A piezoelectric immunosensor using hybrid self-assembled monolayers for detection of Schistosoma japonicum

Background

The parasite Schistosoma japonicum causes schistosomiasis disease, which threatens human life and hampers economic and social development in some Asian countries. An important lesson learned from efforts to reduce the occurrence of schistosomiasis is that the diagnostic approach must be altered as further progress is made towards the control and ultimate elimination of the disease.

Methodology/Principal Findings

Using mixed self-assembled monolayer membrane (mixed SAM) technology, a mixture of mercaptopropionic acid (MPA) and mercaptoethanol (ME) was self-assembled on the surface of quartz crystals by gold-sulphur-bonds. Soluble egg antigens (SEA) of S. japonicum were then cross-linked to the quartz crystal using a special coupling agent. As compared with the traditional single self-assembled monolayer immobilization method, S. japonicum antigen (SjAg) immobilization using mixed self-assembled monolayers exhibits much greater immunoreactivity. Under optimal experimental conditions, the detection range is 1∶1500 to 1∶60 (infected rabbit serum dilution ratios). We measured several infected rabbit serum samples with varying S. japonicum antibody (SjAb) concentrations using both immunosensor and ELISA techniques and then produced a correlation analysis. The correlation coefficients reached 0.973.

Conclusions/Significance

We have developed a new, simple, sensitive, and reusable piezoelectric immunosensor that directly detects SjAb in the serum. This method may represent an alternative to the current diagnostic methods for S. japonicum infection in the clinical laboratory or for analysis outside the laboratory.     

Development of a Disperse Dye Immunoassay Technique for Detection of Antibodies against Neospora caninum in Cattle

In this study a disperse dye immunoassay method was standardized and evaluated for detection of antibodies against Neospora caninum in cattle. Sera from 150 cattle with a recent history of abortion were collected and tested by commercial ELISA kit and a standardized in-house dye immunoassay system. The positivity rate for the sera used in this study was 34.6% for the disperse dye immunoassay (DDIA) compared to 32% obtained by ELISA kit. This study showed no significant difference between DDIA and ELISA. The results indicated that the DDIA provide an economic, simple, rapid and robust test for detection of N. caninum infection in cattle.     

Serological diagnosis of petriellidiosis (allescheriosis) II. Indirect (passive) hemagglutination assay for antibody to polysaccharide antigens of pertriellidium (allescheria) boydii and monosporium apiospermum

The indirect (passive) hemagglutination assay (IHA) was utilized for detection of humoral antibody resulting from an experimental or a natural petriellidiosis. Polysaccharide antigens from three strains ofP. boydii and three strains ofM. apiospermum were isolated from culture filtrates of these fungi by anion-exchange chromatography and were coupled to sheep erythrocytes using chromium chloride. Rabbits were inoculated by the subcutaneous route with viable spores of these fungi and were bled periodically to obtain serum samples. IHA assay of serial specimens revealed varying levels of humoral antibody. Antibody stimulation by the subcutaneous route of inoculation appeared advantageous inasmuch as high IHA titers (1∶1024 to 1∶4096) could be achieved without endangering the life of the animal or exposing investigators to draining lesions. When serum specimens from six human beings with culturally proven petriellidiosis were tested by the IHA method, five were shown to have antibody levels of 1 ∶ 16 to 1 ∶ 512. IHA assay of serum samples obtained from normal human beings showed measurable levels (1 ∶ 4 to 1 ∶ 32) of antibody in 29 of 30 specimens. A serum pool composed of the higher titered specimens from the normal group was adsorbed with killed whole spores ofM. apiospermum, and the attached serum components were eluted by altering the pH. The immune globulin that had adsorbed to the spores was found, by immunoelectrophoresis, to be IgG. An indirect hemagglutination inhibition assay, developed from the IHA assay, provided a means for determining whether heterologous fungus antigens were capable of cross-reacting with petriellidium antibody. Soluble antigens from several other genera of fungi failed to react with petriellidium antibody in the IHA assay, suggesting that the reaction of petriellidium antibody with homologous antigen was genus specific.     

Aptamer-based bio-barcode assay for the detection of cytochrome-c released from apoptotic cells

The recently developed bio-barcode (BBC) assay using polymerase chain reaction (PCR) to generate signals has been shown to be an extraordinarily sensitive method to detect protein targets. The BBC assay involves a magnetic microparticle (with antibody to capture the target of interest) and gold nanoparticle (with recognition antibody and thiolated single-stranded barcode DNAs) to form a sandwich around the target. The concentration of target is determined by the amount of barcode DNA released from the nanoparticles. Here we describe a modification using aptamers to substitute the gold nanoparticles for the BBC assay. In this study, we isolated a 76-mer monoclonal aptamer against cytochrome-c (cyto-c) and this single-stranded DNA in defined 3D structure for cyto-c was used in the BBC assay for both recognition and readout reporting. After magnetic separation, the aptamer was amplified by PCR and this aptamer-based barcode (ABC) assay was sensitive enough to detect the cyto-c in culture medium released from the apoptotic cells after drug treatment at the picomolar level. When compared to the conventional cyto-c detection by Western blot analysis, our ABC assay is sensitive, and time for the detection and quantification with ready-made probes was only 3 h.     

Utilization of ELISA Using Thioredoxin Peroxidase-1 and Tandem Repeat Proteins for Diagnosis of Schistosoma japonicum Infection among Water Buffaloes

Background

The presence of animal reservoirs in Schistosoma japonicum infection has been a major obstacle in the control of schistosomiasis. Previous studies have proven that the inclusion of control measures on animal reservoir hosts for schistosomiasis contributed to the decrease of human cases. Animal surveillance should therefore be included to strengthen and improve the capabilities of current serological tests.

Methodology/Principal Findings

Thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) were initially evaluated against human sera. The previous test showed high sensitivity and specificity for antibody detection against SjTPx-1 and Sj7TR. In this study, the immunodiagnostic potential of these recombinant proteins was evaluated using enzyme-linked immunoassay on 50 water buffalo serum samples collected in Cagayan, the Philippines as compared with the soluble egg antigen (SEA). For specificity, 3 goat serum samples positive with Fasciola hepatica were used and among the antigens used, only SEA showed cross-reaction. Stool PCR targeting the S. japonicum 82 bp mitochondrial NAD 1 gene was done to confirm the true positives and served as the standard test. Twenty three samples were positive for stool PCR. SjTPx-1 and Sj1TR gave the highest sensitivity among the recombinant proteins tested for water buffalo samples with 82.61% and 78.26% respectively which were higher than that of SEA (69.57%).

Conclusions/Significance

These results prove that SjTPx-1 works both for humans and water buffaloes making it a good candidate antigen for zoonotic diagnosis. Sj1TR showed good results for water buffaloes and therefore can also be used as a possible candidate for detecting animal schistosome infection.     

Electrophoresis '82 : Advanced methods. Biochemical & clinical applications. Proceedings of the International Conference on Electrophoresis,Athens, April 1982 Edited by D. Stathakos Walter de Gruyter; Berlin, 1983 xvi + 867 pages. DM 260.00

A competitive solid-phase immunoassay for the determination of testosterone in serum samples using time-resolved fluorescence is described. The solid phase is a testosterone-3-(O-carboxymethyl)-oxime-ovalbumin conjugate coated to polystyrene microtiter strips. Europium-labelled polyclonal and monoclonal antibodies against testosterone-3-(O-carboxymethyl)-oxime-bovine serum albumin were compared. Their behavior was quite similar although the polyclonal antibody was more sensitive, giving a detection limit of 15 fmol testosterone per assay. Correlation with RIA was very good (r = 0.982 and y = ?0.150 + 0.969x).     

Integrated microfluidic magnetic immunosensor for quantification of human serum IgG antibodies to Helicobacter pylori

In this paper, we have developed and characterized a microfluidic magnetic immunosensor coupled to a gold electrode for the rapid and sensitive quantification of human serum IgG antibodies to Helicobacter pylori. This microorganism cause peptic ulcers and chronic gastritis, affecting around the 10% of the world population. The sensor was completely automated and the antibodies detection in serum samples was carried out using a non-competitive immunoassay based on the use of purified H. pylori antigens that are immobilized on magnetic microspheres 3-aminopropyl-modified. The magnetic microbeads were injected into microchannel devices and manipulated for an external removable magnet. The IgG antibodies in human serum sample are allowed to react immunologically with the immobilized antigens, and the bounded antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP and an electroactive product was detected on gold layer electrode at 0.250 V. The response current obtained from the product of enzymatic reaction is directly proportional to the activity of the enzyme and, consequently, to the amount of IgG antibodies to H. pylori in serum samples. The electrochemical detection can be done within 1 min and total assay time was 25 min. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.37 and 2.1 U mL⁻¹, respectively, and the within- and between-assay coefficients of variation were below 5%. Our results indicate the potential usefulness of our fabricated microbiochip for the early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.     

Enzyme-linked immunoassay of ursodeoxycholic acid in serum.

A sensitive and specific enzyme-linked immunoassay for the measurement of ursodeoxycholic acid in human serum was developed. Ursodeoxycholic acid conjugated to alkaline phosphatase (from calf intestine) was used as a tracer. An antiserum to ursodeoxycholic acid serum was raised in rabbits using ursodeoxycholic acid--bovine serum albumin conjugate as an antigen. The binding required 1 hr at 42 degrees C; separation of the bound tracer was achieved by addition of a second antibody, and alkaline phosphatase activity of this bound complex was measured colorimetrically. The ratio of bound to total enzyme activity decreased linearly with a logarithmic increase in ursodeoxycholic acid concentration from 20 to 900 pmol. The specificity and sensitivity of this enzyme-linked immunoassay were similar to those of a radioimmunoassay reported previously. The serum ursodeoxycholic acid levels measured by this method correlated well with those determined by gas--liquid chromatography and radioimmunoassay. Based on these findings, this enzyme-linked immunoassay of bile acid might be useful as a tool for the routine clinical analysis of serum bile acids.     

Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)₂ antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.     

Diagnostic test accuracy for detecting Schistosoma japonicum and S. mekongi in humans: A systematic review and meta-analysis

BackgroundMost of national schistosomiasis elimination programmes in Asia are relying on stool examination, particularly Kato Katz stool examination technique for regular transmission monitoring. However, the Kato-Katz technique has shown low sensitivity for the detection of light-intensity infections, and therefore highly sensitive diagnostic tools are urgently required to monitor prevalence of infection in low transmission settings. The objective of this systematic review was to evaluate and synthesize the performance of diagnostic tests for detecting Schistosoma japonicum and S. mekongi infection in people living in endemic areas.Methodology/Principal findingsWe comprehensively searched these nine electronic databases and other resources until July 2019, with no language or publication limits: PubMed, EMBASE, MEDLINE, Web of Science, BIOSIS Citation Index, HTA, CINAHL PLUS, The Cochrane Library, and PsycINFO. We included original studies that assessed diagnostic performance using antibody, antigen, and molecular tests with stool examination test as a reference standard. Two reviewers independently extracted a standard set of data and assessed study quality. We estimated the pooled estimates of sensitivity and specificity for each index test. We used diagnostic odds ratio to determine the overall accuracy and hierarchical summary receiver operating characteristics (HSROC) curve to assess the index tests performance.Fifteen studies (S. japonicum [n = 13] and S. mekongi [n = 2]) testing 15,303 participants were included in the review. Five studies reported performance of enzyme-linked immunosorbent assay (ELISA), seven studies reported indirect hemagglutination assay (IHA), and four studies reported polymerase chain reaction (PCR) for detecting S. japonicum. The pooled sensitivity and specificity were 0.93 (95% CI: 0.84–0.98) and 0.40 (95% CI: 0.29–0.53) for ELISA, 0.97 (95% CI: 0.90–0.99) and 0.66 (95% CI: 0.58–0.73) for IHA, and 0.89 (95% CI: 0.71–0.96) and 0.49 (95% CI: 0.29–0.69) for PCR respectively. A global summary indicated the best performance for IHA, closely followed by ELISA. We were unable to perform meta-analysis for S. mekongi due to insufficient number of studies.Conclusions/SignificanceIHA showed the highest detection accuracy for S. japonicum. Further studies are needed to determine the suitable diagnostic methods to verify the absence of transmission of S. mekongi and also to compare detection accuracy against more sensitive reference standards such as PCR.     

Immunomagnetic capture and colorimetric detection of malarial biomarker Plasmodium falciparum lactate dehydrogenase

We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/μl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4–6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody.     

Schistosoma japonicum translationally controlled tumour protein,which is associated with the development of female worms,as a target for control of schistosomiasis

Schistosomiasis is a globally important helminthic disease of both humans and animals, and is the second most common parasitic disease after malaria. Although praziquantel is extensively used for treatment of parasitic diseases, drug resistance has been reported. Therefore, new drugs and effective vaccines are needed for continuous control of schistosomiasis. Eggs produced by schistosomes are responsible for the occurrence and spread of schistosomiasis. Revealing the reproductive mechanism of schistosomes will help to control this disease. In this study, the proteomic profiles of single-sex infected female worms and bisexual infected mature female worms of Schistosoma japonicum at 18, 21, 23 and 25 days p.i. were identified with isobaric tags for relative quantitation-coupled liquid chromatography–tandem mass spectrometry. Differentially expressed proteins were subsequently used for bioinformatic analysis. Six highly expressed differentially expressed proteins in mature female worms were selected and long-term interference with small interfering RNA (siRNA) was conducted to determine biological functions. SiRNA against S. japonicum translationally controlled tumour protein (SjTCTP) resulted in the most significant effect on the growth and development of MF worms. Sjtctp mRNA expression gradually increased over time with a high level of expression maintained at 25–42 days p.i., while levels were significantly higher in mature female worms than male and SF worms. The subsequent animal immune protection experiments showed that recombinant SjTCTP (rSjTCTP) reduced the number of adults by 44.7% (P < 0.01), average egg burden per gram of liver by 57.94% (P < 0.01), egg hatching rate by 47.57% (P < 0.01), and oviposition of individual females by 43.16%. rSjTCTP induced higher levels of serum IgG, IL-2, and IL-10 in mice. Collectively, these results show that SjTCTP is vital to reproduction of female worms and, thus, is a candidate antigen for immune protection.     

A human kringle domain-based fluorescence-linked immunosorbent assay system

As a non-immunoglobulin protein scaffold, human kringle domain (KD) has attractive properties such as high specificity, stability, and production in bacterial hosts. Here, we developed a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) system using a fluorescent kringle domain (fluoKD), a fusion protein of a green fluorescent protein (GFP), and a kringle domain variant (KD548). Two kinds of fluoKDs in which KD was fused to the N terminus of GFP (N-fluoKD) or the C terminus of GFP (C-fluoKD) were constructed and characterized. In Escherichia coli host, both fluoKDs were produced in high yield and solubility and were successfully purified by a simple procedure. The purified fluoKDs exhibited strong fluorescent activities and high affinities to the target antigen. Furthermore, it was successfully demonstrated that the FLISA with purified fluoKDs allowed for more rapid detection of target antigens with higher sensitivity compared with conventional enzyme-linked immunosorbent assay (ELISA), indicating that a simple, rapid, and sensitive immunoassay system could be developed by using KD instead of antibody or antibody fragments.     

Sensitive and rapid detection of Schistosoma japonicum DNA by loop-mediated isothermal amplification (LAMP)

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Schistosoma japonicum DNA in faecal and serum samples of rabbits, and serum samples of humans infected with S. japonicum. This LAMP assay was based on the sequence of highly repetitive retrotransposon SjR2, and was able to detect 0.08 fg S. japonicum DNA, which is 10⁴ times more sensitive than conventional PCR. The LAMP assay was also highly specific for S. japonicum and able to detect S. japonicum DNA in rabbit sera at 1 week p.i. Following administration of praziquantel, detection of S. japonicum DNA in rabbit sera became negative at 12 weeks post-treatment. These results demonstrated that LAMP was effective for early diagnosis of, and evaluation of therapy effectiveness for, S. japonicum infection. Both PCR and LAMP assays were then used to detect S. japonicum DNA in 30 serum samples from S. japonicum-infected patients and 20 serum samples from healthy persons. The percentage sensitivity of LAMP was 96.7%, whereas that of PCR was only 60%, indicating that LAMP was more sensitive than conventional PCR for clinical diagnosis of schistosomiasis cases in endemic areas. The established LAMP assay should provide a useful and practical tool for the routine diagnosis and therapeutic evaluation of human schistosomiasis.     

Strongy Detect: Preliminary Validation of a Prototype Recombinant Ss-NIE/Ss-IR Based ELISA to Detect Strongyloides stercoralis Infection

BackgroundStrongyloides stercoralis (Ss) is the etiological agent of strongyloidiasis, a neglected tropical disease of global concern. Laboratory diagnosis of strongyloidiasis is most often based on detection of antibodies against antigens in an enzyme linked immunosorbent assay (ELISA). Herein, we report a preliminary validation study of newly developed IgG4- and/or IgG- based ELISAs to detect strongyloidiasis (Strongy Detect, InBios) incorporating a cocktail of 2 previously described recombinant antigens, Ss-NIE and Ss-IR.MethodsThe sensitivity and specificity were determined by using the assay in 150 cryopreserved serum samples from humans known to be Ss infected (n = 74), helminth uninfected (n = 47), or infected with a helminth other than Ss [n = 29). The treatment associated dynamics of antibody detection were then assessed using 35 paired samples obtained before and after definitive therapy.ResultsThe IgG and IgG4 assays were 99% and 96% sensitive, respectively, and 99% and 100% specific, respectively. Neither the IgG or IgG4 assay showed cross reactions with sera from those infected with other helminths. Although ELISA values did decline post-treatment few returned to levels below the cutoff for infection.ConclusionStrongy Detect is the most sensitive and specific commercialized immunoassay for detection of strongyloidiasis. The assay remains positive for greater than a year post-treatment.     

Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.     

Production and Characterization of Monoclonal Antibodies Against a Highly Immunogenic Fraction of Entamoeba histolytica (NIH:200) and Their Application in the Detection of Current Amoebic Infection

Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme-linked immunotransfer blot technique and by the enzyme-linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free-living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal-monoclonal antibody-based enzyme-linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb-based enzyme-linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool.     

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H₂O₂ reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.     

Tumstatin,a Matrikine Derived from Collagen Type IVα3, is Elevated in Serum from Patients with Non–Small Cell Lung Cancer

OBJECTIVES: Fibrosis and cancer are characterized by extracellular matrix (ECM) remodeling. The basement membrane is mainly composed by collagen type IV and laminin. Tumstatin is a matrix metalloproteinase-9 (MMP-9) generated matrikine of collagen type IV α3 chain. We evaluated the potential of tumstatin as a diagnostic biomarker of lung disorders. METHODS: A monoclonal antibody was raised against the neo-epitope tumstatin. A novel competitive enzyme-linked immunosorbent assay for detection of tumstatin (TUM), was developed and technically characterized. Levels of TUM were measured in serum of patients with idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and non–small cell lung cancer (NSCLC) belonging to two cohorts. RESULTS: The developed TUM enzyme-linked immunosorbent assay (ELISA) was technically robust. In cohort 1, levels of TUM were significantly higher in NSCLC compared to healthy controls, IPF, and COPD (P = 0.007, P = 0.03 and P = 0.001, respectively). The area under the receiver operating characteristics (AUROC) for separation of patients with NSCLC from healthy controls was 0.97, for separation of NSCLC and IPF patients was 0.98, and for separation of NSCLC and COPD patients was 1.0. In cohort 2, levels of TUM were also significantly higher in patients with NSCLC compared to healthy controls (P = 0.002), and the AUROC for separation of NSCLC and healthy controls was 0.73. CONCLUSIONS: We developed a technically robust competitive ELISA targeting the fragment tumstatin. The level of TUM in circulation was significantly higher in patients with NSCLC compared to patients with IPF, COPD and healthy controls. The assay provided high diagnostic accuracy in separating NSCLC patients from other lung disorders and from healthy controls.     

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum

Current clinically assays, such as enzyme‐linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high‐throughput analysis. A novel assay based on magnetic beads and time‐resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti‐HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium‐labeled anti‐HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02–700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra‐ and interassay coefficients of variation were 4.7–8.7% and 3.8–7.5%, respectively. The performance of this assay was further assessed against a well‐established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X – 0.017, R = 0.989). In the current study, we demonstrated that this novel time‐resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high‐throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus. Copyright © 2013 John Wiley & Sons, Ltd.