases. VZV glycoprotein E (gE) is the major and most immunogenic membrane protein that plays important roles in vaccine efficacy. A quantitative assay for gE content is desirable for the VZV vaccine process monitoring and product analysis. In this study, 70 monoclonal antibodies (mAbs) were obtained after immunizing mice with purified recombinant gE (rgE). The collection of mAbs was well-characterized, and a pair of high-affinity neutralization antibodies (capture mAb 4A2 and detection mAb 4H10) was selected to establish a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the native and recombinant gE. The detection limit of this assay was found to be 1.95ng/mL. Furthermore, a reasonably good correlation between the gE content (as measured by the mAb-based quantitative ELISA) and the virus titer (as measured by the "gold standard" plaque assay) was observed when both assays were performed for tracking the kinetics of virus growth during cell culture. A total of 16 batches of lyophilized VZV vaccine were tested using the newly developed quantitative ELISA and classical plaque assay, demonstrating reasonably good correlation between gE content and virus titer. Therefore, this mAb-based gE quantitative assay serves as a rapid, stable, and sensitive method for monitoring viral antigen content, one additional quantitative method for VZV vaccine process and product characterization. This quantitative ELISA may also serve as a complementary method for virus titering.