Agreement of anti-neutrophil cytoplasmic antibody measurements obtained from serum and plasma.

Serum and plasma are used interchangeably to measure anti-neutrophil cytoplasmic antibodies (ANCA), even though the release of ANCA target antigens during the preparation of serum could affect ANCA assays and cause discrepancies between the results obtained from serum and plasma. To what extent ANCA test results obtained from serum agree and correlate with results from plasma remains unknown. Therefore, a comprehensive comparison was performed using serum and plasma samples which were collected in 175 patients with active Wegener's granulomatosis at enrollment of a recent randomized trial. These paired serum and plasma samples were subjected to parallel ANCA testing by standard indirect immunofluorescence on ethanol-fixed neutrophils, a direct enzyme-linked immunoassay (ELISA) for proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA, and two different capture ELISAs for PR3-ANCA. The concordance of categorical serum and plasma ANCA results was assessed using kappa-coefficients. These were > 0.8 for all assays, indicating a very good concordance between positive and negative serum and plasma results. Spearman's correlation coefficients for serum and plasma PR3-ANCA values obtained by direct ELISA and both capture ELISAs were > or = 0.95 (P < 0.0001). Our study shows that serum and plasma samples can be used interchangeably for measuring ANCA.

Detection of antinuclear antibodies: comparative evaluation of enzyme immunoassay and indirect immunofluorescence methods.

OBJECTIVE: To evaluate a commercial microtiter enzyme immunoassay for antinuclear antibodies (ANA) by comparing the results of tests performed with this assay to an established indirect immunofluorescence method performed on human epitheliod cell substrate slides. DESIGN: Both analytical methods were used to test for the presence and levels of ANA in stored sera from 313 patients previously shown to have detectable ANA and from 102 healthy control subjects. Follow-up tests for specific autoantibodies (anti-dsDNA antibodies and antibodies to extractable nuclear antigens [ENA]) were performed on all sera from patients. The medical histories of all patients were reviewed to determine the presence of systemic rheumatic diseases (SRDs). Different cut-off levels of positivity were examined to determine the sensitivity and predictive values of positive results on the enzyme immunoassay for detecting patients with SRDs or sera with positive tests for specific autoantibodies. RESULTS: Among patients with clinically diagnosed SRDs (n = 197), the enzyme immunoassay was positive for ANA (> or =1 U) in 100% and the indirect immunofluorescence method was positive (titer > or =40) in 95.4% of cases. Among ANA-positive patients with no SRDs (n = 116), testing by enzyme immunoassay and indirect immunofluorescence yielded positive results in 97.6% and 75.6% of cases, respectively. Among healthy control subjects, each of the two methods was positive in 15% of cases. As expected, most patients with SRDs had higher levels of ANA than did ANA-positive patients with other clinical diagnoses. A cut-off level of > or =3 U on the enzyme immunoassay correctly classified 77% of patients with a SRD as "positive" and 88% of patients with other clinical diagnoses as "negative." The probability of detecting a positive result for specific autoantibodies on second-order testing increased directly with the level of ANA. A cut-off level of > or =3 U had a sensitivity of 92% for identifying sera with positive specific autoantibodies, and results > or =3 U had a predictive value of 52% for a positive second-order test result. CONCLUSION: Enzyme immunoassay is substantially equivalent to indirect immunofluorescence for detecting clinically important ANA. Cut-off levels for positive results on the enzyme immunoassay can be established that optimize the usefulness of this method in diagnostic algorithms for specific autoantibodies.

Serum IgG4 concentrations and allergen-specific IgG4 antibodies compared in adults and children with asthma and nonallergic subjects.

We describe the development of specific immunoassays for IgG4 protein and for allergen-specific IgG4 antibodies. We also measured the concentrations of IgG4 protein and determined the frequencies of detectable IgG4 antibodies to several common allergens in sera from adults and children with asthma and from nonallergic subjects. Serum concentrations of IgG4 protein increase with age but are not different in children with asthma and nonallergic children, nor does a raised serum concentration predict a severe clinical course in childhood asthma. IgG4 antibodies to milk and egg are common in children and adults and are more common in children with asthma than in nonallergic children less than 3 years of age. The presence of detectable IgG4 antibodies or a raised concentration of IgG4 protein in serum is not useful empirically as a diagnostic indicator of asthma but more likely results from antigen exposure occurring at mucosal surfaces.

Measurement of secretory IgA in serum by radioimmunoassay in patients with chronic nonalcoholic liver disease or carcinoma.

The authors describe the development of a double antibody radioimmunoassay specific for human secretory IgA (sIgA), and they report the results of measurements of serum sIgA concentrations in patients with chronic, nonalcoholic liver disease or carcinoma. Above-normal sIgA concentrations (greater than 25 micrograms/mL) were found in 22 of 38 sera from patients with chronic active liver disease and in 37 of 40 sera from patients with primary biliary cirrhosis. Markedly increased concentrations (greater than 118 micrograms/mL) were specific for primary biliary cirrhosis. Above-normal sIgA concentrations were found frequently in patients with colorectal (29/86, 34%), pancreatic (38/70, 54%), gastric (11/30, 37%), or mammary (6/46, 13%) carcinoma. An above-normal concentration of sIgA was more specific for hepatic metastases than an above-normal alkaline phosphatase activity in each type of carcinoma. The authors conclude that measurement of sIgA in serum is a useful diagnostic test in patients with chronic liver disease suspected of having primary biliary cirrhosis or in patients with carcinoma suspected of having hepatic metastases.

Analytic accuracy of specific immunoglobulin E antibody results determined by a blind proficiency survey.

Analytic variability affects the accuracy of measurements of specific IgE antibodies, but the frequency of false results attributable to analytic variability is not well documented. We have monitored the accuracy of the results generated in our laboratory by testing aliquots of positive serum pools and a negative serum pool submitted blindly for the measurement of IgE antibodies to 16 different allergens, including foods; weed, grass, and tree pollens; mites, molds, and epithelia; and Hymenoptera venoms. Positive serum pools were prepared to contain modest amounts of IgE antibodies. Tests were performed by immunometric assays with microcrystalline cellulose allergen immunosorbents. Frank false-positive and false-negative results were very uncommon when binding levels were classified by a ratio-based reporting scheme. False-borderline results were more common. A borderline result is truly equivocal and may or may not indicate the presence of low levels of IgE antibodies. Analytic variability adds uncertainty to the measurement of small quantities of IgE antibodies regardless of the classification scheme used to report test results.

Measurement of anti-IgA antibodies by a two-site immunoradiometric assay.

To enable the detection of IgG class, anti-IgA antibodies (IgG-aIgA) and to investigate the possible occurrence of IgE class, anti-IgA antibodies (IgE-aIgA), we developed a solid phase immunoradiometric assay (IRA), which uses purified IgA coupled covalently to microcrystalline cellulose as an immunosorbent. Radiolabeled, Fc specific anti-IgG and anti-IgE antibodies were used to detect specific aIgA after incubation of test sera or controls with the immunosorbent. IgG-aIgA were detected by the IRA in 100 and 67 per cent of control sera with class specific and limited specificity aIgA. The IRA was sensitive to approximately two ng of class specific IgG-aIgA. IgG-aIgA also were detected by IRA in 7.9 per cent of sera from patients with urticarial transfusion reactions and 73 per cent of sera from patients with ataxia telangiectasia and IgA deficiency. Sera from 50 normal blood donors did not have detectable IgG-aIgA. Tests for IgE-aIgA were negative in all cases, including control sera with class specific IgG-aIgA. We conclude that the IRA is a sensitive and reproducible method for detection of class specific and limited specificity IgG-aIgA, and that IgE-aIgA do not mediate urticarial transfusion reactions.

Radioimmunoassay of creatine kinase B-isoenzymes in serum of patients with azotemia, obstructive uropathy, or carcinoma of the prostate or bladder.

We measured the concentrations of creatine kinase B-isoenzymes by radioimmunoassay in 271 serum specimens from patients with azotemia, benign prostatic hyperplasia, adenocarcinoma of the prostate, and transitional cell carcinoma of the bladder. There was no correlation between the concentrations of B-isoenzymes and creatinine in the sera of azotemic patients. Above-normal concentrations of B-isoenzymes were found in sera from three patients with acute renal failure, but in only two of 28 specimens from patients with chronic renal failure. Above-normal concentrations of B-isoenzymes also were found in sera from three of 18 patients with untreated carcinoma of the prostate, 10 of 25 patients with treated carcinoma, 20 of 135 patients with benign prostatic hyperplasia, and 10 of 33 patients and with transitional cell carcinoma of the bladder. An above-normal concentration of B-isoenzymes in serum has a low predictive value for adenocarcinoma of the prostate, was not a sensitive indicator of the presence of carcinoma, and was noted paradoxically in six patients with treated carcinoma who had normal acid phosphatase activities in serum. We conclude that routine measurement of B-isoenzymes is not useful to establish the diagnosis of adenocarcinoma of the prostate.

Creatine kinase radioimmunoassay and isoenzyme electrophoresis compared in the diagnosis of acute myocardial infarction.

We compared, in 116 patients, the relative usefulness of results of tests for creatine kinase B isoenzymes, as measured by radioimmunoassay, and the MB isoenzyme, as measured by electrophoresis, in diagnosis of acute myocardial infarction. The radioimmunoassay was specific for isoenzymes of creatine kinase containing the B subunit. All patients with acute transmural infarcts had positive test results by both techniques, but concentrations of B-isoenzymes were more frequently above normal than were MB bands in the case of patients with acute subendocardial infarcts and in the case of all patients with acute myocardial infarcts from whom sera were collected more than 24 h after onset of chest pain. Concentrations of B-isoenzymes also were increased, even when MB bands were not electrophoretically detectable, in specimens from several patients without documented actue myocardial infarcts. These abnormal results presumably were caused by increased concentrations of the BB isoenzyme in serum. Accordingly, an increased concentration of B-isoenzymes had less diagnostic specificity and predictive value for acute myocardial infarction than did a detectable MB band. Results of isoenzyme electrophoresis were more reliable for establishing this diagnosis, but the results of radioimmunoassay were more reliable for excluding it in patients with chest pain as the primary symptom.

Metastable creatine kinase MM and complexes of creatine kinase BB and immunoglobulin G, "atypical" isoenzymes with similar electrophoretic mobility.

Metastable creatine kinase MM isoenzyme was isolated and partially purified from homogenates of myocardium and skeletal muscle by gradient elution on carboxymethyl cellulose. This variant isoenzyme migrated between the MM and MB isoenzymes on agarose electrophoresis, accounted for 3.5% of the total creatine kinase activity in each tissue, was not a macromolecule, and had stable electrophoretic mobility only in borate buffer (0.02 mol/L). By comparison, the creatine kinase isoenzymes with similar "atypical" electrophoretic mobility in serum specimens were complexes of the BB isoenzyme and immunoglobulin G. These complexes were measured by a radioimmunoassay specific for the creatine kinase B-subunit and eluted predominantly with the MB isoenzyme in a commercial anion-exchange reagent system.

Immunoglobulin E antibodies to pollen allergens account for high percentages of total immunoglobulin E protein.

The quantities of immunoglobulin E (IgE) antibodies to grass or ragweed allergens were measured by an immunoabsorption in the serums of patients sensitive to one of these allergens. IgE antibodies to grass or ragweed allergens accounted for means of 30 and 29 percent of the total IgE protein. After the ragweed pollination season, the levels of serum IgE antibodies to ragweed allergens rose dramatically and in postpollination serums they accounted for 39 percent of the total IgE protein with a range from 13 to 50 percent.