Commercial Immunoassay Research Articles

Imbalances in tissue inhibitors of metalloproteinases differentiate choroidal neovascularization from geographic atrophy.

Tissue inhibitor of metalloproteinase (TIMP) is known to play a role in age-related macular degeneration (AMD). We wished to investigate alterations in different late stages of AMD: neovascular AMD and geographic atrophy (GA). This was a prospective case-control study. A total of 125 participants were included consecutively during a period of 18months. We included 46 patients with neovascular AMD, 46 patients with GA without any sign of choroidal neovascularization in either eye, and 33 healthy aged controls. Patients with immune-affecting disorders were not included. Commercial immunoassay kits were used to quantify levels of TIMP-1, TIMP-3, MMP-2 and MMP-9 in blood plasma. We found that patients with neovascular AMD had lower plasma concentration of TIMP-3 (p=0.028) than healthy controls. Patients with GA had higher plasma levels of TIMP-1 (p<0.001) and MMP-9 (p=0.022) compared to healthy controls. Also, we found that TIMP-1 levels in patients with GA increased with age (Spearman's rho=0.04, p=0.006). Matrix metalloproteinases (MMPs) and TIMPs, which are known to be involved in age-related changes in Bruch's membrane, are significantly altered systemically, suggesting the presence of an imbalance in the homeostasis of the extracellular matrix. These imbalances may explain differences in the clinical manifestation of late AMD.

A New ELISA to Overcome the Pitfalls in Quantification of Recombinant Human Monoclonal Anti-HBs, GC1102, by Commercial Immunoassays

Several methods for the quantification of human anti-HBs, an antibody to hepatitis B surface antigen (HBsAg), have been developed based on enzyme reaction, chemiluminescence, fluorescence, and radioactivity for application to human serum or plasma. Commercial anti-HBs immunoassay kits use a sandwich method in which a bridge is formed by the anti-HBs between a HBsAg immobilized solid matrix and the labeled HBsAg. However, this direct sandwich enzyme-linked immunosorbent assay (ELISA) is insufficient to accurately evaluate the activity of the human monoclonal anti-HBs, GC1102. As an alternative, we developed an indirect anti-HBs ELISA (anti-HBs qELISA_v.1) that improved detection of anti-HBs. In this current study, we further optimized this indirect method to minimize nonspecific binding of human serum, by employing incubation buffers containing animal serum, Tween 20, skim milk, and a low pH washing buffer. This new and improved method, termed anti-HBs qELISA_v.2, showed accurate quantification of plasma-derived hepatitis B immune globulin (HBIG) and was comparable to results obtained with commercial ELISA (r = 0.93) and RIA (r = 0.85) kits. Further, the GC1102 in human serum could be precisely measured using the anti-HBs qELISA_v.2 without limitations of nonspecific binding.

Mechanisms of death in structurally normal stillbirths.

Objectives To investigate mechanisms of in utero death in normally formed fetuses by measuring amniotic fluid (AF) biomarkers for hypoxia (erythropoietin [EPO]), myocardial damage (cardiac troponin I [cTnI]) and brain injury (glial fibrillary acidic protein [GFAP]), correlated with risk factors for fetal death and placental histopathology. Methods This retrospective, observational cohort study included intrauterine deaths with transabdominal amniocentesis prior to induction of labor. Women with a normal pregnancy and an indicated amniocentesis at term were randomly selected as controls. AF was assayed for EPO, cTnI and GFAP using commercial immunoassays. Placental histopathology was reviewed, and CD15-immunohistochemistry was used. Analyte concentrations >90th centile for controls were considered "raised". Raised AF EPO, AF cTnI and AF GFAP concentrations were considered evidence of hypoxia, myocardial and brain injury, respectively. Results There were 60 cases and 60 controls. Hypoxia was present in 88% (53/60), myocardial damage in 70% (42/60) and brain injury in 45% (27/60) of fetal deaths. Hypoxic fetuses had evidence of myocardial injury, brain injury or both in 77% (41/53), 49% (26/53) and 13% (7/53) of cases, respectively. Histopathological evidence for placental dysfunction was found in 74% (43/58) of these cases. Conclusion Hypoxia, secondary to placental dysfunction, was found to be the mechanism of death in the majority of fetal deaths among structurally normal fetuses. Ninety-one percent of hypoxic fetal deaths sustained brain, myocardial or both brain and myocardial injuries in utero. Hypoxic myocardial injury was an attributable mechanism of death in 70% of the cases. Non-hypoxic cases may be caused by cardiac arrhythmia secondary to a cardiac conduction defect.

Development and Use of an Endpoint Titration Assay To Characterize Mumps IgG Avidity following Measles, Mumps, and Rubella Vaccination and Wild-Type Mumps Infection.

Waning mumps IgG antibody and incomplete IgG avidity maturation may increase susceptibility to mumps virus infection in some vaccinees. To measure mumps IgG avidity, serum specimens serially diluted to the endpoint were incubated on a commercial mumps-specific IgG enzyme immunoassay and treated with the protein denaturant diethylamine (60 mM, pH 10). End titer avidity indices (etAIs [percent ratio of detected diethylamine-resistant IgG at endpoint]) were calculated. Unpaired serum specimens (n = 108) from 15-month-old children living in a low-incidence setting were collected 1 month and 2 years after the first measles, mumps, and rubella vaccine dose (MMR1) and tested for mumps avidity. Per the receiver operating characteristic curve, the avidity assay is accurate (area under the curve, 0.994; 95% confidence interval [CI], 0.956 to 1.000), 96.5% sensitive (95% CI, 87.9 to 99.6%), and 92.2% specific (95% CI, 81.1 to 97.8%) at an etAI of 30%. When 9 sets of paired serum specimens collected 1 to 60 months post-MMR1 were tested for mumps and measles IgG avidity using comparable methods, the mumps etAI increased from 11% to 40 to 60% in 6 months. From 6 to 60 months, avidity was sustained at a mean etAI of 50% (95% CI, 46 to 54%), significantly lower (P < 0.0001) than the mean measles etAI of 80% (95% CI, 74 to 86%). Mean etAIs in children 2 years post-MMR1 (n = 51), unvaccinated adults with distant mumps disease (n = 29), and confirmed mumps cases (n = 23) were 54, 62, and 57%, respectively. A mumps-specific endpoint avidity assay was developed and validated, and mumps avidity was determined to be generally sustained at etAIs of 40 to 60%, reaching etAIs of >80% in some individuals.IMPORTANCE Numerous outbreaks of mumps have occurred in the United States among two-dose measles-mumps-rubella (MMR)-vaccinated populations since 2006. The avidity of mumps-specific IgG antibodies may affect susceptibility to mumps virus infection in some vaccinated individuals. To accurately measure mumps avidity, we developed and validated a mumps-specific IgG avidity assay that determines avidity at the endpoint titer of serially diluted serum specimens, providing results that are independent of IgG concentration. At low antibody titers, endpoint methods are considered more accurate than methods that determine avidity at a single dilution. We determined that 6 months after the first MMR dose, mumps IgG avidity is high and generally sustained at avidity indices of 40 to 60%, reaching values of >80% in some individuals. Additionally, 4% (4/103) of individuals had avidity indices of ≤30% (low avidity) 2 years after vaccination. Inadequate mumps avidity maturation may be one factor influencing susceptibility to mumps virus infection among previously vaccinated or naturally infected individuals.

Post-mortem in situ stability of serum markers of cerebral damage and acute phase response.

The aim of the given study was to test the in situ stability of biochemical markers of cerebral damage and acute phase response in the early post-mortem interval to assess their usability for forensic pathology. A monocentric, prospective study investigated post-mortem femoral venous blood samples at four time points obtained within 48h post-mortem starting at the death of 20 deceased, using commercial immunoassays for the ten parameters: S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), C-reactive protein (CRP), procalcitonin (PCT), ferritin, soluble tumor necrosis factor receptor type 1 (sTNFR1), and lactate dehydrogenase (LDH). Significant changes in serum levels were observed only later than 2h after death for all markers. Inter-laboratory comparability was high, and intra-assay precision was sufficient for most markers. Most of the biomarker levels depended on the severity of hemolysis and lipemia but were robust against freeze-thaw cycles. Serum levels increased with longer post-mortem intervals for S100B, NSE, ferritin, sTNFR1, and LDH (for all p < 0.001) but decreased over this period for CRP (p = 0.089) and PCT (p < 0.001). Largely unchanged median values were found for GFAP (p = 0.139), BDNF (p = 0.106), and IL-6 (p = 0.094). Serum levels of CRP (p = 0.059) and LDH (p = 0.109) did not differ significantly between the final ante-mortem (resuscitation) and the first post-mortem sample (moment of death). Collecting the post-mortem blood sample as soon as possible will reduce the influence of post-mortem blood changes. Serum GFAP for detection of cerebral damage as well as serum IL-6 and CRP as proof of acute phase response seemed to be preferable due to their in situ stability in the first 2days after death.

Accuracy evaluation of Roche and Siemens tacrolimus and cyclosporine assays in comparison with liquid chromatography-tandem mass spectrometry

Therapeutic drug monitoring of tacrolimus and cyclosporine is crucial to the success of organ transplantation. We evaluated the analytical performances and accuracy of two commercially available tacrolimus and cyclosporine assays (Roche ISD and Siemens) in comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 342 leftover whole blood samples requested for tacrolimus or cyclosporine assays were stored at −20 °C until analysis. Repeatability and between-run imprecision were evaluated using quality control materials provided by the manufacturer. Ring trial samples were used for the assessment of recovery. The results of the Roche ISD assay were compared with those of Siemens tacrolimus and cyclosporine assays and LC-MS/MS. Repeatability and between-run imprecision were 2.1–5.3% and 2.6–7.5%, respectively. Recovery of Roche ISD was 85.7 − 90.6% for cyclosporine and 96.2–98.5% for tacrolimus. The two immunoassays showed slight positive biases relative to LC-MS/MS for cyclosporine. For tacrolimus, Roche ISD produced virtually identical results to those of LC-MS/MS, whereas Siemens showed proportional differences, especially in patients receiving kidney transplantation. The analytical performances of Roche ISD were generally acceptable, especially regarding accuracy. Clinical laboratory staff should be aware of the strengths and weaknesses of commercial immunoassays in order to ensure accurate results.

Current Thyrotropin Immunoassays Recognize Macro-Thyrotropin Leading to Hyperthyrotropinemia in Females of Reproductive Age.

Macro-thyrotropin (macro-TSH) is a high molecular weight form of TSH that leads to hyperthyrotropinemia. This study was undertaken to examine the prevalence and nature of macro-TSH in females of reproductive age. Blood samples were taken from 1794 female patients who visited the Hamada Obstetrics and Gynecology Hospital in Tokyo, Japan, complaining of infertility. The serum of 305 patients with TSH concentrations >2.5 mIU/L was screened for macro-TSH by the polyethylene glycol method. Samples with TSH precipitation ratios by polyethylene glycol >70% were further analyzed using gel filtration chromatography (GFC), protein G columns, and 125I-TSH binding experiments. Screening of the 305 patients revealed that 63 had serum TSH precipitation ratios >70%. GFC revealed that immunoreactive TSH, with a molecular weight of approximately 150 kDa, eluted at higher ratios (79.6 ± 24.4%) in 27 of the 63 patients compared to 0.4 ± 2.0% in the control group. Serum TSH concentrations in 24 of the 27 patients were spuriously elevated due to human anti-mouse antibodies. Macro-TSH was found in the other three patients, and one of them had detectable anti-TSH autoantibodies. Eight of the remaining 36 patients who did not have high-molecular-weight TSH assessed by GFC had immunoglobulin G-associated TSH. Three commercially available TSH immunoassays (Elecsys®, Centaur®, and Architect®) all recognized macro-TSH leading to the elevated serum TSH concentrations. Macro-TSH was present in 0.17% of infertile women. Commercial TSH immunoassays recognized macro-TSH, resulting in the diagnosis of hyperthyrotropinemia.

Validating the use of a commercial enzyme immunoassay to measure oxytocin in unextracted urine and saliva of the western lowland gorilla (Gorilla gorilla gorilla).

The neuroendocrine hormone oxytocin, which is an important physiological driver of social behavior and bonding, is increasingly being measured in conjunction with behavior to better understand primate sociality. To date no data are available on oxytocin concentrations within the genus Gorilla; however, as a result of their close genetic relatedness to humans, and tolerance-based social system, Gorilla represents an important group of study. The purpose of this study was to validate the measurement of urinary and salivary oxytocin in western lowland gorillas (Gorilla gorilla gorilla) to help facilitate future study of the interaction between oxytocin and behavior within the subspecies. The primary validation procedure was an intranasal challenge. Elevated oxytocin concentrations were observed in saliva samples taken 15-120min post challenge. Urine levels remained within baseline range approximately 30 and 90min following the challenge; however, elevated levels were observed 24h post challenge. No diurnal variation was found in salivary samples taken at regular intervals throughout the day; however, morning urine samples had higher concentrations than afternoon samples. In addition, samples were collected opportunistically following three social events: play, breeding, and the death of a conspecific. Following the play bouts, salivary oxytocin was almost three times greater than baseline. Salivary oxytocin was also significantly higher 15min post breeding compared to match-control samples. Following the death of a conspecific, the group mate's urinary oxytocin concentrations decreased by half compared to a baseline period when the group was intact. This study provides a biological validation of the measurement of urinary and salivary oxytocin in western lowland gorillas. These results suggest that urinary oxytocin measurements are suitable for establishing baseline levels, as they represent the build up of the previous day's concentrations, and salivary oxytocin measurements are suitable for assessing changes following specific events.

Diagnostic performance of commercial IgM and IgG enzyme-linked immunoassays (ELISAs) for diagnosis of Zika virus infection

BackgroundSerologic detection of Zika virus (ZIKV) infections is challenging because of antigenic similarities among flaviviruses.ObjectiveTo evaluate the sensitivity and specificity of commercial ZIKV IgM and IgG enzyme-linked immunoassay (ELISA) kits.MethodsWe used sera from febrile patients with RT-PCR-confirmed ZIKV infection to determine sensitivity and sera from RT-PCR-confirmed dengue cases and blood donors, both of which were collected before ZIKV epidemics in Brazil (2009–2011 and 2013, respectively) to determine specificity.ResultsThe ZIKV IgM-ELISA positivity among RT-PCR ZIKV confirmed cases was 0.0% (0/14) and 12.5% (1/8) for acute- and convalescent-phase sera, respectively, while its specificity was 100.0% (58/58) and 98.3% (58/59) for acute- and convalescent-phase sera of dengue patients, and 100.0% (23/23) for blood donors. The ZIKV IgG-ELISA sensitivity was 100.0% (6/6) on convalescent-phase sera from RT-PCR confirmed ZIKV patients, while its specificity was 27.3% (15/55) on convalescent-phase sera from dengue patients and 45.0% (9/20) on blood donors’ sera. The ZIKV IgG-ELISA specificity among dengue confirmed cases was much greater among patients with primary dengue (92.3%; 12/13), compared to secondary dengue (7.1%; 3/42).ConclusionsIn a setting of endemic dengue transmission, the ZIKV IgM-ELISA had high specificity, but poor sensitivity. In contrast, the ZIKV IgG-ELISA showed low specificity, particularly for patients previously exposed to dengue infections. This suggests that this ZIKV IgM-ELISA is not useful in confirming a diagnosis of ZIKV infection in suspected patients, whereas the IgG-ELISA is more suitable for ZIKV diagnosis among travelers, who reside in areas free of flavivirus transmission, rather than for serosurveys in dengue-endemic areas.

Dual-mode fluorescent and colorimetric immunoassay for the ultrasensitive detection of alpha-fetoprotein in serum samples

We present a novel dual-mode fluorescent and colorimetric immunosensor based on conventional immunoassay platforms by utilizing a gold nanoflower (AuNF)-loaded fluorescein molecule (AuNF@Fluorescein) as signal output. The AuNFs were modified with thiolated carboxyl ligand, which consisted of a hydrophobic alkane chain as hydrophobic wallet for fluorescein encapsulation, a tetra (ethylene glycol) unit for biocompatibility and solubility, and a functional carboxyl group for the conjugation of biorecognition molecules for biosensing. The resultant AuNFs showed a high loading capacity of 3.74 × 106 fluorescein molecules per AuNF because of its flower-like shape with many complex branches. By adjusting the solution pH to 8.0, the fluorescein molecules can almost entirely be released from the hydrophobic wallet of AuNF@Fluorescein, which led to strong fluorescent-signal amplification. Under the optimal detection conditions, the proposed immunoassay based on fluorescent signal exhibited ultrahigh sensitivity for alpha-fetoprotein (AFP) detection, with a limit of detection (LOD) of 29 fg/mL. This value is approximately 9.3 × 103-fold lower than that of corresponding horseradish peroxidase (HRP)-based immunoassay (LOD = 270 pg/mL). The fluorescein molecule also had intrinsic peroxidase-like activity to catalyze 3,3′,5,5′-tetramethylbenzidine oxidation with hydrogen peroxide for colorimetric signal. The proposed method with colorimetric mode further exhibited a sensitivity with a LOD of 17.7 pg/mL, which is about 15-fold lower than that of conventional HRP-based immunoassay. The recoveries of the proposed dual-mode immunoassay for AFP spiked serum samples ranged within 89.85%–100.0%, with the coefficient of variations ranging from 0.5% to 2.4%, indicating acceptable accuracy and precision for AFP quantitative detection. The reliability of the developed dual-mode immunoassay was further compared with a commercial chemiluminescence immunoassay kit by analyzing 20 clinical serum samples, showing that the two methods well agreed with each other, with high correlation coefficients of 0.997 and 0.986 based on recorded fluorescence and colorimetric signals, respectively. In summary, the proposed method was highly suitable for the ultrasensitive analysis of biomarkers or infectious diseases by fluorescence mode and can be used for routine clinical diagnosis by colorimetric mode.

Prevalence of hepatitis E virus viremia and antibodies among healthy blood donors in India

Hepatitis E virus (HEV) is transmitted primarily through contaminated water and food. Recently, HEV viremia in blood donors and transfusion-related transmission of HEV have been reported, leading to calls to screen donated blood for this virus. However, these data are from regions where genotype 3 HEV is predominant. In India, where human infections are caused only by genotype 1 HEV, the frequency of subclinical HEV viremia is unknown. Minipools of sera prepared from three donor units each from our institution's blood bank in Lucknow, India, were tested for HEV RNA using a sensitive amplification-based assay. A randomly selected subset was also tested for IgG anti-HEV antibodies using a commercial (Wantai) immunoassay. Sera from 1799 donors (median [range] age 30 [18-63] years; 1746 [97.0%] men) were collected (June-July 2016, 900; November-December 2016, 899). Of these, 17 (0.95%), 16 (0.90%), and 3 (0.17%) tested positive for HBsAg, anti-HCV, and anti-HIV antibodies, respectively. None of the donors tested positive for HEV RNA. Of 633 randomly selected donors (age 30 [18-63] years, 613 [96.8%] male) tested for IgG anti-HEV, 383 (60.5%) tested positive. Seropositivity rate increased with age, being 70/136 (52%), 177/299 (59%), 100/154 (65%), 30/34 (88%), and 6/10 (60%) in the 18-24, 25-34, 35-44, 45-54, and 55years or olderage groups, respectively. In healthy blood donors from northern India, HEV viremia is infrequent though anti-HEV antibody prevalence is high. This suggests that asymptomatic HEV viremia may be less frequent in areas with genotype 1 predominance than those with genotype 3 predominance.

Changes in synovial fluid and serum concentrations of cartilage oligomeric matrix protein over 5 years after anterior cruciate ligament rupture: an exploratory analysis in the KANON trial

To monitor longitudinal changes of cartilage oligomeric matrix protein (COMP) in synovial fluid (sf) and serum (s) over 5years after acute anterior cruciate ligament (ACL) rupture, and to compare results from two commercial COMP immunoassays. Bio-fluids were collected from 121 patients on six occasions over 5 years after acute ACL injury, and from 25 knee healthy reference subjects. Concentrations of sf- and sCOMP were measured by AnaMar (sCOMP-Ana) and by BioVendor (sf- and sCOMP-Bio) immunoassays; other biomarkers were previously assessed. We used ANCOVA for group comparisons and linear mixed models for associations between biomarkers over 5-years with P<0.05 considered a statistically significant difference or association. Compared to the reference group, sfCOMP-Bio concentrations were 2-fold elevated within 6 weeks after ACL injury and remained elevated 5 years thereafter, whereas sCOMP-Bio and sCOMP-Ana concentrations were no different from reference levels at any time point. Over the 5-year period, there was an association between sCOMP-Bio and sCOMP-Ana concentrations, although neither sCOMP-Bio nor sCOMP-Ana associated with sfCOMP-Bio. sfCOMP-Bio associated with SF ARGS-aggrecan, urine type I and II collagens (uNTX-I and uCTX-II) and SF cytokines, while sCOMP-Bio associated inversely with uCTX-II, uNTX-I and SF cytokines. The local process after an acute ACL injury generates increased SF COMP concentrations in the injured knee up to 5years after injury. This response is not detected in serum. Discrepancies in associations between sCOMP measured by BioVendor and AnaMar immunoassays with other biomarkers indicate differences in detected COMP fragments.

Low prevalence of HTLV1/2 infection in a population of immigrants living in southern Italy.

AimsTo assess the prevalence of HTLV-1 and HTLV-2 infections in a cohort of immigrants living in southern Italy.FindingsWe screened for antibody to HTLV-1/2 infection 1,498 consecutive immigrants born in endemic areas (sub-Saharan Africa or southern-Asia) by a commercial chemiluminescent microparticle immunoassay. If confirmed in a Western blot assay, which differentiates anti-HTLV-1 from anti-HTLV-2, the positive sera were tested for specific HTLV RNA by a home-made PCR. The immigrants investigated were more frequently males (89.05%), young (median age 26 years), with a low level of education (median schooling 6 years), born in sub-Saharan Africa (79.70%). They had been living in Italy for a median period of 5 months. Only one (0.07%) subject was anti-HTLV-1 -positive/HTLV-1 RNA-negative; he was an asymptomatic 27-year-old male from Nigeria with 6 years’ schooling who stated unsafe sexual habits and unsafe injection therapy.ConclusionsThe data suggest screening for HTLV1 and HTLV-2 infections all blood donors to Italy from endemic countries at least on their first donation; however, a cost-effectiveness study is needed to clarify this topic.

Development of a simple, rapid and high-throughput fluorescence polarization immunoassay for glycocholic acid in human urine

In this paper, a simple, rapid and high-throughput fluorescence polarization immunoassay (FPIA) based on polyclonal antibodies (PAb) is described for the determination of glycocholic acid (GCA) in human urine. Three fluorescein-labeled GCA (tracers) with different structures and spacer bridges were synthesized and purified by thin-layer chromatography (TLC). The structure effect of tracers on the assay was investigated and the sensitivity of best tracer in the optimized FPIA demonstrated an IC50 value of 306 ng/mL. The working range of FPIA was 36 ∼ 2 600 ng/mL and the limit of detection (LOD) was 9 ng/mL. The developed FPIA was time-saving that could be completed within 10 min. Human urine samples spiked with GCA were analyzed by this method, followed by confirmation with commercial enzyme immunoassay analysis (EIA). Excellent recoveries and correlation between these two methods were observed (R2 = 0.996), suggesting the developed FPIA could be applied to screening of GCA in human urine samples without complicated cleanup.

Current and Emerging Multianalyte Assays with Algorithmic Analyses-Are Laboratories Ready for Clinical Adoption?

Over the past decade, there have been a rising number of clinically used tests that combine 2 or more biochemical or molecular assays, demographics, and clinical information into an algorithm to generate diagnostic, prognostic, or predictive information for a specific disease. The concept of multianalyte analyses is relatively new in the field of laboratory medicine. Dating back to the 1980s, prenatal screening for fetal abnormalities, such as Down syndrome, by use of maternal biomarkers is among the pioneer tests that use algorithmic analyses for risk assessment. Yet, the number of multianalyte algorithms used clinically remains modest. The American Medical Association provides current procedural terminology (CPT)8 codes for 20 multianalyte assays with algorithmic analyses (MAAAs.) Among these, 9 consist of biochemical markers detected by immunoassay or mass spectrometry, with or without other clinical information; 11 use molecular genetics markers; and 1 is for generic use. In this Q&A, we refer to multivariable tests with risk scores as MAAAs, although not all of them have an associated MAAA CPT code. Generally speaking, MAAAs aim at improving diagnostics for diseases in which single biomarkers have limited clinical validity. The Prostate Health Index (Beckman Coulter) and the 4Kscore® (GenPath) exemplify some of these strategies for prostate cancer detection. Likewise, multianalyte analyses such as the Risk of Ovarian Malignancy Algorithm (ROMA®) and OVA1® and its second-generation OVERATM (Vermillion Inc.) improve upon the suboptimal performance of the tumor marker CA125 in the differential diagnosis and likelihood of ovarian carcinoma in women presenting with a pelvic mass. While both have Food and Drug Administration (FDA) clearance, ROMA is a nonproprietary algorithm cleared on various commercial immunoassays (Fujirebio Diagnostics, Inc.; Abbott Laboratories; Roche Diagnostics). Early identification of acute kidney injury is another area in which an FDA-approved multianalyte test, Nephrocheck® (Astute Medical), …

Elevated levels of adrenomedullin in eutopic endometrium and plasma from women with endometriosis

To test adrenomedullin (Adm, ADM) as a downstream target of signal transducer and activator of transcription 3 (STAT3) in endometrial cells and to test midregional proadrenomedullin (MR-proADM) as a biomarker of endometriosis. Cross-sectional analysis of Adm expression in eutopic endometrium and of MR-proADM in plasma from women with and without endometriosis; and prospective study of MR-proADM levels in women with endometriosis undergoing surgical resection of ectopic lesions. Academic medical centers. Fifteen patients with endometriosis and 11 healthy control subjects who donated eutopic endometrial biopsies; and 28 patients with endometriosis and 19 healthy control subjects who donated plasma for MR-proADM analysis. None. Adm mRNA levels according to quantitative real-time polymerase chain reaction after activation of STAT3 by interleukin-6 (IL-6) in Ishikawa cells; immunohistochemistry for ADM in eutopic endometrial biopsies from women with endometriosis compared with healthy donors; and MR-proADM levels measured by commercial immunoassay in plasma from healthy women and women with endometriosis who subsequently underwent surgical resection of ectopic lesions. Activation of STAT3 by IL-6 up-regulated Adm mRNA expression in Ishikawa cells. ADM protein levels were elevated in the eutopic endometrium of women with endometriosis. MR-proADM concentrations were higher in women with endometriosis but were not correlated with disease stage, corrected by surgery, or predictive of fertility outcome. MR-proADM may be able to serve as a biomarker of endometriosis, but it is unknown whether elevated MR-proADM levels are secondary to the estrogenic and inflammatory properties of endometriosis or an inciting pathogenic factor.

Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum.

The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method for quantification of INSL3 by mass spectrometry. We developed an assay in which the INSL3 A-chain is released from the INSL3 A-B heterodimer by chemical reduction and alkylation. The alkylated INSL3 A-chain is quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as substitute for serum INSL3. The method was compared to a validated and sensitive in-house serum INSL3 immunoassay using 97 serum samples from 12 healthy boys during pubertal transition. Adult levels were determined based on sera from 72 adult healthy males aged 18-40 years. An LC-MS/MS assay with limit of detection and limit of quantification (LOQ) of 0.06 and 0.15 ng/mL, respectively, and intra-assay CVs <9% in the relevant ranges was obtained. The LC-MS/MS compared well with the in-house immunoassay (Deming regression slope: 1.28; Pearson correlation: R=0.86). INSL3 concentrations increased with pubertal maturation in healthy boys. INSL3 concentrations were above the LOQ in all samples from the adult men. The mean (±2 SD range)for serum INSL3 concentrations in the adult men was 2.2 (0.5-3.9) ng/mL. We have developed a robust and sensitive method suitable for quantitation of serum INSL3 in a clinical setting using LC-MS/MS instrumentation available in modern clinical laboratories. The method paves the way for future studies into the clinical role of serum INSL3 measurements.

Significance of HBV NAT Among HBs Antigen-Negative Blood Donors in Saudi Arabia.

Bloodborne hepatitis B virus (HBV) transmission from asymptomatic donors with acute HBV infections who have undetectable surface antigen of HBV (HBsAg), or from donors with chronic infections in whom serological markers were not detected, could cause residual infections leading to relevant transfusion-transmitted infections (RTTIs). HBV nucleic acid testing (NAT) can detect HBV DNA in the HBsAg-negative and total hepatitis B core antibody (anti-HBc)-negative window period of infection and in chronic cases. To assess the presence or absence of HBV DNA in blood donors with HBsAg negativity. We collected 3014 blood specimens from volunteer blood donors at the blood bank of King Khalid University Hospital in Riyadh, Saudia Arabia. Specimens from each donor were tested for HBsAg, anti-HBc, and hepatitis B surface antibody (anti-HBs) by commercial immunoassays and for qualitative assessments of HBV-DNA by HBV-NAT testing. Of the 3014 donors, 7 (0.23%) tested positive for HBsAg and anti-HBc, 1 for HBsAg (0.03%) only, and of those 264 donors (8.8%) for anti-HBc. Of these last, 6.9% also tested positive for anti-HBs and 1.9% tested negative for anti-HBs. HBV-NAT testing was reactive in 75.0% of subjects who tested HBsAg positive, and nonreactive in 100% of subjects who tested anti-HBc positive/HBsAg negative (with or without anti-HBs). Among 2742 donors who tested seronegative, 1 specimen was determined to be reactive via HBV-NAT testing. The frequency of HBV DNA in blood donors who tested seronegative was low. This finding may indicate the significance of the HBV NAT technique in reducing the residual risk of transfusion-transmitted HBV infection.

Simultaneous quantitation of carbohydrate antigen 125 and carcinoembryonic antigen in human serum via time-resolved fluoroimmunoassay

In clinical diagnosis of cancer, immunology assay with single tumor marker often lead to a false and missed inspection. A quantitative method with a high degree of accuracy, sensitivity, and effectiveness is required for its diagnosis. We developed a dual-label time-resolved fluoroimmunoassay (TRFIA) to simultaneously detect carbohydrate antigen 125 (CA125) and carcinoembryonic antigen (CEA) in human serum to aid the diagnosis and prognosis of gastric cancer. The method was based on a microplate sandwich immunoassay using europium-labeled anti-CA125 antibodies and samarium-labeled anti-CEA antibodies as fluorescent reporters. The assay detection range was widely, and the limit of detection was sufficiently for detecting clinical sample. The intra- and inter-assay coefficients of variation were below 6%, and recoveries ranged from 90% to 110%. No significant statistical difference in sensitivity or specificity was observed between dual label-TRFIA and commercial chemiluminescent immunoassays in serum samples. These results demonstrate the successful development of an effective, reliable, and convenient novel TRFIA method for the simultaneous detection of CA125 and CEA, which can be used for clinical blood screening to monitor the occurrence and development of tumors to facilitate early treatment.

Use of urinary 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) concentrations to diagnose pregnancy and predict parturition in the giant panda (Ailuropoda melanolecua).

Pregnancy determination is difficult in the giant panda (Ailuropoda melanolecua), representing a challenge for ex situ conservation efforts. Research in other species experiencing pseudopregnancy indicates that urinary/fecal concentrations of 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) can accurately determine pregnancy status. Our objective was to determine if urinary PGFM concentrations are associated with pregnancy status in the giant panda. Urinary PGFM concentrations were measured in female giant pandas (n = 4) throughout gestation (n = 6) and pseudopregnancy (n = 4) using a commercial enzyme immunoassay. Regardless of pregnancy status, PGFM excretion followed a predictable pattern: 1) baseline concentrations for 11–19 weeks following ovulation; 2) a modest, initial peak 14–36 days after the start of the secondary urinary progestagen rise; 3) a subsequent period of relatively low concentrations; and 4) a large, terminal peak at the end of the luteal phase. Pregnant profiles were distinguished by an earlier initial peak (P = 0.024), higher inter-peak concentrations (P < 0.001), and a larger terminal peak (P = 0.003) compared to pseudopregnancy profiles. Parturition occurred 23 to 25 days from the initial PGFM surge and within 24 hours of the start of the terminal increase. These pattern differences indicate that urinary PGFM monitoring can be used to predict pregnancy status and time parturition in the giant panda. Furthermore, this is the only species known to exhibit a significant PGFM increase during pseudopregnancy, suggesting a unique physiological mechanism for regulating the end of the luteal phase in the giant panda.