Serodiagnosis of infectious diseases with antigen microarrays.

AIMS: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. METHODS AND RESULTS: Antigens were printed on activated glass slides using high-speed robotics. The slides were incubated with serum samples and subsequently with fluorescently labelled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected using confocal scanning microscopy and quantified with internal calibration curves. The microarray assay could detect as little as 0.5 pg of both IgG and IgM bound onto the glass surface. Precision profiles ranged from 1.7 to 18.5% for all the antigens. Microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Overall >80% concordance was obtained between microarray and ELISA kits in the classification of sera. CONCLUSIONS: These results indicate that the microarray is a suitable assay format for the serodiagnosis of infectious diseases. SIGNIFICANCE AND IMPACT OF STUDY: Antigen microarrays can be optimized for clinical use, their performance is equivalent to ELISA but they offer significant advantages in throughput, convenience and cost.

SR1 assays of alpha-fetoprotein, carcinoembryonic antigen, and prostate-specific antigen compared with corresponding established commercial assays.

We compared results obtained with the newly developed alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA) assays for the fully-automated Serono SR1 analyzer with those obtained with the major, established methods for these analytes: Serono Serozyme and Bridge, Abbott IMx, Kodak Amerlite, Boehringer Mannheim ES600, Pharmacia Delfia, Hybritech Tandem-E and Tandem-R, Ciba Corning ACS180, and DPC IRMA-Count. The correlations were good for all methods studied (r > or = 0.94). For AFP, numerical agreement was good, with linear regression slopes of 0.88 to 1.15; for CEA, correlation slopes of 1.03 to 1.16 were observed. The SR1 PSA assay agreed well with five of the seven methods studied (slopes of 0.98 to 1.22), but the Ciba Corning ACS180 PSA assay gave results higher than all other methods studied (slope 0.54 vs SR1 at low doses) and the Abbott IMx PSA assay gave results lower than all other methods studied (slope 1.47 vs SR1).

Measurement of prostate-specific antigen in serum using four different immunoassays.

This paper summarises the results of a comparison of the Serono SR1, Ciba Corning ACS 180, Abbott IMx and Hybritech Tandem-R prostate-specific antigen assays. One hundred serum pools were assayed using the four methods. Linear regression analysis of the data showed that, although overall correlations were good, different assays gave different prostate-specific antigen concentrations. Tandem-R and SR1 assays gave very similar prostate-specific antigen values; in general, the ACS assay gave higher prostate-specific antigen values than the IMx assay gave lower prostate-specific antigen values than the established Tandem-R assay. Following fractionation of serum from prostate cancer patients, all immunoassays detected several immunoreactive prostate-specific antigen forms. The major immunoreactive form (> 88% of immunoreactivity) had an apparent molecular size of M(r) approximately 100,000 and is likely to be a complex of prostate-specific antigen with alpha 1-antichymotrypsin; two minor forms had apparent molecular sizes of M(r) approximately 30,000 (probably free prostate-specific antigen) and 200,000 (probably prostate-specific antigen complexed to high molecular mass anti-proteases). From this study there is no evidence that polyclonal/monoclonal antibody immunoassays are to be preferred to monoclonal/monoclonal antibody immunoassays for the determination of free prostate-specific antigen in serum.

Measurement of alpha-fetoprotein, carcinoembryonic antigen and prostate-specific antigen in serum and heparinised plasma by enzyme immunoassay on the fully automated serono SR1 analyzer.

This paper describes the new enzyme immunoassays for the measurement of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA) in serum and heparinised plasma as developed for the fully automated Serono SR1 analyzer. Each assay incorporates two monoclonal antibodies in an immunoenzymetric sandwich format using alkaline phosphatase as the label and magnetic solid phase separation. All processing steps are performed by the SR1. The assays have good analytical performance with respect to detection limits (typically 0.24 microgram/l, 1.0 microgram/l and 0.1 microgram/l respectively for AFP, CEA and PSA), precision (within and between run CVs less than 7 and 12% respectively) and recovery of added analyte (100 +/- 10%). Regression analysis of linearity on dilution gives correlation coefficients (r) of 0.9984-1.0000. Tumour marker concentrations measured with these assays are in good agreement with concentrations determined by established immunoassays (r > 0.96). Values measured in samples of healthy probands and samples of patients with malignant and non-malignant diseases are as would be expected for clinically valid assays. The study demonstrates that the measurement of AFP, CEA and PSA in serum and heparinised plasma on the fully automated SR1 analyzer is suitable for the clinical diagnosis, monitoring and management of certain cancers and, in the case of AFP, for prenatal screening of maternal serum.

A fully automated enzyme immunoassay for the measurement of cortisol in biological fluids.

This paper describes a fully automated assay on the Serono SR1 for the measurement of cortisol in serum, heparinised plasma and urine. The assay incorporates a specific polyclonal antibody to cortisol and cortisol conjugated to alkaline phosphatase as a label. Following immunoincubation, bound and free labelled cortisol are separated by magnetic sedimentation of the antibody complex. Phenolphthalein is liberated by the enzymatic hydrolysis of the substrate (phenolphthalein monophosphate) and the absorbance generated is measured as the assay end-point. All processing steps are performed by the SR1. The assay has good analytical performance with respect to precision (within and between run CVs less than 10 and 11.5% respectively), detection limit (less than 5 nmol/l) and recovery of added cortisol (100 +/- 10%). The assay agrees well with cortisol concentrations determined by ID-MS and by established immunoassays (r > 0.96). Reference ranges of normal urine samples (pretreated by solvent extraction) are in good agreement with accepted values. The study demonstrates that the SR1 Cortisol assay on the SR1 analyzer is suitable for the routine determination of cortisol in serum, heparinised plasma and urine samples.