Two-step Immunoassay Research Articles

Verification of the analytical performance of the anti-cyclic citrullinated peptide assay on ALINITY ci ® experience from the biochemistry laboratory of Mohammed VI University Hospital in Oujda

The aim of our work was to evaluate the analytical performance of the anti-cyclic citrullinated peptide determination by a two-step immunoassay using microparticle chemiluminescence immunoassay (CMIA) technology, in accordance with the Scope A criteria of the guide of the verification/validation of medical biology methods. We evaluated the repeatability and the intermediate precision of the assay. The results obtained are very satisfactory for the two levels (low and high) both for intermediate fidelity, with coefficients of variation (CV) of CV1= 1.9% and CV2=1.8% respectively, and for repeatability, with coefficients of variation of CV1 = 1.7%, CV2= 1.61%. respectively. The results obtained made it possible to verify the method's performance and compare it with the analytical objectives set in order to meet the regulatory and normative requirements set by the supplier and learned societies.

Verification of the analytical performance of the serum CA 125 assay on the Abbott Alinity ci®: Experience of the biochemistry laboratory of the Mohammed VI university hospital of Oujda

The aim of our work was to evaluate the analytical performance of CA 125 determination by a two-step immunoassay using microparticle chemiluminescence immunoassay (CMIA) technology, in accordance with the Scope A criteria of the guide of the verification/validation of medical biology methods. We evaluated the repeatability and the intermediate precision of the CA 125 assay.The results obtained are very satisfactory for the three levels (low, medium and high), both for intermediate fidelity, with coefficients of variation (CV) of 2.68%, 1.62% and 2.11% respectively, and for repeatability, with coefficients of variation of CV1 = 2.17%, CV2 = 2.04%, and CV3 = 1.38% respectively. The results obtained made it possible to verify the method's performance and compare it with the analytical objectives set in order to meet the regulatory and normative requirements set by the supplier and learned societies. The achieved results facilitated the verification of the method's performance and its comparison with the analytical objectives established, aligning with regulatory and normative requirements outlined by the supplier and relevant professional learned societies.

Complement C1q in plasma induces nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticle antibody conjugates

Upconverting nanoparticles are attractive reporters for immunoassays, because their high specific activity and lack of autofluorescence background enable their detection at extremely low concentrations. However, the sensitivity achieved with heterogeneous sandwich immunoassays using nanoparticle reporters is generally limited by the nonspecific binding of nanoparticle antibody conjugates to solid supports. In this study, we characterized plasma components associated with elevated nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticles in heterogeneous two-step sandwich immunoassays. Plasma was consecutively fractionated using various chromatographic methods by selecting after each step the fractions producing the highest nonspecific binding of upconverting nanoparticle conjugates in an immunoassay for cardiac troponin I. Finally, the proteins in the fractions associated with highest amount of nonspecific binding were separated by gel electrophoresis and identified with mass spectrometry. The results indicated that complement component C1q was present in the fractions associated with the highest signal from nonspecific binding. The interference was not limited to only poly(acrylic acid)-coated nanoparticles or certain antibody combination, but occurred more generally. The interference was removed by increasing the ionic strength of the assay buffer in the sample incubation step or by adding a negatively charged blocker to bind on positively charged C1q, suggesting that the interaction is mostly electrostatic. Hence, we assume that the interference is likely to affect various negatively charged nanoparticles. The identification of complement component C1q as the major interfering protein allows for more rational design of countermeasures in future immunoassay development utilizing nanoparticle reporters.Graphical abstract

Serum concentrations of IL-31 in dogs with nonpruritic mast cell tumours or lymphoma.

The aim of this study was to compare serum interleukin (IL)-31 concentrations in dogs with lymphoma and mast cell tumours (MCT) without pruritus to those of healthy dogs. To determine if IL-31 plays a role in tumour pathogenesis and if IL-31 could be a biological marker for disease progression. Forty-eight healthy dogs and 36 dogs with neoplasia [multicentric lymphoma (14), MCT (15) and cutaneous lymphoma (7)] were included in the study. Dogs with neoplasia were assigned to three different groups. Group 1 consisted of patients with multicentric lymphoma, which were diagnosed by cytological, histopathological and clonality investigations. Thoracic radiographs, ultrasound examination of the abdominal cavity, and fine-needle aspirates from liver and spleen were used to determine the lymphoma stage Patients with cutaneous lymphoma, diagnosed by cytological and histopathological findings, were included in Group 2. Patients with MCT, diagnosed by cytological and histopathological findings, were included in Group 3. Serum was frozen at -80ºC before measuring the concentration of IL-31 via a Simoa ultra-sensitive, fully automated two-step immunoassay. Serum concentrations of IL-31, regardless of the disease and its staging, were within the normal range in all patients; there was no difference between any of the different tumour groups and healthy dogs. IL-31 is not likely to be involved in the pathogenesis of canine MCT or lymphoma without pruritus.

Development and Technical Validation of an Immunoassay for the Detection of APP669-711 (Aβ-3-40) in Biological Samples.

The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.

Prenatal serum screening - a summary of our experience with high risk reporting

Objective The spectrum of pregnancy tests for screening of chromosomal disorders in the unborn child includes maternal serum testing through double, triple and quadruple marker as well as the emerging cell free fetal DNA analysis based noninvasive prenatal test. The aim of this short communication is to summarize our finding’s pertaining to high risk serum screening cases with reference to clinical indications and history. Methods Data of cases reported high-risk by serum screening including a total of 3368 women with median age of 32 years was included. Serum testing was done in the laboratory using the technology of Chemiluminescence micro particle two-step immunoassay for sensitive quantification of hormones routinely tested for in the double, triple and quadruple marker. Risk estimation using values of hormone levels and the resulting MoM was done using the PRISCA 5.0.2.37. Results A total of 16,608 samples were tested for serum screening in the study period and specific high risk analysis detected a total of 20% reported as high risk. Trisomy 21 was detected to be the most common finding at 86% followed by neural tube defects at 9%. Advanced maternal age (≥35 years) accounted for a total of 34% of the reported high risk cases. Conclusion The gap between absence of a nation-wide screening mandate, and guidelines for agencies involved in prenatal screening tests can be reduced with studies which focus on trends in recommendation and developing an understanding of the clinical backdrop leading to high risk screening results.

Familial dysalbuminaemic hyperthyroxinaemia interferes with current free thyroid hormone immunoassay methods.

ObjectiveFamilial dysalbuminaemic hyperthyroxinaemia (FDH), most commonly due to an Arginine to Histidine mutation at residue 218 (R218H) in the albumin gene, causes artefactual elevation of free thyroid hormones in euthyroid individuals. We have evaluated the susceptibility of most current free thyroid hormone immunoassay methods used in the United Kingdom, Europe and Far East to interference by R218H FDH.MethodsDifferent, one- and two-step immunoassay methods were tested, measuring free T4 (FT4) and free T3 (FT3) in 37 individuals with genetically proven R218H FDH.ResultsWith the exception of Ortho VITROS, FT4 measurements were raised in all assays, with greatest to lowest susceptibility to interference being Beckman ACCESS > Roche ELECSYS > FUJIREBIO Lumipulse > Siemens CENTAUR > Abbott ARCHITECT > Perkin-Elmer DELFIA. Five different assays recorded high FT3 levels, with the Siemens CENTAUR method measuring high FT3 values in up to 30% of cases. However, depending on the assay method, FT4 measurements were unexpectedly normal in some, genetically confirmed, affected relatives of index FDH cases.ConclusionsAll FT4 immunoassays evaluated are prone to interference by R218H FDH, with their varying susceptibility not being related to assay architecture but likely due to differing assay conditions or buffer composition. Added susceptibility of many FT3 assays to measurement interference, resulting in high FT4 and FT3 with non-suppressed TSH levels, raises the possibility of R218H FDH being misdiagnosed as resistance to thyroid hormone beta or TSH-secreting pituitary tumour, potentially leading to unnecessary investigation and inappropriate treatment.

Multiple myeloma masquerading as T3 toxicosis

We report a case of a 52-year-old man who was referred by his primary care physician to rule out thyroid illness. His thyroid function tests (TFTs) revealed isolated elevation of total T3 (TT3) with normal total T4 (TT4), free T3 (FT3), free T4 (FT4), and unsuppressed thyroid stimulating hormone (TSH) raising the suspicion of antibody interference or abnormal thyroid hormone-binding proteins. To identify antibody interference, we repeated the immunoassay using varied platforms and assay methods. The total T3 levels which were high in one-step immunoassay normalized in two-step immunoassay. On further evaluation, the patient was diagnosed with multiple myeloma and the antibody interference was secondary to IgG monoclonal antibody binding specifically to analog T3 leading to spurious results. Ours is the first report from India and the fourth case in literature to report a multiple myeloma antibody interfering with TFTs.

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

MON-612 Familial Dysalbuminemic Hyperthyroxinemia Cases in Five Unrelated Japanese Families and the Influences on Free T4 Measurement

Familial dysalbuminemic hyperthyroxinemia (FDH) is caused by abnormal human serum albumin (HSA) with an increased thyroxine (T4) affinity leading to euthyroid hyperthyroxinemia. In Japanese FDH patients with the HSAR218P mutation, not only one-step but also two-step immunoassays for free T4 (FT4) can yield false-positive results. Therefore, it is difficult to distinguish FDH from syndrome of inappropriate secretion of TSH, even when multiple assays are used. Patient 1 was a 29-year-old pregnant woman. A radiographic examination and hormone stimulation test were not performed. We suspected that the patient had FDH because of her family history such as abnormal thyroid function and hyperthyroxinemia (Total T4 level was greater than the measurement sensitivity). Genetic analysis using direct sequencing for HSA revealed that she was consistent with FDH. She experienced a normal delivery. Other patients were performed the thyroid test with Chief complaints of infertility or depression and their thyroid function abnormalities were pointed out. Thyroid function tests revealed elevated FT4 and FT3 levels; their TSH level was within the reference range in all patients. To investigate T4 to HSA binding, gel filtration high-performance liquid chromatography was used for the biochemical analyses. Furthermore, FT4 was measured in FDH patients using one-step method LUMIPULSE (Fujirebio) using T3 as an antigen and an anti-T4 antibody as a label. The genomic analysis revealed a heterozygous missense mutation in HSA(R218P). In FDH patient sera, the albumin effluent corresponded to the peaks for total T4 (TT4); approximately 60% of the T4 in the effluent was detected as FT4. Mean FT4 level measured with LUMIPULSE was 1.28 ng/dL within the reference value range. In the FDH patients, a relatively larger quantity of T4 was bound to abnormal HSA. This bound T4 was measured as FT4 during the analysis. When thyroid function abnormality without clinical findings is observed, it is considered preferable to understand the antigen and antibody to be used and to select measurement method.

A two-step immunoassay for the simultaneous assessment of A\u03b238, A\u03b240 and A\u03b242 in human blood plasma supports the A\u03b242/A\u03b240 ratio as a promising biomarker candidate of Alzheimer\u2019s disease

BackgroundThe quantification of amyloid-beta (Aβ) peptides in blood plasma as potential biomarkers of Alzheimer’s disease (AD) is hampered by very low Aβ concentrations and the presence of matrix components that may interfere with the measurements.MethodsWe developed a two-step immunoassay for the simultaneous measurement of the relative levels of Aβ38, Aβ40 and Aβ42 in human EDTA plasma. The assay was employed for the study of 23 patients with dementia of the Alzheimer’s type (AD-D) and 17 patients with dementia due to other reasons (OD). We examined relationships with the clinical diagnosis, cerebral Aβ load as quantified by amyloid-positron emission tomography, apolipoprotein E genotype, Aβ levels and Tau protein in cerebrospinal fluid.ResultsPreconcentration of plasma Aβ peptides by immunoprecipitation substantially facilitated their immunological measurements. The Aβ42/Aβ40 and Aβ42/Aβ38 ratios were statistically significantly lower in the AD-D patients than in the OD group. The areas under the receiver operating characteristic curves reached 0.87 for the Aβ42/Aβ40 ratio and 0.80 for the Aβ42/Aβ38 ratio.ConclusionsThe measurement of plasma Aβ peptides with an immunological assay can be improved by preconcentration via immunoprecipitation with an antibody against the Aβ amino-terminus and elution of the captured peptides by heating in a mild detergent-containing buffer. Our findings support the Aβ42/Aβ40 ratio in blood plasma as a promising AD biomarker candidate which correlates significantly with the validated core biomarkers of AD. Further studies will be needed for technical advancement of the assay and validation of the biomarker findings.

Evaluation of free thyroxine determination based on one-step fluorometric immunoassay technique and the suboptimal concordance with two-step immunoassay

Free thyroxine (FT4) quantification is continuing to be a concern. The purpose of the following study was to evaluate the analytical performance of Tosoh AIA900 based on a one-step technique and its comparison to Access 2 (two-step technique) over different clinical contexts (euthyroid, thyroid disorders, uncontrolled diabetes, renal failure and pregnancy). The protocol established by the French society of Clinical Biology was used to evaluate: imprecision, limit of detection, trueness, linearity, interferences and method comparisons. Within-run variation of 3.1%, 5.7% and 4.4% were found for the low, medium and high controls, respectively. Between-run was 5.8% for low control, 5.7% for medium control and 7.1% for high control. Common interferences did not affect one-step immunoassay FT4 results. The linearity was checked up to 86 pmol/L. The limit of detection was 5.5 pmol/L. The concordance correlation coefficient (CCC) showed a low agreement (0.6) between both methods. Bland-Altman plot revealed that AIA 900 one-step immunoassay technique provides a significant higher values ((+2.8 ± 2.7 pmol/L;p < 0.0001). The Passing-Bablok regression demonstrated both proportional and systematic differences in comparison to Access 2. The lowest association was noted in subjects with impaired renal function (CCC = 0.27). At the time of the study, the results of on-step immunoassay are not directly comparable with Access 2.

A Direct Assay for Measuring Free 25-Hydroxyvitamin D.

Recent studies suggest that the concentration and genotype of vitamin D binding protein (VDBP) are important factors that determine the bioavailability of 25-hydroxyvitamin D [25(OH)D] in blood. Accumulating data indicate that, e.g., in pregnant women, hemodialysis patients, chronic kidney disease, liver failure, and bladder and pancreatic cancers, the measurement of free 25(OH)D in serum provides more relevant diagnostic information than measurement of total 25(OH)D. The aim of this study was to develop and validate an ELISA for direct measurement of free 25(OH)D in serum. A simple and direct ELISA was developed, based on a two-step immunoassay procedure performed in a microtiter plate. The assay has been characterized in terms of precision (4-10% CV, according to concentration), sensitivity (limits of blank = 0.5-1.0 pg/mL and LODs = 1.3-1.8 pg/mL), accuracy (correlation to dialysis, ELISA = 0.99xdialysis-0.5 pg/mL, r2 = 0.74), cross-reactivity of the antibody for the D2 form (77%), and addition of both VDBP and albumin (35-38% recovery upon addition of VDBP, 53-58% upon addition of albumin). The assay has already been used in multiple studies, including its comparison with calculation methods and in studies of patients with liver failure, different ethnic groups, supplemented mice, respiratory diseases, and obesity. The free 25(OH)D ELISA can be used in studies as a valuable tool to establish the clinical relevance of free 25(OH)D.

White light reflectance spectroscopy biosensing system for fast quantitative prostate specific antigen determination in forensic samples

A label-free biosensor based on white light reflectance spectroscopy for the determination of PSA as semen indicator in forensic samples is presented. The sensor is based on a two-step immunoassay which employs the same polyclonal anti-PSA antibody as capture and detection antibody followed by reaction with streptavidin as a signal enhancement step. The whole assay time was set to 10min; 5min reaction of immobilized antibody with the PSA calibrators or the samples, 3min reaction with the biotinylated anti-PSA antibody and 2min reaction with streptavidin. Following this protocol, a detection limit of 0.5ng/mL was achieved and the assay's linear response range extended up to 500ng/mL. Thus, taking into account the quantification limit of 1.0ng/mL and the average PSA concentration in semen (0.2–5.5mg/mL), semen quantities of a few nanoliters could be detected. The accuracy of the sensor developed was demonstrated through recovery (% recovery ranged from 89.6 to 106) and semen dilution experiments. A linear correlation was found for semen dilutions ranging from 5000 to 360,000. The lack of interference by other bodily fluids was confirmed by analysing stains of blood, urine and saliva prior to and after the addition of semen. Finally, the sensor was evaluated by analysing 51 forensic casework samples which were also analysed with a semi-quantitative membrane strip test (Seratec® PSA), through microscopic detection of spermatozoa, and male DNA identification through detection of Y chromosome. The results obtained with the sensor were in excellent agreement with those provided by an immunoradiometric assay kit (PSA-RIACT) and in complete agreement with the findings using the membrane strip assay, spermatozoa and Y chromosome detection. The excellent analytical performance and small size of the instrument make the sensor developed an attractive tool for use in forensic evidence screening for semen detection.

Abstract 437: Lumipulse G HE4 assay for monitoring of ovarian cancer recurrence and progression

Abstract Human epididymis protein 4 (HE4), a member of Whey acidic four-disulfide core protein (WFDC) family, was demonstrated as one of the most useful biomarkers for ovarian cancer (HellstrÖm I, et al. 2003; Drapkin R, et al. 2005; Moore RG, et al. 2007).This study was to evaluate the use of Lumipulse G HE4 assay with patient serum for monitoring recurrence and progression of epithelial ovarian cancer. Recently, Lumipulse G HE4 assay was cleared by US FDA as an aid in monitoring recurrence or progressive disease in patients with epithelial ovarian cancer. Methods: Lumipulse G HE4 is a Chemiluminescent Enzyme Immunoassay (CLEIA) for the quantitative determination of HE4 in human serum and plasma on the Lumipulse G System by a two-step sandwich immunoassay method. In the assay, serum was added to and incubated with an anti-HE4 monoclonal antibody (MAb)-linked magnetic particles. The particles were then washed and rinsed to remove unbound materials. Alkaline phosphatase-labeled 2nd anti-HE4 MAb was added to and incubated with the HE4-bound particles. The particles were then washed and rinsed again to remove unbound materials. Substrate 3-(2’-spiroadamantane)-4-methoxy-4-(3”-phosphoryloxy) phenyl-1, 2-dioxetane disodium salt (AMPPD) solution was then added to and mixed with the particles. Luminescence signals were thus generated by the cleavage of dephosphorylated AMPPD and converted into the amount of HE4 in the serum. Results: In the monitoring study, changes in HE4 levels in serial serum samples collected in SST tubes from 72 subjects with epithelial ovarian cancer were compared to changes in disease status, that is, progression or no progression. A total of 330 observations were undertaken with an average number of 5.6 observations per subject. A positive change in the HE4 value was defined as an increase in the observation value that was at least 18% greater than the previous observation value. Of the 61 samples with a positive change, 49% of them correlated with the progression of epithelial ovarian cancer while 80% of the 269 subject serial samples with no significant change in the HE4 value correlated with no progression. The total concordance was 74%, positive predictive values (PPV) 35%, and negative predictive value (NPV) 87%. In addition, a comparison of Lumipulse G HE4 with the predicate device, HE4 EIA, was carried out using specimens consistent with CLSI Protocol EP09-A3 and weighted Deming regression analysis. The slope and correlation coefficient (r) obtained were 1.03 and 0.9891, respectively, for the tested specimens (n = 143) which ranged from 33.4 - 969.5 pmol/L, and slope and r of 1.03 and 0.9917, respectively, for the tested specimens (n = 168) ranged from 33.4 - 4602.0 pmol/L. Conclusion: The Lumipulse G HE4 assay has demonstrated to be useful in monitoring the course of disease in women with epithelial ovarian cancer and well correlated with the predicate device HE4 EIA. Citation Format: Rachel Radwan, Anders Öhrvik, Katherine Falcone, Sara Gannon, Candice Felman, Natalya Benina, Natascha Svensson, Savitha Raju, Sharee Jones, Catherine Peacock, Julianna Young, Zhong-Qian Li, Christian Fermer, Diana Dickson. Lumipulse G HE4 assay for monitoring of ovarian cancer recurrence and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 437.

Universal quantum dot-based sandwich-like immunoassay strategy for rapid and ultrasensitive detection of small molecules using portable and reusable optofluidic nano-biosensing platform

A universal sandwich-like immunoassay strategy based on quantum-dots immunoprobe (QD-labeled anti-mouse IgG antibody) was developed for rapid and ultrasensitive detection of small molecules. A portable and reusable optofluidic nano-biosensing platform was applied to investigate the sandwich-like immunoassay mechanism and format of small molecules, as well as the binding kinetics between QD immunoprobe and anti-small molecule antibody. A two-step immunoassay method that involves pre-incubation mixture of different concentration of small molecule and anti-small molecule antibody, and subsequent introduction of QD immunoprobe into the optofluidic cell was conducted for small molecule determination. Compared with the one-step immunoassay method, the two-step immunoassay method can obtain higher fluorescence signal and higher sensitivity index, thus improving the nano-biosensing performance. Based on the proposed strategy, two mode targets, namely, microcystin-LR (MC-LR) and Bisphenol A (BPA) were tested with high sensitivity, rapidity, and ease of use. A higher concentration of small molecules in the sample led to less anti-small molecule antibody bound with antigen-carrier protein conjugate immobilized onto the sensor surface, and less QD immunoprobes bound with anti-small molecule antibody. This phenomenon lowered the fluorescence signal detected by nano-biosensing platform. Under optimal operating conditions, MC-LR and BPA exhibited a limit of detection of 0.003 and 0.04 μg/L, respectively. The LODs were better than those of the indirect competitive immunoassay method for small molecules via Cy5.5-labeled anti-small molecule antibody. The proposed QD-based sandwich-like immunoassay strategy was evaluated in spiked water samples, and showed good recovery, precision and accuracy without complicated sample pretreatments. All these results demonstrate that the new detection strategy could be readily applied to the other trace small molecules in real water samples.

Autoantibodies against cardiac troponin I in patients presenting with myocarditis

Summary Background Autoantibodies against cardiac troponin I (cTnI) play an important role in the pathogenesis of experimental cardiomyopathy. We developed a new method to measure anti-cardiac troponin I autoantibody (Anti-cTnIAAB) in patients with myocarditis with or without HCV infection. Methods Patients with heart failure for up to 2 years, without a distinct cause, were enrolled in the Myocarditis Treatment Trial between 1986 and 1990. Frozen blood samples were available from 1315 to 2233 enrolled patients. Anti-cTnIAAB was determined by a two-step immunoassay. Results The mean (±SEM) value of serum Anti-cTnIAAB titer in the 1315 patients was 0.067 ± 0.003 arbitrary unit (AU), significantly higher than in 1115 healthy volunteers (0.053 ± 0.002 AU, P Conclusions Elevated autoantibody titers against cTnI were detected in patients with myocarditis, and were higher in HCV-infected patients. The presence of Anti-cTnIAAB might correlate with inflammation and viral infection of the heart.

Peginterferon for chronic hepatitis B: Predicting success with on‐treatment benchmarks

Peginterferon for chronic hepatitis B: Predicting success with on‐treatment benchmarks

Three Minutes-Long Electrophoretically Assisted Zeptomolar Microfluidic Immunoassay with Magnetic-Beads Detection

An ultrasensitive, two-step immunoassay method was developed for rapid assay of proteins and viruses. In the first step, electrophoresis was used to quickly bring soluble analytes from a flowing solution to a microarray of probe molecules immobilized on a semipermeable membrane. In the second step, the captured analyte was detected by scanning the microarray with functionalized magnetic beads passed over the array surface by shear flow and pressed to the array surface by a magnetic field. We demonstrated that this method routinely detected analytes in concentrations as low as 20 aM (500−1000 molecules, or 1−2 zeptamoles) within 2−3 min in the presence of a ∼1011-fold excess of other protein molecules in the sample.

Use of the Architect-i2000 estradiol immunoassay during in vitro fertilization.

In women undergoing in vitro fertilization and embryo transfer (IVF-ET), serial measurements of 17β-estradiol (E2) can be used to monitor follicular growth, and serum E2 has been shown to be correlated with the number and diameter of preovulatory follicles observed on transvaginal ultrasound scan (1)(2)(3)(4)(5)(6). The serum E2 concentration is therefore an essential variable for evaluating the progression of stimulation, adjusting daily gonadotropin therapy, predicting the optimal day for induction of ovulation \[administration of human chorionic gonadotropin (hCG)\] (7), preventing ovarian hyperstimulation syndrome (8)(9), and ensuring that pituitary function is adequately suppressed if a long-acting gonadoliberin-releasing hormone agonist (GnRHa) is used before stimulation (10). Another major use of E2 measurements is to evaluate ovarian function at day 3 of the menstrual cycle to determine the prognosis of IVF-ET (11). Because results must be available within a few hours, rapid and automated assays are required. In this study, we measured E2 with a new automated assay that could be used in random, continuous access mode and assessed its usefulness for monitoring ovulation stimulation for IVF-ET. The Architect-i2000 E2 (Abbott Laboratories) assay is a competitive two-step immunoassay based on chemiluminescent microparticle immunoassay technology and can be performed in 29 min. The ACS-180 E2 assay (Bayer Diagnostics) is a competitive one-step immunoassay based on solid-phase antigen-linked technology and chemiluminescence detection and can be completed in 15 min. The luminescence reaction and the calibration procedure are identical for the two E2 immunoassays. The two methods are linear up to 3670 pmol/L and use monoclonal antibody derived by coupling the E2 molecule at the specificity-enhancing sixth position. The within- and between-run imprecision, detection limits, and functional sensitivities of these assays have been reported …