When Assay Format Matters: a Case Study on the Evaluation of Three Assay Formats to Support a Clinical Pharmacokinetic Study

Data generated using various immunoassay methods are an integral part of the development of protein therapeutics. These assays are used in clinical and preclinical studies to establish the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics as well as to assess the immunogenicity properties of a therapeutic. PK assays measure therapeutic levels post-administration which is essential for understanding the effective dose and dose regimen for a therapeutic. Anti-OX40L is a fully humanized monoclonal antibody designed for the potential treatment of an autoimmune disease. The anti-OX40L human PK assay is required to be sensitive, robust, and precise. To address challenges due to assay sensitivity and reproducibility, as well as assay technology limitations, during development of the anti-OX40L human PK assay, three different assays, including an MSD-based electrochemiluminescence assay (ECLA), a fluorometric enzyme-linked immunosorbent assay (ELISA), and a colorimetric ELISA, were evaluated. The MSD-based assay was the most sensitive but posed risk of inter-well signal crosstalk. The fluorescence ELISA fell short on reproducibility. The colorimetric ELISA was ultimately chosen for supporting sample analysis. This paper presents characterization data obtained from each of these assay formats, challenges that were encountered in the development of the assay, and the rationale for selecting the ultimate assay format.     

肺癌标志物流式微球阵列的分析性能评价

目的 通过探讨检测下限、精密度、特异性、方法学比对和线性范围,评价基于流式细胞仪的肺癌标志物流式微球阵列的分析性能。方法 利用流式细胞仪,测试肺癌标志物流式微球阵列试剂盒检测血清中肺癌标志物癌胚抗原（CEA）、细胞角蛋白19片段（Cyfra21-1）和神经元特异性烯醇化酶（NSE）的检测下限、精密度、特异性和线性范围;以蛋白质印迹（Western blotting）法验证抗体识别抗原的专一性;检测血红蛋白、三酰甘油、胆红素对CEA、Cyfra21-1和NSE检测的干扰作用;通过与电化学发光免疫分析法比对,考察了肺癌标志物流式微球阵列的准确性。结果 CEA、Cyfra21-1和NSE的检测下限分别为1.71、3.97、2.27pg/mL,批内精密度均≤10%,批间精密度均≤15%;特异性结果显示,CEA、Cyfra21-1、NSE的配对抗体能分别专一识别抗原,CEA与同源类似物癌胚抗原相关黏附分子6（CEACAM6）、cyfra21-1与重组人细胞角蛋白18（CK18）、NSE与非神经元特异性烯醇化酶（NNE）无明显交叉反应;三酰甘油、胆红素对血清样本检测无显著干扰作用,500 ng/mL的血红蛋白能够明显干扰Cyfra21-1（P<0.05）和NSE（P<0.05）的检测;流式微球阵列和电化学发光免疫分析的CEA、Cyfra21-1、NSE检测结果的相关系数值分别为0.9842、0.9622、0.982 0;CEA、Cyfra21-1、NSE的线性范围分别为355.76 pg/mL～367.74 ng/mL、87.89 pg/mL～107.8 ng/mL、90.12 pg/mL～86.07 ng/mL。结论 肺癌标志物流式微球阵列的分析性能符合要求。     

Development and validation of immunoassays to quantify the half-antibody exchange of an IgG4 antibody, natalizumab (Tysabri®) with endogenous IgG4

Natalizumab is a humanized IgG4 monoclonal antibody which binds human α4 integrin and is approved for treatment of multiple sclerosis and Crohn's disease. Assessment of the in vivo disposition of natalizumab presents a unique assay development challenge due to the ability of human IgG4 antibodies to undergo half-antibody exchange in vivo. Such exchange generates IgG4 molecules of mixed specificity comprising a natalizumab heavy-light chain pair coupled to an IgG4 heavy-light chain pair of unknown specificity. Since exchanged and non-exchanged species cannot be quantified independently using a single enzyme linked immunosorbent assay (ELISA), a novel quantitation strategy was developed employing two ELISAs: one measuring total natalizumab including both intact and exchanged molecules, and the second measuring only intact natalizumab. The presence and amount of exchanged natalizumab in serum is calculated by the difference in values obtained in the two assays. To evaluate assay performance, a control reagent was created from natalizumab and an irrelevant humanized monoclonal IgG4 antibody. Subsequent validation demonstrated that both assays are specific, accurate, and precise within the working ranges of the assays (1.5-10μg/mL for total and 0.5-12μg/mL for intact natalizumab assays). The mean accuracy, intra- and inter-assay precision for both assays were 82-113%, ≤9% and ≤20%, respectively. Additionally, the limits of detection of intact and exchanged natalizumab were established using statistical methods. The utility of the two-assay strategy was confirmed by analyzing samples from a pharmacokinetic study in rats using different variants of natalizumab administered along with another human IgG4 antibody as an exchange partner.     

Therapeutic anti-VEGF antibodies

Vascular endothelial growth factor (VEGF-A) is a key cytokine in the development of normal blood vessels as well as the development of vessels in tumors and other tissues undergoing abnormal angiogenesis. Here, we review the molecular engineering of two humanized antibodies derived from a common mouse anti-VEGF antibody--bevacizumab, a full-length IgG1 approved for the treatment of specified cancer indications, and ranibizumab, an affinity-matured antibody Fab domain approved for use in age-related macular degeneration (AMD). In clinical trials and as FDA-approved therapeutics, these two anti-VEGF antibodies, bevacizumab (Avastin anti-VEGF antibody) and ranibizumab (Lucentis anti-VEGF antibody), have demonstrated therapeutic utility in blocking VEGF-induced angiogenesis.     

Evaluation of an immunoassay for human-specific quantitation of therapeutic antibodies in serum samples from non-human primates

Pharmacokinetic characterization of therapeutic antibodies plays an important role during preclinical and clinical development. However, accurate pharmacokinetic evaluation of therapeutic antibodies in serum samples from non-human primates is often complicated by insufficient specificity of the assays to measure drug levels. The present paper describes the use of a murine monoclonal antibody in an immunoassay format to specifically and quantitatively measure human therapeutic antibodies in serum from non-human primates. This murine antibody is directed against a unique epitope on the constant region CH2 domain of all isotypes of human immunoglobulin G (IgG). The antibody, designated anti-human Fcγ-pan: R10Z8E9, does not cross-react with serum from mouse, rat, and the non-human primates marmoset, rhesus macaque, cynomolgus monkey and baboon when using an enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance technology (Biacore) format for measurement of the therapeutic antibody. Use of the antibody anti-human Fcγ-pan: R10Z8E9 as capturing and detection reagent allowed human-specific quantitation of total therapeutic antibody anti-IGF-1R in spiked cynomolgus monkey serum via a Sandwich ELISA format. In contrast, a commercially available polyclonal antibody (PAB) directed to the Fcγ fragment of human IgG only specifically measured the therapeutic antibody in buffer samples, but not in serum from cynomolgus monkeys. This generic human IgG assay was already applied in several pharmacokinetic studies in cynomolgus monkeys to determine serum levels of different therapeutic antibodies, including the anti-IGF-1R. Validation of the assay for a humanized IgG1 therapeutic antibody against a membrane protein revealed a lower limit of quantitation of 8 ng/mL in undiluted serum. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 15%. Dilutional linearity was evidenced by a recovery of 98.7–114% of expected concentrations. In conclusion, the monoclonal antibody anti-human Fcγ-pan: R10Z8E9 provides a standard means for human-specific quantitation of therapeutic antibodies with high sensitivity in serum samples from non-human primates in a generic human IgG assay.     

心源性哮喘与肺源性哮喘患者BNP水平的比较及意义

目的探讨脑钠肽(BNP)在心源性哮喘与肺源性哮喘鉴别诊断中的价值。方法选取2005年10月至2008年6月上海市青浦区中医医院收治的心源性哮喘36例与肺源性哮喘患者23例及22例无心肺疾病患者为对照,采用电化学发光免疫测定技术检测患者血浆中BNP的水平。结果心源性哮喘、肺源性哮喘患者与对照组血浆中的BNP水平分别为(1338.96±900.88)、(147.84±56.08)、(72.47±23.18)pg/mL,心源性哮喘与肺源性哮喘患者间差异有统计学意义(P<0.05)。结论血浆BNP水平检测有助于快速准确地诊断心源性哮喘和肺源性哮喘。     

Quantification of oltipraz using liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in rat plasma

An assay method for the determination of oltipraz, a candidate drug for the treatment of liver fibrosis and liver cirrhosis, was developed in rat plasma using a fast-flow protein precipitation (FF-PPT) method coupled with LC-MS/MS for quantification to reduce the labor and to improve the speed of analysis. The applicability of the assay to pharmacokinetic studies was also evaluated. Oltipraz and ethyl-oltipraz, an internal standard (IS), were analyzed by multiple reaction monitoring (MRM) at m/z transitions of 227 → 193 and 241 → 174, respectively. A lower limit of quantification (LLOQ) of 20 ng/mL was observed, with a linear dynamic range from 20 to 4000 ng/mL (R > 0.997). The accuracy, precision, dilution, recovery, and stability of the assay were deemed acceptable according to FDA guidelines. Oltipraz concentrations were measured successfully in plasma samples up to 12 h post-dose in rats that had received an oral dose of 60 mg/kg. The findings indicate that the assay method is rapid and sensitive to oltipraz, showing applicability for pharmacokinetics (PK) studies of oltipraz in other small animals, including rats.     

Preclinical pharmacokinetics, interspecies scaling, and tissue distribution of humanized monoclonal anti-IL-13 antibodies with different IL-13 neutralization mechanisms

Numerous animal and in vitro studies suggest that neutralization of IL-13 is an attractive approach for therapeutic intervention in asthma. In this paper we describe preclinical pharmacokinetics (PK), interspecies scaling, and biodistribution of two humanized anti-IL-13 IgG1 monoclonal antibodies, Ab-01 and Ab-02, with different IL-13 neutralization mechanisms. PK parameters of Ab-01 and Ab-02 following IV or SC dosage to mouse, rat, cynomolgus monkey, and sheep, were similar. After IV administration, the elimination of anti-IL-13 antibodies was slow in all species tested and the serum clearance ranged from 0.13 mL/h/kg in monkeys to 0.81 mL/h/kg in mice. Both anti-IL-13 antibodies appeared to be confined primarily to the vascular space, as volume of distribution was relatively small (<120 mL/kg) in all species and tissue-to-serum concentration ratios (in mice and rats) were low (<0.5) in the tissues examined. The elimination half-life ranged from 3-6 days in mice to 14-17 days in monkey and sheep. In monkeys, PK parameters appeared to be approximately linear in the 1-100 mg/kg dose range. Following SC administration, the bioavailability of anti-IL-13 antibodies was 60-90% in all species tested. PK profile of Ab-02 in the model of acute airway inflammation (induced by Ascaris challenge) was, in general, similar to that in unchallenged monkeys; however, volume of distribution and clearance tended to decrease in Ascaris-challenged animals. Allometric scaling suggested that anti-IL-13 antibodies would likely to have a favorable PK profile, such as slow clearance and long terminal half-life, following IV or SC administration to humans.     

Generation of monoclonal antibodies against a soluble form of lectin-like oxidized low-density lipoprotein receptor-1 and development of a sensitive chemiluminescent enzyme immunoassay

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), expressed prominently in atherosclerotic lesions, is cleaved and released as a soluble LOX-1 (sLOX-1), which is a specific biomarker to diagnose acute coronary syndrome (ACS) at an early stage. Although sLOX-1 levels in patient's blood were successfully measured with our previously established enzyme-linked immunosorbent assay (ELISA), the assay was not sensitive enough to detect normal serum levels of sLOX-1 in healthy human subjects. We therefore developed sensitive and specific monoclonal antibodies (mAbs) against sLOX-1 in order to establish a more sensitive immunoassay. Mice were immunized with recombinant human LOX-1 extracellular domain. mAbs were subsequently generated by standard myeloma cell fusion techniques with a novel screening method using time-resolved fluorescence immunoassay. Using two anti-human sLOX-1 mAbs and alkaline phosphatase as a label, a sandwich chemiluminescent enzyme immunoassay (CLEIA) was developed. In total, nine mAbs were obtained. The dissociation constant (K_d) values of these mAbs for sLOX-1 were 0.12–1.32 nM. Characteristics of these mAbs were estimated and the best combination for CLEIA was selected. The newly established CLEIA could determine sLOX-1 levels as low as 8 pg/mL, and thus, was sensitive enough to measure serum sLOX-1 levels in normal human subjects and to evaluate subtle differences. Values for sLOX-1 measured by monoclonal CLEIA and polyclonal ELISA were highly correlated (r² = 0.7594, p < 0.0001). Area under the curve values of the receiver-operating characteristic curves in detecting ACS were 0.948 and 0.978 for monoclonal CLEIA and polyclonal ELISA, respectively. Thus, a more sensitive sLOX-1 CLEIA was established using newly developed mAbs against sLOX-1. In addition to its advantage in early diagnosis of ACS, this assay may also be useful in predicting cardiovascular disease risk in disease-free subjects.     

A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity

Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC₈₀)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.KEY WORDS: ADCC, benralizumab, cell-based assay, mechanism of action, neutralizing antibody     

Development and Validation of Electrochemiluminescence Assays to Measure Free and Total sSLAMF7 in Human Serum in the Absence and Presence of Elotuzumab

Elotuzumab is a first in class humanized IgG1 monoclonal antibody for the treatment of multiple myeloma (MM). Elotuzumab targets the glycoprotein signaling lymphocyte activation molecule family 7 (SLAMF7, also described as CS1 or CRACC) which is expressed on the surface of myeloma cells and a subset of immune cells, including natural killer cells. A soluble version of SLAMF7 (sSLAMF7) has also been reported in MM patients but has not been evaluated as a potential biomarker following therapeutic intervention. In order to measure serum levels of sSLAMF7, two immunoassays were developed to monitor changes in circulating sSLAMF7 before and after elotuzumab treatment. Free (drug-unbound) and total (drug-bound and unbound) electrochemiluminescence (ECL) ELISA assays were developed and validated following a fit for purpose (FFP) methodology. Both assays met analytical acceptance criteria for precision, drug interference, dilution linearity, spike recovery, parallelism, and stability. Both exhibited the range and sensitivity necessary to measure clinical samples with an LLOQ of 51.2 pg/mL and ULOQs of 160 (free) and 800 ng/mL (total). Previously described assays were unable to detect sSLAMF7 in healthy individuals. However, due to the increased sensitivity of these new assays, low but measurable sSLAMF7 levels were detected in all normal healthy sera evaluated and were significantly elevated in MM patients. Cohort statistics revealed a significant increase of circulating sSLAMF7 in MM patients versus normal controls and both significant decreases in free and increases in total levels of protein post-elotuzumab treatment.     

Investigation of the mechanism of drug-induced autoimmune hemolytic anemia in cynomolgus monkeys elicited by a repeated-dose of a humanized monoclonal antibody drug

We investigated the mechanism of hemolytic anemia detected in a repeated-dose toxicity study using cynomolgus monkeys that were treated with a humanized antibody drug. This drug was an IgG1 monoclonal antibody (MoAb) that binds to the human HM1.24 antigen named anti-HM1.24 MoAb. The presence of the HM1.24 antigen on the erythrocyte membranes and the erythrocyte agglutination following the addition of anti-HM1.24 MoAb was examined. In addition, an indirect Coombs' test, a hemolysis assay and the measurement of anti-single stranded-DNA antibodies were performed using test animal serum or plasma. The specific binding of FITC- and 125I-labeled anti-HM1.24 MoAb to the erythrocyte membrane was not observed. HM1.24 antigen was not identified on the erythrocyte membranes. However, a high concentration (more than 713 microg/mL) of anti-HM1.24 MoAb hemagglutinated the erythrocyte suspensions. The cause of this agglutination was unclear, but it is assumed that the non-specific binding and/or adhesion caused the direct agglutination. In the examination using test serum from the anemic monkeys, a positive reaction in the indirect Coombs' test was noted. Moreover, in these Coombs' test-positive animals, the production of anti-single stranded-DNA antibodies was sequentially increased. In the female monkey sacrificed in extremis due to severe anemia, an in vitro hemolytic reaction was detected attributable to complement activation. From these results, the hemolytic anemia detected in the repeated-dose toxicity study was diagnosed as a drug-induced autoimmune hemolytic anemia (AIHA) and the primary cause was assumed to be production of IgG class anti-erythrocyte autoantibodies.     

Targeted pharmacotherapy of retinal diseases with ranibizumab

Diseases of retinal and/or choroidal blood vessels are the most prevalent causes of moderate and severe vision loss in developed countries. Vascular endothelial growth factor (VEGF)-A plays a critical role in the pathogenesis of many of these diseases. Ranibizumab is a humanized antigen-binding fragment that binds all isoforms of VEGF-A. Intraocular injections of ranibizumab cause significant visual improvement in approximately 40% of patients with choroidal neovascularization due to age-related macular degeneration (AMD). Pilot trials have indicated that intraocular injections of ranibizumab also provide benefits in patients with macular edema due to diabetic retinopathy or retinal vein occlusions. Based upon several case series, bevacizumab, a full-length humanized monoclonal antibody that binds all isoforms of VEGF-A, improves vision in patients with choroidal neovascularization due to AMD and other diseases. Case series also suggest that bevacizumab can cause regression of retinal neovascularization in patients with proliferative diabetic retinopathy. Taken together, results with ranibizumab and bevacizumab suggest that potent antagonists of VEGF will provide the foundation of treatment for a wide variety of diseases complicated by retinal or choroidal neovascularization, or by excessive vascular leakage leading to macular edema.     

The association of serum FGF23 and non‐alcoholic fatty liver disease is independent of vitamin D in type 2 diabetes patients

Recent studies have shown that circulating fibroblast growth factor (FGF) 23 and vitamin D levels are closely correlated with insulin resistance. The aim of this study was to investigate the relationship among serum FGF 23 levels, serum 25‐hydroxyvitamin D [25(OH)D] levels, and non‐alcoholic fatty liver disease (NAFLD) in Chinese patients with type 2 diabetes mellitus (T2DM). This study enrolled 331 hospitalized T2DM patients (209 patients with NAFLD and 122 patients without NAFLD). Serum FGF23 levels were measured using a sandwich enzyme‐linked immunosorbent assay. Serum 25(OH)D levels were determined by an electrochemiluminescence immunoassay. NAFLD was diagnosed by hepatic ultrasound, and the fatty liver index (FLI) was calculated to quantify hepatic steatosis. Results showed that T2DM patients with NAFLD had significantly higher serum FGF23 levels (44.17 [37.92‐51.30] pg/mL vs 40.21 [34.07‐48.33] pg/mL, P = .002), but lower serum 25(OH)D levels (16.43 [12.70‐21.37] ng/mL vs 19.59 [13.78‐26.26] ng/mL, P = .002) than those without NAFLD. Moreover, the incidence rate of NAFLD increased with increasing serum FGF23 levels and decreased with increasing 25(OH)D levels (both P < .05). Logistic regression analysis showed that both serum FGF23 and 25(OH)D levels were independent factors for NAFLD (both P < .05). Furthermore, a multiple stepwise regression analysis also revealed that both serum FGF23 and 25(OH)D levels were independently correlated with FLI (both P < .01). In conclusion, both high FGF23 and low vitamin D levels showed an independent relationship with NAFLD in Chinese T2DM patients, indicating that FGF23 and vitamin D function via different regulatory pathways in the liver.     

Characterization of antibody–polyol interactions by static light scattering: Implications for physical stability of protein formulations

In this study, the nature of interactions between monoclonal antibodies and polyols was studied using static light scattering. Solutions of mAb-U and mAb-P (4–12 mg/mL) were analyzed using static light scattering in buffer, 10% w/v trehalose and ethylene glycol solutions at pH 5.0, 7.0 and 9.0. Mechanical stress studies were conducted by shaking the mAb-U solutions (5 mg/mL, pH 5.0, 7.0 and 9.0) and mAb-P solutions (5 mg/mL, pH 7.0) at 200 rpm for 5 days at 25 °C. Addition of trehalose and ethylene glycol resulted in a decrease in the attractive interactions between mAb-U molecules at pH 7.0 and 9.0, and at pH 9.0 between mAb-P molecules. At a higher ionic strength (300 mM, pH 5.0) trehalose and ethylene glycol decreased attractive interactions for both mAbs. Mechanical stress studies showed higher aggregation of mAb-U in trehalose solutions than ethylene glycol and buffer solutions at pH 7.0 and 9.0. A converse trend was seen for mAb-P at pH 7.0. This study showed that polyols, conformational stabilizers or destabilizers, decrease attractive interactions between protein molecules. The decrease is a result of masking of the hydrophobic sites on a protein as polyols can have favorable hydrophobic interactions with the surface exposed hydrophobic groups.     

Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion

An HPLC procedure was validated for determining the purity with respect to the charge variant distribution of the recombinant monoclonal antibody (MAb) IDEC-C2B8 by high-performance ion-exchange chromatography. Papain was used to fragment the molecule into Fab and Fc fragments prior to chromatographic analysis. Fragmentation allowed the resolution of the variants arising from the cyclization of glutamine to pyroglutamate at the amino-terminus of the light and heavy chains (Fab-pE/Q variants) from the variants resulting from the processing of the carboxy-terminal lysine residues of the heavy chains (Fc-Lys variants). The assay demonstrated good linearity, yielding correlation coefficients of >0.99 for total protein, Fc-Lys variants and Fab-pE/Q variants. Recovery of total protein from the column was 95.7%. Sample recovery studies demonstrated a mean accuracy of 102% for a Fab fragment over the range 2–10% of the total protein. The limit of detection was 0.2 μg and 0.1 μg for Fc and Fab variants, respectively. The repeatability of the assay and intermediate precision had relative standard deviation (RSD) values of <1%. Parameters of the papain digest (time, digest stability, reagent stability, pH and papain vendor) and of the chromatography (mobile phase pH, stability, buffer concentration, and column lot and aging) were evaluated for robustness and determined to be acceptable. Data are presented demonstrating the suitability of the assay for determining the product purity of a recombinant MAb.     

Simultaneous determination of efavirenz, rifampicin and its metabolite desacetyl rifampicin levels in human plasma

A simple and rapid isocratic, high performance liquid chromatography (HPLC) assay employing solid phase extraction (SPE) for the simultaneous determination of the anti HIV drug, efavirenz, the anti-tuberculosis drug, rifampicin and the desacetyl metabolite of rifampicin in plasma from HIV/tuberculosis infected patients has been developed. Using a Zorbax SB-Phenyl reverse-phase analytical column with UV detection, good separation and detection of the drugs was attained within a 10 min run time. Intra- and inter-assay precision RSD values were found to be less than 15% at the concentrations examined (0.1-20 μg/mL). The LOQ was found to be 0.1 μg/mL for each agent and the assay was found to generate a linear response up to 20 μg/mL.This low cost assay can accurately detect efavirenz and rifampicin concentrations within a clinically relevant concentration range using standard chromatography equipment, making it particularly applicable to resource-limited settings.     

Development of sensitive colorimetric capture elisas for Clostridium botulinum neurotoxin serotypes A and B.

Sensitive and specific enzyme-linked immunosorbent assays were developed to detect Clostridium botulinum neurotoxin serotypes A (BoNT A) and B (BoNT B) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kDa C-fragments of each toxin. Standard curves were linear over the range of 0.1-10 ng mL. Detection was possible at 0.2 ng mL (20 pg/well) and accurate quantitation at 0.5 ng/mL (50 pg well) in assay buffer and 10% human serum. Variations between triplicates was typically 5-10%. Less than 1% cross reactivity occurred between other serotypes when each assay was performed against serotypes A, B and E. When tested against toxins complexed to their associated nontoxic proteins, interference was absent (BoNT B) or < 25% (BoNT A). These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other reported assays of similar sensitivity.     

Ranibizumab: in macular oedema following retinal vein occlusion

Ranibizumab (Lucentis?), a recombinant humanized IgG(1) κ isotype monoclonal antibody fragment, is approved in the US for the treatment of macular oedema following retinal vein occlusion (RVO). It binds to the receptor-binding site of active forms of vascular endothelial growth factor-A, inhibiting its biological activity. In two large, well designed, phase III trials in patients with macular oedema following branch RVO (BRVO) or central RVO (CRVO), monthly intravitreal injections of ranibizumab 0.5 mg were associated with significantly greater improvement from baseline in mean best-corrected visual acuity letter score (measured on the Early Treatment Diabetic Retinopathy Study chart) in the study eye at 6 months (primary endpoint) than sham injections. Moreover, ranibizumab was significantly more effective than sham injections with regard to improvements in central foveal thickness at 6 months, as well as several other visual acuity measures. Ranibizumab was generally well tolerated in patients with macular oedema following CRVO or BRVO. Overall, the most common adverse events with ranibizumab were consistent with the adverse event profile previously reported in patients with age-related macular degeneration.     

Leptin and IL-8: Two novel cytokines screened out in childhood lead exposure

Lead is a toxic heavy metal with many recognized adverse health side effects. The central nervous system is the main target of lead toxicity. Although many studies on lead toxicity were conducted, the mechanism of lead toxicity remains uncertain. One possible attribution is the immature blood–brain barrier that causes lead exposure in children. Few studies have investigated the cytokine changes caused by this exposure. Novel cytokines were detected by RayBio^® Human Cytokine Antibody Array and validated by enzyme-linked immunosorbent assay. Several children were admitted to West China Second University Hospital, after a serious lead pollution event in longchang, Sichuan, China. A total of 4 children with elevated blood lead levels (BLLs) and 4 children with low BLLs were randomly chosen in the discovery set, and 40 children with elevated BLLs and 40 children with low BLLs were included in the validation set. Leptin and interleukin-8 (IL-8) were identified to be significantly different between children with elevated and low BLLs via RayBio^® Human Cytokine Antibody Array. In the validation set, IL-8 was higher in children with elevated BLLs [median(P₂₅–P₇₅), 117.69(52.31–233.63) pg/mL] than in children with low BLLs [median(P₂₅–P₇₅): 17.70(10.75–26.52) pg/mL] (p = 0.000). Leptin was lower in children with elevated BLLs [median(P₂₅–P₇₅): 1658.23(1421.86–2606.55) pg/mL] than in children with low BLLs [median(P₂₅–P₇₅): 4168.68(3246.32–4744.94) pg/mL] (p = 0.000). In children with low BLLs, leptin was higher in children with BLLs < 3 μg/dL (N = 7) [median(P25–P75): 7220.86(4265.72–7555.15) pg/mL] than in children with BLL ≥ 3 μg/dL (N = 33) [median(P₂₅–P₇₅): 4103.86(3163.40–4678.34) pg/mL] (p = 0.026); IL-8 was significantly different in children with BLL < 4 μg/dL (N = 13) [median(P₂₅–P₇₅): 12.49(8.25–14.86) pg/mL] than in children with BLL ≥ 4 μg/dL (N = 27) [median(P₂₅–P₇₅): 21.98(13.64–33.50) pg/mL] (p = 0.013). The results defined specific changes in cytokine expressions to lead exposure, which can be used to explore the mechanism of lead toxicity and monitor lead exposure.