Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays.

The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively binds to the middle part of human GAD65 (rFab b96.11). In the assay the biotinylated rFabs are recognized by Europium labeled streptavidin. The obtained time-resolved fluorescence (TRF) is directly proportional to the concentration of GAD65 over a large measuring range (0.1 to >100 ng/mL). Based on total error estimation including both bias and imprecision, the lower limit of quantitation (LLOQ) of GAD65 in cell extracts is 0.33 ng/mL with the N-GAD65 TRFIA, and 0.10 ng/mL with the b96.11 TRFIA, but the latter is suitable for human GAD65 only, whereas the N-GAD65 TRFIA has equal sensitivity with rat and human GAD65. Specificity was further checked with GAD65/67 fusion proteins, confirming that the presence of intact capture as well as detection epitope on the analyte is a prerequisite for recognition in both assays. We show that the beta cell-specific marker GAD65 can be quantified in pancreatic cell extracts and in serum, allowing studies on discharge during cell death in vitro as well as in vivo.

Negative interference of bilirubin and hemoglobin in the MEIA troponin I assay but not in the MEIA CK-MB assay.

Troponin I is a sensitive and specific marker for the diagnosis of myocardial infarction. Several commercially available immunoassays measure the concentration of troponin I in serum. The microparticle enzyme immunoassay (MEIA) for troponin I (Abbott Laboratories, Abbott Park, IL) is widely used in clinical laboratories, including our hospital laboratory. We studied the effect of bilirubin and hemolysis on the MEIA for troponin I and compared our assay with a newly available chemiluminescent assay (CLIA) for troponin I (Bayer Diagnostics, Tarrytown, NY). We also measured CK-MB concentration using the MEIA CK-MB assay. One serum pool was prepared by combining several specimens of one patient with elevated troponin I and with a diagnosis of myocardial infarction. Other serum pools were prepared by combining sera with similar troponin I values. All serum pools showed normal bilirubin concentrations and had no hemolysis. Then we supplemented aliquots of serum pools with various concentrations of bilirubin (5.0, 10.0, 15.0, and 20.0 mg/dL). After supplementation, troponin I concentrations were measured again using the MEIA and CLIA. We observed a statistically significant decrease in troponin I concentration in the presence of bilirubin with the MEIA. For example, in serum pool 1, the troponin I concentration was 16.3 (bilirubin: 0.8 mg/dL). In the presence of 5.0, 10.0, 15.0 and 20.0 mg/dL of added bilirubin, the cardiac troponin I concentrations were 13.9, 13.4, 13.3 and 13.0 ng/ml respectively. We observed similar negative interference of bilirubin in troponin I measurement by the MEIA in other pools. The troponin I value decreased slightly (not statistically significant) in one pool and did not change in two other pools in the presence of bilirubin when we measured troponin I concentration using the CLIA. Interestingly, bilirubin did not interfere with the MEIA CK-MB assay. Moderate hemolysis did not have any effect on the troponin I assay using either the MEIA or CLIA. However, gross hemolysis (hemoglobin > 40 mg/dL) interfered with both assays for troponin I.

Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB.

BACKGROUND: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. METHODS: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. RESULTS: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. CONCLUSIONS: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.

Influence of isoenzyme patterns on commutability in enzyme determinations.

The use of recommended methods has very much improved the precision and accuracy of enzymatic determinations. The use of 'enzyme reference materials' allows us to overcome the remaining difficulties. The possibility of extending standardization by using calibration materials to convert results obtained by different methods requires an essential property: 'commutability'. The relationship between patients' specimens and reference materials are conditioned by analytical causes (relative to the used methods) and clinical causes (relative to the condition of the human samples to be examined). Among the clinical causes, the different isoenzyme content of patients sera, induced by different pathologies, was considered. The effect of this influence has been investigated with an analytical approach of the isoenzymes in the individual clinical samples. The results obtained on determination of alkaline phosphatase (ALP), creatine kinase (CK), and lactate dehydrogenase (LD), are discussed according to a model of investigation which can be utilized for the experimental protocols in the assessment of commutability.

Development of a reference material for alkaline phosphatase.

In developing a Reference Material for alkaline phosphatase, we studied the stability, kinetic properties, and commutability of separate preparations of the purified enzyme from human liver, intestine, bone, and placenta. The Michaelis constants (Km) for the preparations from liver, bone, and intestine agreed well with the Km values we obtained for five human serum specimens, whereas that for the placental isoenzyme differed significantly. The first three isoenzymes exhibited nearly identical response-surface patterns, which closely paralleled those observed for 12 human serum specimens (commutability), but not that of the placental isoenzyme. Thus, we believe that a reference material could equally well consist of either the bone, intestinal, or liver isoenzyme. All four isoenzymes were satisfactorily stable in temperature-accelerated degradation studies. We chose the liver isoenzyme as an appropriate reference material because liver tissue is easier to obtain than bone or intestine and the isoenzyme is abundant in liver, is easy to extract, and is the one most commonly increased in human serum. This material is stable at -20 degrees C, is free of interfering and degradative enzymes and, being of human origin, is commutable with the enzyme in human serum.

Activity of human and nonhuman amylases on different substrates used in enzymatic kinetic assay methods--a pitfall in interlaboratory quality control.

Many commercial kits have been marketed recently for the determination of amylase activity in clinical specimens by enzymatic kinetic methods. Oligosaccharides (e.g., maltotetraose or maltopentaose) or limit dextrin are used as substrates. Hydrolysis of the substrate is coupled through a series of enzymes to convert NAD+ to NADH which is measured at 340 nm. Commercially available controls and standards for the amylase test consist of pooled human sera supplemented with human, porcine, or bovine amylase. The authors tested various control sera and standards by six commercial kits. Sera supplemented with porcine or bovine pancreatic amylase gave significantly lower values when assayed by methods using maltotetraose as substrate than when assayed by methods using maltopentaose or other oligosaccharides as substrate. Sera supplemented with human salivary amylase gave comparable results by five of the six methods. Results were comparable by all six methods for serum specimen supplemented with human pancreatic amylase. These kinetic methods are superior to the older amyloclastic, saccharogenic, or dye-coupled starch methods and are expected to gain popularity among clinical laboratories. The authors recommend that quality control programs designed to evaluate interlaboratory performances consider the use of human pancreatic amylase to supplement their serum specimens.