Specificity Serum Research Articles

Photoluminescent and multi-phonon resonance Raman scattering dual-mode immunoassays based on CdS nanoparticles for HIgG detection.

A dual-mode immunoassay strategy based on CdS nanoparticles as signal probes with both of photoluminescent (PL) and multi-phonon resonance Raman scattering (MRRS) properties was developed. Simplified structural design and preparation were achieved due to the intrinsic integration of PL and MRRS dual signals in the single-unit CdS nanoprobes. Human immunoglobulin G (HIgG) was sensitively and specifically detected using the proposed PL-MRRS dual-mode strategy. The linear relationship between the HIgG concentration and the intensity of 707nm PL peaks/300cm-1 MRRS peaks under the excitation of 488nm laser wasestablished. The limit of detection was 0.93fgmL-1 for PL and 1.10fgmL-1 for MRRS. In comparison with previous IgG detection methods, the proposed method exhibited prominent advantages in detection sensitivity and workingrange with good stability and repeatability. An internal self-calibration was realized which ensured the accuracy and reliability of detection results. Both results of specificity experiments and serum sample analysis further confirmed the feasibility of thedesigned immunoassay strategy in practical serological detection.

Switchable assembly and function of antibody complexes in vivo using a small molecule

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function invivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities invivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.

Modulation of PTH1R signaling by an extracellular binding antibody.

Parathyroid hormone receptor 1 (PTH1R) is a class B G-protein coupled receptor with key roles in bone development. The receptor signals through both the Gs and Gq G-proteins as well as through β-arrestin in a G-protein independent manner. Current treatments for bone disorders, such as osteoporosis, target the PTH1R but are suboptimal in their efficacy. Monoclonal antibodies represent a major growth area in therapeutics as a result of their superior specificity and long serum half-life. Here, we discovered antibodies against the extracellular domain (ECD) of PTH1R from a phage display library. One of these antibodies, ECD-ScFvhFc, binds PTH1R with high affinity and although it has little or no effect on G-protein dependent receptor signaling, it does reduce PTH1R mediated β-arrestin signaling. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the ECD-ScFvhFc binding site overlapped partially with that of the cognate ligand, PTH. The results of this study demonstrate the suitability of PTH1R as a target for therapeutic antibody development.

An evaluation of ischaemia-modified albumin levels in the development of diabetic foot ulcer.

This study investigated the status of serum ischaemia-modified albumin (IMA) levels in the development of diabetic foot ulcer (DFU) in patients with diabetes mellitus (DM) and in predicting ulcer formation and ulcer grading. Thirty patients with DM, 30 with DFU and 30 healthy controls were included in the study. All participants' demographic characteristics and serum IMA, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and white blood cell (WBC) levels and DFU infection grades were recorded. Nine (30%) patients with DFU were grade 2 according to the grading of International Working Group of the Diabetic Foot, 14 (46.7%) were grade 3 and seven (23.3%) were grade 4. Significant, powerful and positive correlation was determined between serum IMA and albumin-adjusted IMA levels and degrees of DFU (r=0.878 and r=0.846, P<.001 for both). Serum IMA levels in the DFU group were significantly higher than in the DM and control groups (P<.001). The optimal cut-off values for IMA in predicting DFU was 23.5ng/mL (sensitivity 96%, specificity 87% and AUC=0.97, P<.001). Additionally, at a cut-off value of 20.6ng/mL, serum albumin-adjusted IMA differentiated cases of DFU from healthy individuals with 90% sensitivity and 83% specificity (AUC=0.95, P<.001) Serum IMA levels exhibited significant, positive correlation with CRP, ESR, WBC, fasting plasma glucose and HbA1c (r=0.575, r=0.592, r=0.597, r=0.68 and r=0.74, respectively, P<.001). Serum albumin levels were significantly negatively correlated with IMA, CRP, ESR and WBC values (r=-0.49, r=-0.56, r=-0.62 and r=-0.53, respectively, P<.001). Our study findings indicate that together with CRP, ESH, WBC and albumin, increased IMA levels in patients with DM can be useful in the early prevention of DFU development and in predicting the severity of DFU infection.

Spectroscopy goes viral: Diagnosis of hepatitis B and C virus infection from human sera using ATR-FTIR spectroscopy

The development of a new fast, portable and reagent-free diagnostic technique for hepatitis B (HBV) and hepatitis C (HCV) viruses would be an enormous benefit to society. Here, we evalulate the ability of Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy combined with multivariate data analysis to classify human serum samples based on the presence of HBV and HCV infection. Sera samples were prepared using three different methodologies: i) Sera depsoited onto glass cover slips, airdried and placed onto the ATR crystal. ii) Whole serum dried directly onto the ATR crystal. iii) Serum separated into high and low molecular weight compounds using a filtration approach and the high molecular weight fraction placed directly onto the ATR-FTIR diamond window and dried. For methodology i) the Partial Least Squares Discriminate Analysis (PLS-DA) calibration set included 313 (70 %) samples and the validation set 93 (30 %) samples. For HBV vs control the sensitivity and specificity was found to be 69.4 % and 73.7 % (10 latent variables (LV)), respectively. For HCV vs control the sensitivity and specificity was 51.3 % and 90.9 % (LV 11), respectively. In the second set of experiments the serum samples were dried directly onto the ATR diamond. PLS-DA models were constructed using 144 (70 %) samples for the calibration set and tested using an independent test set containing 62 (30 %) samples. For HBV versus control the sensitivity and the specificity was 84.4 % and 93.1 %, respectively (LV 8). For HCV versus control the sensitivity and specificity was 80.0 % and 97.2 %, respectively (LV 9). For HBV versus HCV the sensitivity and the specificity was 77.4 % and 83.3 %, respectively (LV 5). To increase the sensitivity and specificity serum sample was fractionated into high and low molecular weight components. In PLS-DA cross validated model (LV 8) the sensitivity and specificity was 87.5 % and 94.9 %, respectively for HBV vs control (high molecular concentrate). The PLS-DA cross-validated model (LV 8) for HCV vs control high molecular fraction produced a sensitivity and specificity of 81.6 % and 89.6 %, respectively. No linear correlation was observed for sera samples spiked with known viral loads using Partial Least Squares Regression (PLS-R) modelling. Spectra of positive serum (HBV and HCV) showed a strong band observed at 1631 cm −1 , which was absent in the spectra of controls and assigned to the β-pleated sheet protein marker of immunoglobulin (Ig). A band at 1093 cm −1 , observed in spectra of HBV infected sera, was assigned to C C and CO modes of polysaccharide N-glycan from hepatitis B surface antigen (HBsAg). The assignment was confirmed by atomic force microsocpy infrared (AFM-IR) spectroscopy of the isolated protein. This band represents a unique marker for HBV infection. In summary, ATR-FTIR spectroscopy is a powerful tool to study blood composition and identify potential disease markers but care must be taken to ensure that the modelling is not biased by inflammation markers, which may confound diagnosis.

Electrochemical procalcitonin immunoassay based on Au@Ag heterojunction nanorods as labels and CeO2-CuO nanorods as enhancer

Abstract Procalcitonin (PCT) is a kind of protein exists in the human blood. If the human bodies are infected with bacteria, fungi or parasites, the value of PCT in blood will be significantly raised. Thereby, we report an electrochemical immunosensor for measurement of PCT using gold nanoparticle (Au NP) decorated CeO2-CuO (CeO2-CuO-Au) as the platform and thionine modified Au@Ag heterojunction nanorods (Au@Ag-Th) as signal labels. Au NP decorated CeO2-CuO exhibits high surface area, high activity and good biocompatibility, which can immobilize more primary antibodies and promote electron transfer. The Au@Ag-Th provides good active sites to link secondary antibodies, further effectually amplify the current signal. This immunosensor exhibits a well-pleasing response with a limit detection of 0.17 pg mL−1 and linear detection between 0.5 pg mL−1 and 50 ng mL−1. Meanwhile, the high sensitivity, excellent specificity and acceptable human serum samples testing results of this method lead to potential applications in diagnosis of bacterial infections.

Discovery and Characterization Of a Highly Specific Antibody Inhibitor Of Plasma Kallikrein

Abstract The plasma-kallikrein kinin (contact) system contributes to the physiological and pathophysiological reactions of vascular biology. Activation of this pathway causes the release of the potent nonapeptide vasodilator bradykinin following proteolytic cleavage of high-molecular weight kininogen (HMWK) by the serine protease plasma kallikrein (pKal). Normal vascular homeostasis requires regulation of pKal activity by interactions with the C1-inhibitor (C1-INH). This is most apparent in individuals with hereditary angioedema (HAE), a disease characterized by a genetic deficiency in C1-INH that results in persistent pKal activity and consequent bradykinin release. These events can ultimately manifest as unpredictable and potentially fatal attacks of subcutaneous and mucosal edema. Inhibition of pKal proteolytic activity has proven to be a viable therapeutic option for HAE, however there remains an unmet medical need for a long-lasting prophylactic treatment for this disease. Given the potential for target specificity and long serum half-life with antibody therapeutics, we used phage display to select a fully human antibody inhibitor (DX-2930) specific for pKal. In vitro enzyme inhibition and affinity assays demonstrate that DX-2930 is a potent antibody inhibitor of pKal (Ki = 125 pM) that binds the active form of pKal, but not the proenzyme form (prekallikrein) or any other serine protease tested. DX-2930 binding consequently prohibits pKal from cleaving bradykinin out of HMWK and thereby prevents the activation of the bradykinin receptor B2. A 2.1Å resolution X-ray crystallographic structure of pKal complexed to a DX-2930 Fab construct supports these findings, demonstrating that the pKal proteolytic active site is intimately bound - and thereby occluded - by the Fab. This structural analysis provides both a rationale for the potency and specificity of DX-2930, and demonstrates the utility of using antibodies to specifically target an antigen among a family of related proteins (e.g. serine proteases). To further address the functional activity of DX-2930, we demonstrate that subcutaneous dosing of DX-2930 effectively reduces carrageenan-induced paw edema in vivo in rats when injected 24 hours prior to challenge. Combined with our finding that DX-2930 has a prolonged serum residence time in cynomolgus monkeys (t1/2 = 301 hours, SC), the data presented here demonstrates the potential of DX-2930 for the prophylactic inhibition of pKal-mediated diseases, such as HAE. Disclosures: Kenniston: Dyax Corp: Employment. Sexton:Dyax Corp: Employment. Martik:Dyax Corp: Employment, former employee of Dyax Corp Other. Faucette:Dyax Corp: Employment. Viswanathan:Dyax Corp: Employment. Kastrapeli:Dyax: Employment. Kopacz:Dyax Corp: Employment. Conley:Dyax Corp: Employment. Lindberg:Dyax Corp: Employment. Cosic:Dyax Corp: Employment. Comeau:Dyax Corp: Employment. Mason:Dyax Corp: Employment. DiLeo:Dyax Corp: Employment. Chen:Dyax Corp: Employment. Ladner:Dyax Corp: Employment. Edwards:Emerald Biostructures: Employment. TenHoor:Dyax Corp: Employment. Nixon:Dyax Corp: Employment. Adelman:Dyax Corp: Employment.

Research topic: An Investigation into the Specificity or Antiplatelet Serum Prepared in Rabbits

This summer I spent six weeks in the Immunopathology Laboratory in the Pathology Department of the University, investigating this antiplatelet serum. This antiscrum had been prepared by multiple immunisation of several rabbits with human platelets, and I was trying to find out, among other things, if this antiserum was specific for platelets, or if it reacted with other tissues and organs of the human body.

A Comparative Study on Serum Immunoglobulin and Tumor Marker Levels in the Patients with Autoimmune Pancreatitis and Pancreatobiliary Malignancies

Autoimmune pancreatitis (AIP) often occurs with obstructive jaundice in old age in cases of weight loss, mimicking pancreatobiliary cancer. This study aimed to determine the sensitivity and specificity serum IgG, IgG4 and CEA, CA 19-9 levels for the diagnosis of AIP and their ability to distinguish AIP from pancreatobiliary cancer. The level of serums IgG, IgG4 and CEA, CA 19-9 were measured in 413 patients including 125 with AIP, 201 with pancreatic cancer, and 87 with cholangiocarcinoma. Among AIP patients, 43.2% (54/125) showed elevated IgG levels (≥1,800 mg/dL) and 52% (65/125) showed elevated IgG4 levels (≥135 mg/dL). Sensitivity and specificity of elevated serum IgG for diagnosis AIP were 43% and 88% respectively, and 52% and 97%, respectively for elevated serum IgG4. When the cut-off value of serum IgG4 was raised to 270 mg/dL (twice the upper limit of normal), the specificity improved to 100%. About 25% of the AIP patients showed an increased level of CA 19-9 at >37 U/mL and about 12.2% of them showed an increased level of CA 19-9 at >100 U/mL. On the contrary, only 1.8% of the AIP patients showed an increased level of CEA at >6.0 ng/mL. To avoid unnecessary surgeries resulting from a misdiagnosed pancreatobiliary cancer as opposed to AIP, it is necessary to consider both serum immunoglobulin and tumor marker. In particular, because high level of IgG4 (≥270 mg/dL) and CA19-9 (>100 U/mL) are relatively rare in pancreatobiliary cancer and AIP, respectively, they will be helpful in differential diagnosis.

Novel roles for the IgG Fc glycan

IgG antibodies trigger leukocyte activation and inflammation by forming immune complexes that crosslink activating Fcγ receptors (FcγRs). This is essential to combat infection, but detrimental if antibodies target or cross-react with autoantigens. The high specificity and long serum half-life of IgG antibodies confers tremendous therapeutic potential. Indeed, antibodies have been successfully employed to target cancers, autoreactive B cells, and pro-inflammatory cytokines. Conversely, IgG antibodies can also initiate anti-inflammatory responses. In the form of intravenous immunoglobulin (IVIG), IgGs are routinely administered to treat inflammatory autoimmune diseases. Importantly, the N-linked glycans on the IgG Fc are absolutely required for initiating these IgG effector functions. In fact, the Fc glycan composition dictates IgG affinity to individual FcγRs, and in a broader sense, binding to different FcγRs classes: activating, inhibitory, and anti-inflammatory (dendritic cell-specific ICAM-3 grabbing nonintegrin, DC-SIGN). The Fc glycan requirements to initiate and suppress inflammation will be discussed herein.

IgE antibody-specific activity in human allergic disease

IgE antibody concentration, affinity, clonality and specific activity (also known as the allergen-specific IgE to total IgE ratio) influence the translation of IgE responses into clinically evident allergic symptoms following allergen exposure. Reported IgE-specific activity levels >3-4% place allergic individuals undergoing anti-IgE (Omalizumab((R))) therapy at a disadvantage for poor resolution of their allergy symptoms following manufacturer's recommended dosing schemes. We investigated the hypothesis that the specific activity of the IgE antibody response is highly variable with respect to age, allergen specificity and an individual's total serum IgE level. Second, we investigated whether the IgE-specific activity level influences the extent and rate of loss of effector cell mediator release. IgE-specific activity distributions were plotted against age, allergen specificity and total serum IgE using 18,950 paired total IgE and allergen-specific IgE antibody data obtained from the analysis of sera from 3,614 allergic subjects and covering 182 allergen specificities. The fraction of specific IgE antibody of the total serum IgE was dependent on age of the individual, epitope specificity (clonality) and total serum IgE. The youngest group of allergic individuals with the lowest total serum IgE levels tended to have the highest allergen-specific IgE to total IgE ratios. Hymenoptera venom (54%), peanut (33%) and milk (27%) were the three allergen specificities that elicited the highest frequency of IgE-specific activities >4% among sensitized individuals. A prospective double-blind, placebo-controlled clinical study involving anti-IgE treatment of cat-allergic subjects showed IgE-specific activity was remarkably constant over the 16-week course of treatment, despite the up to 8-fold rise in total serum IgE following repetitive Omalizumab administration. Changes in specific and total IgE levels paralleled each other in patients receiving anti-IgE therapy. The fastest rate of reduction in cat allergen-induced basophil histamine release following anti-IgE therapy was observed when the cat-specific IgE to total IgE ratio was <2.5%. This reflected the more rapid loss of surface cat-specific IgE antibody with anti-IgE therapy in allergic individuals who displayed a more diverse IgE antibody repertoire. We conclude that IgE-specific activity is an age-, IgE heterogeneity- and total serum IgE-dependent variable that influences the magnitude of effector cell mediator release, and by inference, ultimate allergic symptom induction.

Increased levels of KL‐6 and subsequent mortality in patients with interstitial lung diseases

KL-6 is a specific marker in patients with interstitial lung diseases (ILDs); however, the relationship between elevated levels of KL-6 and subsequent mortality is not well defined. To determine if elevated serum levels of KL-6 are associated with increased mortality, and to identify the most suitable cut-off level of KL-6 by which to distinguish between good prognosis and poor prognosis, we evaluated the prognostic significance of serum KL-6 levels in patients with stable-state ILDs. Two hundred and nineteen patients diagnosed with ILDs (152 with idiopathic interstitial pneumonia and 67 with collagen disease-associated pulmonary fibrosis) at Tsukuba University Hospital from April 1999 to October 2005 were entered in this study. Serum KL-6 levels in patients with ILDs were measured with a commercially available enzyme immunoassay kit, and these patients were then followed up. During the follow-up period, 58 of the 219 patients died of respiratory failure. Patients who died during this period had higher levels of KL-6 than did those who did not (P = 0.0004). The receiver operating characteristic curve analysis showed 1000 U mL(-1) as the most suitable cut-off level by which to distinguish between the two groups of patients. The 95% specificity serum KL-6 level with poor outcome was 2750 U mL(-1). In univariate and multivariate analysis, elevated serum KL-6 (>1000 U mL(-1)) in the stable state indicated poor prognosis (P = 0.0005, log-rank test; P = 0.0001, Cox proportional hazard model). Elevated KL-6 level may provide simple, yet valuable information by which to identify patients with ILDs who are at increased risk for subsequent mortality.

New Thiocholine Ester Substrates for the Assay of Human Serum Cholinesterase

Several thiocholine alkanoyl esters were newly synthesized and explored as substrates for the assay of human serum cholinesterase after being subjected to the Ellman reaction (Arch Biochem Biophys 1958;74:443-50 and Arch Biochem Biophys 1959;82:70-7). We synthesized thiocholine ester iodides by the method of Renshow et al. (J Am Chem Soc 1938;60:1765-70). We examined solubility in H(2)O, substrate specificity serum for cholinesterase, (spontaneous) self-hydrolysis, storage stability, and reaction conditions for measurement of the activity of the enzyme. Isobutyryl and cyclohexane-carboxyl esters showed the best efficiency for the specific and stable assay of human serum cholinesterase. Aqueous solubility of each was >10 mmol/L, and the reactivity with acetylcholinesterase was negligible. For isobutyryl and cyclohexane-carboxyl esters, respectively, spontaneous hydrolysis in the aqueous phase was approximately 1/25 and approximately 1/175 slower than the enzymatic hydrolysis, and assays with these substrates were linear to 1800 and 3000 U/L, respectively. The K(m) values of these acylthiocholines with human cholinesterase were almost equivalent (6.9 x 10(-3) mmol/L). The substrates were stable in aqueous solution and in the solid state as the iodides for at least 5 years at 5 degrees C. The isobutyrate and cyclohexane-carboxylate of thiocholine are suitable for the specific assay of human serum cholinesterase.

The CA 125 tumour-associated antigen: a review of the literature.

CA 125 is an antigenic determinant on a high-molecular-weight glycoprotein recognized by a monoclonal antibody which was raised using an ovarian cancer cell line as an immunogen. During the last 5 years the studies reviewed in this paper have provided information concerning the nature, distribution and clinical significance of CA 125. The CA 125 determinant is expressed by epithelial ovarian tumours and various other pathological and normal tissues of Müllerian origin. The function of the glycoprotein expressing CA 125 remains unclear but the distribution of the antigen suggests that it may have a physiological role. The highest serum levels of CA 125 are found in ovarian cancer patients, but elevation of serum CA 125 may also be associated with other malignancies and benign and physiological states, including pregnancy, endometriosis and menstruation. Despite limitations of sensitivity and specificity serum CA 125 estimation is of clinical value in the pre-operative diagnosis and monitoring of ovarian malignancy and may be a prognostic indicator for this disease. The role of CA 125 in screening for early-stage ovarian cancer is currently under investigation. Recent reports suggest that serum CA 125 measurement may also be of value as a prognostic indicator in endometrial cancer and as a reflection of disease status in advanced endometriosis.

Monoclonal murine anti-DNP IgE:in vitro histamine release of rat mast cells in the presence of reaginic rat and mouse sera

Mast cells from normal rats and animals reinfected with Nippostrongylus brasiliensis (N.b.) were sensitized in vitro with monoclonal anti-DNP mouse IgE, reaginic mouse serum with ovalbumin (OA) specificity and reaginic rat serum against N.b. The sensitized cells were triggered for the release of histamine with DNP-bovine-serum-albumin (DNP-BSA), OA, N.b.-homogenate and guinea-pig anti-mouse IgE. The histamine release from normal mast cells sensitized with monoclonal mouse IgE was inhibited either with N.b.-reaginic rat serum or OA-reaginic mouse IgE (30 micrograms/10(5) mast cells) completely desensitized mast cells from reinfected rats for specific histamine release in the presence of either N.b.-antigen or DNP-BSA. Anti-mouse IgE which was prepared by monoclonal mouse IgE did not bind to rat mast cells sensitized with rat IgE as was revealed by immunofluorescence experiments. Consequently we observed that anti-mouse IgE failed to trigger the histamine release from mast cells of reinfected rats.