Goldmag-based enzyme-linked immunosorbent assay for determination of α-lactalbumin in milk

In this work, a goldmag-based enzyme-linked immunosorbent assay was developed for determination of α-lactalbumin (α-LA) in milk. The magnetic nanoparticle functionalized with polyetherimide was synthesized by one-pot method and coated with two layers of gold nanoparticles on the surface to synthesize goldmag nanoparticles. Anti-α-LA monoclonal antibody, prepared by hybridoma cell lines via cell fusion, was then bound to this goldmag nanoparticle to develop a capture nanoprobe. The results showed that this developed immunoassay had a good linear range of 2.33–127.1?ng/mL with IC₅₀ of 17.2?ng/mL. Besides, recovery rates for α-LA in four commercial milks were from 86.7% to 109.8%. The coefficients of variation in intra-assay and inter-assay were 3.9–6.8% and 5.5–9.8%, separately, which could meet the requirements to quantify α-LA content in milk. This goldmag-based immunoassay might have considerable potentials in the detection of food allergies.     

Development of an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic assay for hydrocortisone residues in milk

An anti-hydrocortisone (HDS) monoclonal antibody, 2G8, based on a HDS succinic anhydride derivative hapten, was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic assay for the detection of HDS in milk samples. The half inhibitory concentration (IC₅₀) of the antibody was 0.095?ng/mL, its limit of detection was 0.013?ng/mL, its linear range of detection was 0.026–0.356?ng/mL, and its cross-reactivity with HDS analogs was <5%. In spiked samples and a recovery test, the recovery rates ranged from 92% to 98.5%, indicating the suitability of this ic-ELISA for the analysis of HDS in milk. The immunochromatographic strip had a cutoff value of 2?ng/mL in milk and could be used for the semiquantitative analysis of HDS. When milk samples were added to the sample pad of the strip, a bright test line indicated <0.2?ng/mL HDS, a weak test line indicated 0.2–2?ng/mL HDS, and no test line indicated ≥2?ng/mL HDS. Analysis of HDS in milk samples showed that results acquired by the immunochromatographic assay agreed well with results acquired by ic-ELISA. Thus, the ic-ELISA and strip assay developed in this study rapidly and sensitively detect HDS residues in milk samples.     

Determination of α2-antiplasmin-plasmin complex in human plasma with an enzyme-linked immunosorbent assay

A novel enzyme-linked immunosorbent assay (ELISA) for quantification of α₂-antiplasmin-plasmin complex (APP) in undiluted plasma was developed. The assay follows the sandwich principle and uses two different antibodies directed against plasmin-modified α₂-antiplasmin and plasminogen, respectively. The antibodies bind selectively to the corresponding antigen moieties of APP. The assay was calibrated with definite concentrations of preformed purified APP added to APP-poor plasma. The lower limit of sensitivity of the assay was 10ng/ml. Mean coefficients of variation of 5.8% (intraassay) and 7.2% (interassay) were found for APP concentrations between 50 and 5000 ng/ml. A reference range from 80–470 ng/ml was calculated from APP concentration in plasma samples from 178 healthy donors (mean value±SD: 210±88). In plasma samples from patients during thrombolytic therapy, APP was found up to 20000 ng/ml. From our data we conclude that quantification of APP can be a sensitive tool for specific detection of an activation of the fibrinolytic system.     

Production of antibodies and development of an enzyme-linked immunosorbent assay for 17β-estradiol in milk

The residue of 17β-estradiol (E₂) in milk could potentially lead to the occurrence of various reproductive diseases; therefore, a rapid and sensitive method for monitoring E₂ residues in milk was highly necessary. In this study, we produced new polyclonal and monoclonal antibodies using E₂-3-O-carboxymethyl ether as a hapten and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of E₂ in milk. The results showed that the sensitivity of polyclonal antibody was higher than that of monoclonal antibody, providing a half maximum inhibition concentration (IC₅₀) against E₂ of 0.17?ng/mL, high cross-reactivity (CR) to E₂ benzoate (150%) and oestriol (18.02%), and negligible CR with other oestrogen compounds. Under optimized conditions, the developed icELISA based on the polyclonal antibody had a limit of detection values of 0.093?μg/L, which was enough sensitive to detect E₂ in milk. In spiked samples (0.5, 1, and 2?μg/L), the recoveries ranged from 83.12% to 94.58% with coefficients of variation <12.8%. These results indicated that the icELISA method we developed was suitable for screening of E₂ residue in milk.     

Determination of metolcarb residues by a biotin–streptavidin-amplified enzyme-linked immunosorbent assay in vegetables and edible fungus

An effective biotin–streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) to rapidly detect metolcarb (MTMC) residues is reported. Nonspecific adsorption was minimized by using 0.5% skimmed milk powder as a blocking buffer and 0.5% bovine serum albumin/phosphate-buffered saline (PBS) as a buffer for streptavidin–horse radish peroxidase conjugates. The established method is four times sensitive than direct competitive enzyme-linked immunosorbent assay, with the IC₅₀ of 10.0?ng?mL^?1. The samples were prepared from mustard, cucumber and mushroom with simple extraction and dilution methods, including use of PBS without concentration or cleanup steps. The samples prepared from spinach and shiitake were quantified after 2-fold methanol extraction and 20-fold dilution with 2.0% fish glutin/PBS. Good accuracy and precision were obtained with mean recoveries between 80.0% and 95.6%, and mean coefficients of variation below 12.1%. A good correlation (R^2?=?0.9931) between the BA-ELISA and high-performance liquid chromatography was observed for MTMC analysis in different samples. This method could be potentially useful for high-throughput food inspection.     

Development of two enzyme-linked immunosorbent assay formats for thifluzamide residues’ analysis based on distinct polyclonal antibodies

Haptens 2-Methyl-4-(trifluoromethyl)thiazole-5-carboxylic acid and 2,6-Dibromo-4-(trifluoromethoxy)aniline, the two moieties of thifluzamide, were conjugated with carrier proteins for the synthesis of artificial antigens. Two distinct anti-thifluzamide polyclonal antibodies (PAb-1 and PAb-2) were produced from the immunized female Balb/c mice. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) in two formats based on the PAbs was developed for thifluzamide analysis. The concentration of 50% inhibition (IC₅₀) of ELISA-1 was 1.39?mg?L^?1 and its limit of detection (LOD) was 0.082?mg?L^?1. Meanwhile, ELISA-2 had a similar IC₅₀ of 1.96?mg?L^?1 and a LOD of 0.074?mg?L^?1 as ELISA-1. Both the raised PAbs exhibited high specificity to thifluzamide. The recoveries for spiked samples including water and wheat ranged from 72.0% to 128.4%, and the accuracy of ELISA was confirmed by high-performance liquid chromatography. In summary, the ic-ELISA might be a promising tool for simple, sensitive and rapid detection of thifluzamide residues in real samples.     

Unreliable measurement of basophil maximum leukotriene release with the Bühlmann CAST 2000 enzyme-linked immunosorbent assay kit

The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcepsilonRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.     

Diagnostic performance of an enzyme-linked immunospot assay for interferon-γ in skeletal tuberculosis

The aim of this study was to evaluate the diagnostic performance of an enzyme-linked immunospot (ELISPOT) assay for interferon-γ in patients with suspected skeletal tuberculosis (TB). From March 2007 to June 2010, a total of 36 patients with suspected skeletal TB in a tertiary care hospital in Taiwan were enrolled. Twelve patients (35.3%) had culture-confirmed TB, three (8.8%) patients had probable TB, and the remaining 21 (58.3%) patients did not have TB. Fourteen patients with mycobacterial infection had available biopsy or surgical specimens for histopathological examination and 12 (85.7%) specimens had pathological features consistent with mycobacterial infection. Among the 12 patients with positive findings indicating mycobacterial infection, all seven patients with spinal TB and three of five patients with TB arthritis had positive ELISPOT assays. All nine patients with spinal TB had positive ELISPOT assays, but only four of six patients with TB arthritis had positive ELISPOT assays. The sensitivity, specificity, positive predictive value, and negative predictive value for skeletal TB diagnosis by the ELISPOT assay were 86.7%, 61.9%, 61.9%, and 86.7%, respectively. In conclusion, the ELISPOT assay can provide useful support in diagnosing skeletal TB, and spinal TB can be excluded based on a negative ELISPOT assay.     

Serological detection of <Emphasis Type="Italic">Trichinella spiralis</Emphasis> in swine by ELISA (enzyme-linked immunosorbent assay) using an excretory–secretory (E/S) antigen

The enzyme-linked immunosorbent assay (ELISA) method is recommended for farm surveillance programs and may be useful for epidemiological studies in wildlife or for establishing Trichinella-free areas. In this study, our interest was to compare the specificity and the time of seroconversion of excretory–secretory (E/S) antigens prepared from Trichinella spiralis. A group of eight pigs was inoculated with 500 T. spiralis larvae per animal, and blood sampling was performed at 3 and 4-day intervals during all experiments. The numbers of muscle larvae were determined in four different muscles groups. The larvae per gram burden shows that the most heavily parasitized muscles were the diaphragm [mean = 43.7 larvae per gram (lpg)] and the tongue (mean = 16.9 lpg). Antibody responses were detected by any of eight infected pigs of T. spiralis. Using the ELISA method with E/S antigen, antibodies to T. spiralis were first found on the day 21st p.i. The initial detection of antibodies varied from 21st to 31st day p.i., and the peak was reported 42nd day p.i. Dynamic of antibodies was stable or increased slightly throughout the experimental period (60 days post-inoculation). Our results represent important data for validation of a serological test, especially if blood samples are taken during early stages of infection.     

The 65kDa antigen of mycobacteria—a common bacterial protein?

The 65 kilodalton antigen of Mycobacterium tuberculosis and M. leprae is a well-characterized, strongly immunogenic protein eliciting antibody and T-cell responses in infected patients. Recent studies have disclosed regions of cross-reactivity between the 65kDa antigen and proteins in many other bacterial species. These include the product of the ams gene in E. coli which is involved in the processing of RNA. Here Douglas Young and his colleagues discuss these observations, the significance of the 65kDa antigen and its possible role in the pathogenesis of mycobacterial and other diseases.     

Development of a highly sensitive biotin–streptavidin enzyme-linked immunosorbent assay for detecting diethyl phthalate based on a specific polyclonal antibody

An indirect competitive biotin–streptavidin enzyme-linked immunosorbent assay (BA-ELISA) has been established for the determination of diethyl phthalate (DEP) in wine samples. A highly sensitive and specific polyclonal antibody (pAb-DEP) targeting DEP was prepared. Under optimized conditions, good linearity was achieved within a range of 0.021–9.512 μg·L^?1. The limit of detection (IC₁₀) was 0.0079 μg·L^?1 and the median inhibitory concentration (IC₅₀) was 0.443 μg·L^?1. Besides, the BA-ELISA was highly selective, with low cross-reactivity values with DEP analogs (below 5%). Finally, the concentrations of DEP in wine samples ranged from 5.93 μg·L^?1 to 59.15 μg·L^?1 by BA-ELISA. Satisfactory recoveries (89.19–112.33%) and variation coefficient values (5.81–9.43%) were successfully obtained. The consistency between the results of BA-ELISA and gas chromatography–mass spectrometry (GC–MS) was 97.48%, which further confirmed that the proposed BA-ELISA immunoassay is reliable, rapid, sensitive, and accurate for monitoring DEP in wine samples.     

Ex-vivo whole blood secretion of interferon (IFN)-γ and IFN-γ-inducible protein-10 measured by enzyme-linked immunosorbent assay are as sensitive as IFN-γ enzyme-linked immunospot for the detection of gluten-reactive T cells in human leucocyte antigen (HLA)-DQ2·5+-associated coeliac disease

T cell cytokine release assays are used to diagnose infectious diseases, but not autoimmune or allergic disease. Coeliac disease (CD) is a common T cell-mediated disease diagnosed by the presence of gluten-dependent intestinal inflammation and serology. Many patients cannot be diagnosed with CD because they reduce dietary gluten before medical workup. Oral gluten challenge in CD patients treated with gluten-free diet (GFD) mobilizes gluten-reactive T cells measurable by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) or major histocompatibility complex (MHC) class II tetramers. Immunodominant peptides are quite consistent in the 90% of patients who possess HLA-DQ2·5. We aimed to develop whole blood assays to detect gluten-specific T cells. Blood was collected before and after gluten challenge from GFD donors confirmed to have CD (n = 27, all HLA-DQ2·5⁺), GFD donors confirmed not to have CD (n = 6 HLA-DQ2·5⁺, 11 HLA-DQ2·5⁻) and donors with CD not following GFD (n = 4, all HLA-DQ2·5⁺). Plasma IFN-γ and IFN-γ inducible protein-10 (IP-10) were measured by enzyme-linked immunosorbent assay (ELISA) after whole blood incubation with peptides or gliadin, and correlated with IFN-γ ELISPOT. No T cell assay could distinguish between CD patients and controls prior to gluten challenge, but after gluten challenge the whole blood IFN-γ ELISA and the ELISPOT were both 85% sensitive and 100% specific for HLA-DQ2·5⁺ CD patients; the whole blood IP-10 ELISA was 94% sensitive and 100% specific. We conclude that whole blood cytokine release assays are sensitive and specific for detection of gluten-reactive T cells in CD; further clinical studies addressing the utility of these tests in patients with an uncertain diagnosis of CD is warranted.     

Detection of the copy number of plasmid encoding HBsAg in recombinant yeast by PCR relative quantitative assay

Thegeneticrecombinantyeast,Saccharomyces cerevisiae,carryingtheplasmidscontainingthe hepatitisBvirussurfaceantigen(HBsAg)encod ingregionisemployedtoproducetherecombinant hepatitisBvaccine.Someplasmidsoftherecom binantyeastmaybelostduringindustrialferment ationtherebyinfluencingtheexpressionofHB sAg.Eitheratthepracticalfermentationtoex pressHBsAgformakingvaccineorrenovationoffermentationprocessoftherecombinantyeastto enhancetheyieldsofHBsAg,suchasextension ofculturingperiod,theplasmidstat…     

Evaluation of the Helicobacter pylori stool antigen (HpSA) test in routine clinical practice--is it patient-friendly?

A new immunoassay has been developed to detect the presence of Helicobacter pylori antigens in stool specimens. The aim of our study was to assess the accuracy and utility of the H. pylori stool antigen (HpSA) test in routine clinical practice. Dyspeptic patients undergoing endoscopy were studied. H. pylori status was defined before treatment by CLOtest and histology, and by 13C urea breath test (UBT) after eradication therapy. A standard universal container was provided for stool collection and the HpSA test was performed by an investigator blind to the results of the other diagnostic tests. Patients also provided a venous blood sample prior to endoscopy for H. pylori serology. Sixty patients (30 M : 30 F: mean age 47 yr) were enrolled in the study. The pretreatment sensitivity, specificity, positive and negative predictive value of the HpSA test were respectively, 93%, 94%, 96%, and 90%. Twenty five patients returned for post treatment 13CUBT, but only 14 (56%) provided a stool sample for analysis. The post treatment sensitivity, specificity, positive and negative predictive value of the HpSA test were respectively, 67%, 100%, 67%, and 92%. The HpSA test was negative in 19% of the patients found positive for anti-H. pylori antibodies on serology testing. All H. pylori antibody-negative patients had a negative HpSA test. Our results suggest that the HpSA test provides accurate pretreatment diagnosis of H. pylori infection but the reliability of the test after treatment is uncertain. A potential problem with the HpSA test appears to be patient reluctance about stool handling and this could prove a significant obstacle to patient compliance and the acceptability of the test in everyday clinical practice.     

Performance evaluation of a new fourth-generation HIV combination antigen–antibody assay

Education and diagnostic tests capable of early detection represent our most effective means of preventing transmission of human immunodeficiency virus (HIV). The importance of early detection is underlined by studies demonstrating increased life expectancy following early initiation of antiviral treatment. The Elecsys^® HIV combi PT assay is a fourth-generation antigen–antibody combination assay developed to allow earlier detection of seroconversion, and to have increased sensitivity and improved specificity. We aimed to determine how early the assay could detect infection compared with existing assays; whether all HIV variants could be detected; and the assay’s specificity using samples from blood donors, routine specimens, and patients with potential cross-reacting factors. Samples were identified as positive by the Elecsys^® assay 4.9 days after a positive polymerase chain reaction result (as determined by the panel supplier), which was earlier than the 5.3–7.1 days observed with comparators. The analytical sensitivity of the Elecsys^® HIV combi PT assay for the HIV-1 p24 antigen was 1.05 IU/mL, which compares favorably with the comparator assays. In addition, the Elecsys^® assay identified all screened HIV subtypes and displayed greater sensitivity to HIV-2 homologous antigen and antibodies to HIV-1 E and O and HIV-2 than the other assays. Overall, the specificity of the Elecsys^® assay was 99.88 % using samples from blood donors and 99.81 % when analyzing unselected samples. Potential cross-reacting factors did not interfere with assay performance. The Elecsys^® HIV combi PT assay is a sensitive and specific assay that has been granted the CE mark according to Directive 2009/886/EC.     

Evaluation of lymphocyte function by IFN-γ secretion capability assay in the diagnosis of lymphoma-associated hemophagocytic syndrome

Lymphoma-associated hemophagocytic syndrome (LAHS) is a highly life-threatening disease characterized by an uncontrolled immune disorder. Both under-recognition and delayed diagnosis may contribute to aggressive diseases, and a poorer prognosis. Identification of laboratory features specific for LAHS patients may allow for early detection and intervention with improved outcomes. In the present study, 120 lymphoma patients at first diagnosis were recruited and the function of lymphocytes was evaluated by IFN-γ secretion assay at first diagnosis and follow up. During the surveillance period, 20 patients who complicated with hemophagocytic lymphohistiocytosis (HLH) were classified as LAHS group, and 30 patients without infectious diseases during the course of treatment were classified as lymphoma control group. In addition, 20 non-malignant associated HLH patients recruited as HLH control group and 50 healthy control (HC) subjects were also included. The IFN-γ secretion capability of lymphocytes was compared between first diagnosis of lymphoma patients who was complicate with HLH or not in the disease progression. Our results showed that only NK cell activity was decreased in lymphoma control group, but the activities of NK, CD4+ and CD8+ T cells were all significantly decreased at the time of lymphoma diagnosis in those who would progress with HLH. During the course of treatment, lymphocyte function was relatively stable in lymphoma patients but became further decreased when suffering from complication of LAHS. The IFN-γ secretion capability of lymphocytes in LAHS and non-malignant associated HLH patients were all significantly decreased compared with HCs. So the occurrence of HLH was the key factor leading to the impaired activity of lymphocytes. These data suggest that decreased lymphocyte function might be used as a predictor of LAHS, which has critical clinical significance in diagnosis and further understanding the pathogenesis of the disease.     

Evaluation of methods of surfactant administration in the delivery suite?

Surfactant administered in the delivery suite might prevent or reduce the severity of subsequent respiratory distress syndrome. This review describes the evidence for surfactant delivery methods with relationship to their relevance in the delivery suite. The techniques include delivery using a thin catheter with the first breath, by the intubation-surfactant extubation procedure, less invasive surfactant administration (LISA) technique, using a laryngeal mask airway (LMA), or by nebulisation. There have been few randomised trials that have evaluated outcomes using these techniques in the delivery suite, and these were early trials. Currently, practitioners favour use of nasal continuous positive airway pressure with early rescue surfactant. Whether prophylactic surfactant given by the LISA technique or other techniques, such as via a LMA in the delivery suite, is more beneficial merits testing. This will require appropriately designed randomised trials with long-term outcomes.