J. Alan Erickson

Immunoassay for Quantifying Squamous Cell Carcinoma Antigen in Serum

BACKGROUND Although the benefits of quantifying serum squamous cell carcinoma antigen (SCCa) have been reported, SCCa reagents were no longer available in the US by the late 1990s. Because SCCa quantification still has demonstrated clinical utility, we developed and validated a microtiter plate-based ELISA for measuring SCCa in serum. METHODS We coated microtiter strips overnight with capture anti-SCCa monoclonal antibody, washed the wells, added blocking buffer, and lyophilized the strips. For detection, we used a biotinylated anti-SCCa detection antibody, streptavidin/horseradish peroxidase conjugate, and tetramethylbenzidine/H(2)O(2) substrate. A novel blocking reagent against human antimouse antibodies (HAMA) was evaluated. A reference interval was established with sera from healthy individuals and was confirmed in smokers. RESULTS The assay was linear to 40 microg/L SCCa (slope, 1.00; y intercept, 0.695; R(2), 0.996) with a detection limit of 0.3 microg/L. The intraassay imprecision results [mean (CV)] were 2.5 microg/L (3.4%), 18.0 microg/L (3.0%), and 30.7 microg/L (2.4%); interassay imprecision results were 2.0 microg/L (9.9%), 20.0 microg/L (7.6%), and 36.3 microg/L (3.5%). A correlation analysis against an established automated assay generated a slope of 0.976 and a y intercept of -0.193 microg/L (r(2) = 0.916). An upper reference limit of 2.1 microg/L SCCa was established at 95% confidence level, with no difference observed in smokers. No correlation between SCCa concentration and age was observed (r(2) = 0.0003). At a blocking reagent concentration of 5 mg/L, HAMA interference was eliminated in 3 samples known to produce falsely increased SCCa results. CONCLUSIONS This SCCa ELISA demonstrates acceptable performance characteristics for quantifying serum SCCa and is effective in eliminating HAMA interference.

Evaluation of a dimeric inhibin-A assay for assessing fetal Down syndrome: establishment, comparison, and monitoring of median concentrations for normal pregnancies.

CONTEXT Several studies report the role of dimeric inhibin-A in assessing risk for fetal Down syndrome. The majority, however, use the Serotec inhibin-A assay and not the newer Diagnostic Systems Laboratories inhibin-A enzyme-linked immunosorbent assay (ELISA). OBJECTIVES To establish normal gestational age day-specific medians, to compare our results against previous studies pertaining to the inhibin-A ELISA, and to evaluate long-term assay performance. DESIGN Using the inhibin-A ELISA, 100 specimens were assayed for each completed week of gestation for weeks 15 to 20, 50 specimens for 14 weeks, and 54 specimens for 21 weeks or older. Regressed inhibin-A medians were calculated employing a second-degree polynomial fit of the arithmetic medians. Thereafter, inhibin-A ELISA lot comparisons were performed to evaluate consistency. RESULTS Regressed values of 182, 174, 175, 184, 201, and 226 pg/mL resulted for weeks 15 to 20, respectively [pg/mL inhibin-A = 4.1528(gestational age)2 - 136.49(gestational age) + 1294.9]. A comparison with 2 other studies shows our values to be lower overall by 15 +/- 11.4% and 16 +/- 2.6%. However, variability between kit lots was as high as 30%. CONCLUSIONS The equation derived provides for the calculation of gestational age day-specific inhibin-A medians for integration into maternal serum screening programs with a subsequent decrease in false-positives expected and observed. Our medians differ considerably from those of other studies, with limited data, using the Diagnostic Systems assay. However, lot changes since the initial analysis have exhibited similar inconsistencies. Therefore, we recommend that others incorporating the assay into their screening programs carefully establish, monitor, and adjust their medians accordingly as a result of potential variations.

Performance characteristics of an automated assay for the quantitation of CYFRA 21-1 in human serum☆

OBJECTIVES A fragment of cytokeratin 19, CYFRA 21-1, has been reported to be a sensitive tumor marker for non-small cell lung cancer (NSCLC). We describe analytical performance characteristics of a novel CYFRA 21-1 assay and hypothesize that CYFRA 21-1 complements the clinical sensitivity of carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCCa). DESIGN AND METHODS Performance characteristics of a CYFRA 21-1 immunochemiluminescent assay included analytical sensitivity, imprecision, linearity, analyte stability, and reference interval determination. Ninety-two pretreatment NSCLC serum samples were tested for CYFRA 21-1, CEA, and SCCa. Sensitivity was determined for each marker individually and in combination, with regard to tumor stage and histology. RESULTS The analytical sensitivity was 0.01 ng/mL. Total imprecision ranged from 4.0 to 6.3% at 4.9 to 28.4 ng/mL, respectively. The assay was linear from 0.9 to 71.4 ng/mL (slope=0.995, intercept=-0.60, r(2)=0.999). CYFRA 21-1 was stable for 48 h at ambient temperature and 14 days at 4°C. The 97.5th percentile of a reference population was 1.9 ng/mL. Across disease stage, the sensitivities of CYFRA 21-1, CEA, and SCCa were 17-81%, 30-52%, and 24-39%, respectively. CYFRA 21-1 combined with CEA or SCCa increased sensitivity above that of any single marker. CONCLUSIONS An immunochemiluminescent assay for CYFRA 21-1 had favorable performance characteristics. CYFRA 21-1 was complementary to CEA and SCCa and increased clinical sensitivity in patients with NSCLC.

Comparison of three assays for quantifying S-100B in serum

BACKGROUND Serum S-100B has clinical value in monitoring malignant melanoma and in monitoring and predicting outcomes in patients with traumatic brain injury. METHODS Analytical performance characteristic and split-sample method comparison studies for three commercial S-100B immunoassays (CanAg® S100, Sangtec® 100, YK150 Human S-100 β) were performed. Reference intervals (97.5th percentile) for each assay were established by non-parametric analysis of results from healthy volunteers. RESULTS Linearity results were slope=1.014, intercept=65.1, r(2)=0.999 for the Sangtec assay; slope=1.038, intercept=31.1, r(2)=0.999 for the CanAg assay; slope=1.123, intercept=-105.4, r(2)=0.997 for the YK150 assay. Within-run CVs were ≤5.7, ≤6.3 and ≤10.8 for the Sangtec, CanAg and YK150 ELISAs, respectively. Between-run CVs were ≤11.3, ≤5.9 and ≤9.5, respectively. Upper reference interval limits of 141, 96 and 735 ng/l S-100B were established for the Sangtec, CanAg and YK150 ELISAs, respectively. Deming regression generated the following: CanAg vs. Sangtec, slope=0.339, intercept=24.1, r(2)=0.932; YK150 vs. Sangtec, slope=0.266, intercept=-140.0, r(2)=0.690; YK150 vs. CanAg, slope=1.376, intercept=-13.1, r(2)=0.860. CONCLUSIONS The configurations, procedures and performance characteristics of the Sangtec and CanAg S-100B ELISAs are comparable and better than those of the YK150 assay. Poor agreement and large biases prevent interchangeable use of results.

Evaluation of a fecal pancreatic elastase-1 enzyme-linked immunosorbent assay : Assessment versus an established assay and implication in classifying pancreatic function

BACKGROUND Disagreement continues regarding 2 fecal pancreatic elastase-1 (PE-1) ELISAs and their respective capabilities to assess pancreatic function. METHODS The BioServ Diagnostics polyclonal PE-1 ELISA was validated and its performance characteristics compared to the previously validated ScheBo Biotech monoclonal PE-1 ELISA. Split sample study results were analyzed by Deming regression and Bland-Altman plot analysis. Data mining was utilized to explore PE-1 distribution and evaluate PE-1 and fecal fat correlation. RESULTS Analysis demonstrates limited quantitative agreement; slope=0.9640, intercept=10.787, R(2)=0.633. Means were 228.8 and 226.2 microg PE-1/g stool for the polyclonal and monoclonal assays respectively. Bland-Altman analysis showed 91% of paired values within 2 SD of their means. There was good qualitative agreement when interpreted against established intervals with 91% of results equivalent in pancreatic function classification. The remaining 9% varied by one classification level with no bias evident. The distribution of PE-1 concentrations (n=400, 0-25 years) classified 78% of subjects with normal pancreatic function, 7% with moderate pancreatic insufficiency and 15% with severe insufficiency. There was little agreement between PE-1 and fecal fat results. CONCLUSIONS The polyclonal PE-1 ELISA is an acceptable alternative to the monoclonal PE-1 ELISA. PE-1 is a potential substitute for fecal fat for evaluating pancreatic function.

Performance characteristics of the Access® Inhibin A assay

BACKGROUND Second trimester maternal screening using AFP, uE3, hCG, and inhibin A has shown a detection rate for Downs syndrome of 81% with a 5% false positive rate. Inhibin A may also have utility as a serum tumor marker in postmenopausal women with ovarian cancer and men with testicular stromal tumors. METHODS The Beckman Coulter Access Inhibin A assay was evaluated for limit of blank, dilution linearity, imprecision, interferences, reference intervals, and comparison to an inhibin A ELISA. RESULTS The limit of blank was 0.1 ng/l. The assay was linear from 0.2 to 1347 ng/l. Total inter-assay CVs were <5% for control levels ranging from 24.6 ng/l to 811 ng/l. Interference studies showed recoveries of inhibin A within 10% of expected values at interferent concentrations of 10 g/l hemoglobin and 22 g/l triglycerides. No significant interference was observed at a bilirubin concentration of 400 mg/l. The 97.5th percentile upper reference limits were 6.8 ng/l for postmenopausal women and 3.0 ng/l for men. The Access assay compared to an ACTIVE ELISA showed a slope of 0.88, an intercept of -3.7 ng/l, S(y/x)=40 ng/l, and r=0.98. CONCLUSIONS The analytical performance of the Access inhibin A assay is acceptable for routine laboratory testing.

A chromogranin A ELISA absent of an apparent high-dose hook effect observed in other chromogranin A ELISAs

BACKGROUND Routine testing for chromogranin A (CgA) using an established commercial ELISA revealed an apparent high-dose hook effect in approximately 15% of specimens. Investigations found the same effect in two additional ELISAs. We hypothesized that a CgA derived peptide(s) at high concentrations was responsible but experiments were inconclusive. Here we describe the analytical performance characteristics of the Chromoa™ CgA ELISA that did not display the apparent high-dose hook effect. METHODS Performance characteristics of the Chromoa ELISA were assessed. The reference interval was established utilizing healthy volunteers. Specimens producing the apparent high-dose hook effect in other assays were evaluated using the Chromoa ELISA. RESULTS The limit of detection was 8ng/ml. Linearity was acceptable (slope=1.04, intercept=18.1 and r(2)=0.997). CVs were ≤4.6 and ≤9.3% for repeatability and within-laboratory imprecision, respectively. CgA was stable at ambient and refrigerated temperatures for a minimum of two and 14days, respectively. An upper reference interval limit of 95ng/ml was established. Specimens demonstrating the apparent high-dose hook effect in other ELISAs did not exhibit the phenomenon using the Chromoa ELISA. CONCLUSIONS The Chromoa ELISA demonstrates acceptable performance for quantifying serum CgA. The apparent high-dose hook effect exhibited in other ELISAs was absent using the Chromoa assay.

Quantitative Spectrophotometric Microplate Assay for Angiotensin-converting Enzyme in Cerebrospinal Fluid

Angiotensin-converting enzyme (ACE; EC 3.4.15.1) catalyzes the formation of angiotensin II by cleaving the C-terminal histidylleucine dipeptide from angiotensin I (1). Indications are that ACE is affiliated with an autonomous renin-angiotensin system of the brain that participates in physiologic processes inside the brain (2)(3). In addition, studies suggest that changes in ACE concentrations in brain tissue, caused by various neurologic disorders, are reflected by alterations in ACE activity in cerebrospinal fluid (CSF) (4). For example, increased ACE concentrations in CSF are associated with neurosarcoidosis (4)(5)(6)(7), with affected patients generally having activities approximately twofold or more higher than those of healthy individuals (4)(6)(7). Increased CSF ACE has also been implicated in neurologic diseases, such as bacterial and viral meningitis and Behcet disease (4)(5)(6)(7). Decreased concentrations have been reported in patients with Alzheimer disease, Parkinson disease, and progressive supranuclear palsy (8)(9). The spectrophotometric assays customarily used for serum ACE lack the sensitivity for measuring the concentrations typically found in CSF. Consequently, more sensitive and costly methodologies are routinely used, such as fluorometric assays or HPLC (4). To provide a more economic assay to measure CSF ACE for the screening of neurosarcoidosis, several attempts were made at modifying commercially available reagents and protocols used specifically for spectrophotometric measurement of serum ACE. A successful assay was eventually established that utilizes the ability of ACE to hydrolyze the tripeptide N -[3-(2-furyl)acryloyl]-l-phenylalanylglycylglycine to furylacryloylphenylalanine and glycylglycine (10). However, the sample volume (0.6 mL) required to achieve acceptable sensitivity was impractical. The assay was therefore reformatted for a 96-well microplate. This dramatically decreased sample and reagent volumes and increased accuracy by making duplicate measurements viable for practically all specimens received in our laboratory. …