Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization.

Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury. We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa. The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies. Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the MutS strain KM71H. We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation. Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases. Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography. The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification. Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies.

Standardization of creatine kinase-MB (CK-MB) mass assays: the use of recombinant CK-MB as a reference material.

BACKGROUND: The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays. METHODS: Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples. RESULTS: Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer's sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer's sample diluent showed a 13% between-manufacturer bias. CONCLUSION: Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays.

Development, characterization, and use of monoclonal antibodies made to antigens expressed on the surface of fetal nucleated red blood cells.

BACKGROUND: Current methods for obtaining fetal cells for prenatal diagnosis are invasive and carry a small (0.5-1.0%) but definite risk of miscarriage. An attractive alternative would be isolation of fetal cells from peripheral maternal blood using antibodies with high specificity and avidity. METHODS: To generate antibodies, we purified nucleated red blood cells (NRBCs) from fetal livers and used them as the immunogen to generate monoclonal antibodies (mAbs) directed against surface antigens. RESULTS: The four antibodies recognized at least two conformationally sensitive epitopes of the transferrin receptor. Isolation of NRBCs from 252 maternal blood samples using these antibodies in magnetic activated cell sorting after an initial density gradient centrifugation yielded 0-419 NRBCs per 25 mL of maternal blood. One antibody, 2B7.4, not only isolated the highest number of NRBCs (>10 in 90% of the samples) but also isolated these NRBCs in 78 consecutive maternal samples. CONCLUSION: Antibody 2B7.4 shows promise for the isolation of NRBCs from maternal blood and should allow studies concerning the source of these cells, fetal vs maternal, and the factors controlling their prevalence.

Patterns and potential value of cardiac troponin I elevations after pediatric cardiac operations.

BACKGROUND: Perioperative myocardial injury is a major determinant of postoperative cardiac dysfunction for congenital heart disease, but its assessment during this period is difficult. The objective of this study was to determine the suitability of using postoperative serum concentrations of cardiac troponin I (cTnI) for this purpose. METHODS: Cardiac troponin I levels were measured serially in the serum of patients undergoing uncomplicated repairs of atrial septal defect (n = 23), ventricular septal defect (n = 16) or tetralogy of Fallot (n = 16). The concentrations were correlated with intraoperative parameters (cardiopulmonary bypass time, aortic cross-clamp time, and cardiac bypass temperature), and postoperative parameters (magnitude of inotropic support, duration of intubation, and postoperative intensive care and hospital stay). RESULTS: Postoperative absolute cTnI levels were lesion specific, with a pattern of increase and decrease similar for each lesion. For the total cohort, significant correlations between postoperative cTnI levels at all times (r = 0.43 to 0.83, p < 0.05) until 72 hours were noted for all parameters, except for cardiac bypass temperature. When evaluated as individual procedure groups, no significant relationships were noted in the atrial septal defect group, whereas postoperative cTnI levels were more strongly correlated with all intraoperative and postoperative parameters in the ventricular septal defect group than in the tetralogy of Fallot group. CONCLUSIONS: This study suggests that cTnI values immediately after operation reflect the extent of myocardial damage from both incisional injury and intraoperative factors. Cardiac tropinin I levels in the first hours after operation for congenital heart disease are a potentially useful prognostic indicator for difficulty of recovery.

Cardiac troponin I in pediatrics: normal values and potential use in the assessment of cardiac injury.

OBJECTIVE: To establish normal values and determine the impact of congenital or acquired heart disease on serum cardiac troponin I (cTnI). METHODS: Concentrations of cTnI were measured in two groups of children. Group A represented ambulatory pediatric patients with no apparent cardiac disease (n = 120) and patients in stable condition with known congenital or acquired cardiac abnormalities (n = 96); group B was composed of patients admitted to intensive care units with normal echocardiograms (n = 16), with abnormal echocardiograms (n = 36), and those with blunt chest trauma who were thought to have cardiac contusions (n = 7). RESULTS: The cTnI concentrations were generally less than 2.0 ng/ml in group A and frequently below the level of detection for the assay (1.5 ng/ml). There was no statistical difference between the two outpatient subgroups (p = 0.66). Nine intensive care patients had cTnI values greater than 2.0 ng/ml. Six of these patients, all with abnormal echocardiograms, had values less than 7.7 ng/ml. All improved and had subsequent normal cTnI concentrations. None of the three remaining patients (two with systemic illness (trauma and sepsis) and one with severe pulmonary hypertension), all with values greater than 8.0 ng/ml, survived. Three of the four patients with high likelihood of cardiac contusion had cTnI concentrations greater than 2.0 ng/ml (including one patient who died). CONCLUSIONS: Cardiac troponin-I values are generally not elevated in children with stable cardiac disease or general pediatric conditions. In the context of severe acute illness, significant elevation of cTnI may be an indicator of poor outcome. Elevation of cTnI may also have diagnostic value in cases when cardiac contusion is suspected.

Differential reactivity of cardiac and skeletal muscle from various species in a cardiac troponin I immunoassay.

To identify a blood test that can differentiate cardiac from skeletal muscle injury in animals, we compared tissue reactivities for various species with the use of an immunoassay for human cardiac troponin I (cTnI). Tissue reactivity varied as a function of the homology of tissue troponin with human cTnI. Cardiac reactivity in large mammals was equivalent to cTnI, 9.8 +/- 0.6 mg/g, and was 2-fold, 10-fold, and 100-fold greater than in small mammals, birds, and fish, respectively. Skeletal muscle reactivity was equivalent to cTnI, 5.1 +/- 0.6 micrograms/g, in all species except fish, in which it was 50% lower. The ratio of reactivities of cardiac and skeletal muscle was: 1800 in large mammals, 1100 in small mammals, 230 in birds, and 43 in fish. We conclude that cTnI is a powerful candidate in mammals, a possible candidate in birds, but unlikely to be of use in fish as a sensitive and tissue-selective diagnostic test for cardiac injury.

Comparative sensitivity of cardiac troponin I and lactate dehydrogenase isoenzymes for diagnosing acute myocardial infarction.

Criteria for the retrospective diagnosis of acute myocardial infarction rely heavily on increases in lactate dehydrogenase (LD) isoenzymes. However, increases of LD isoenzyme activities are not specific for myocardial injury. Recently, increased concentrations of cardiac troponin I (cTnI) have been shown to be highly specific for myocardial damage and to have sensitivity comparable with that of creatine kinase MB isoenzyme for detecting cardiac injury. Furthermore, increases of cTnI persist in plasma for at least several days. The present study was designed to determine the relative sensitivities of cTnI and LD isoenzymes over time for the diagnosis of infarction. The results indicate that cTnI values are at least as sensitive as LD isoenzymes: 90% of patients with myocardial infarction had above-normal concentrations of cTnI on the 4th day after admission to the coronary care unit. Criteria based on cTnI should improve the accuracy of retrospective diagnoses of acute myocardial infarction.

Creatine kinase brain isoenzyme (BB-CK) presence in serum distinguishes osteopetroses among the sclerosing bone disorders.

Creatine kinase (CK) isoenzyme BB-CK is predominantly found in brain and is not normally detected in the blood. A few recent reports, however, have described BB-CK in serum from several patients with osteopetrosis (OP). To evaluate the presence and specificity of BB-CK in serum in the osteopetroses among disorders that increase skeletal mass, we quantitated total CK activity and CK isoenzymes in 15 patients representing the five major clinical forms of OP (2 infantile, 3 intermediate, 7 adult [2 type I, 5 type II], and 3 carbonic anhydrase II [CA II] deficiency cases) and in 22 patients representing 14 other types of sclerosing bone disease. All OP patients (except the two adult type I subjects) had BB-CK readily detected in their serum. Conversely, only 1 of the 22 patients with other sclerosing bone disorders had detectable BB-CK in serum (1 of 3 patients with fibrodysplasia [myositis] ossificans progressiva who had barely measurable activity). In three OP patients (one of two with the infantile form and two of five with adult, type II disease), BB-CK values were sufficiently high that serum total CK activity was elevated. In a newborn with malignant OP, both cord blood plasma and peripheral blood serum had substantial amounts of BB-CK. In three subjects (with adult type II OP), who were restudied 2-6 years later, BB-CK was still elevated in their blood. BB-CK in serum appears to distinguish the osteopetroses among the sclerosing bone disorders. Absence of serum BB-CK in adult type I disease suggests that this condition may not be a genuine form of OP. Assay of BB-CK in fetal blood could be studied as a means for prenatal diagnosis of malignant OP. Why the osteoclast failure that characterizes all true forms of OP is associated with BB-CK in the circulation is a new question for skeletal biologists.

Early monitoring of serum cardiac troponin I for assessment of coronary reperfusion following thrombolytic therapy.

The authors report the use of cardiac troponin I (cTnI) for the early, noninvasive determination of coronary reperfusion following thrombolytic therapy. Cardiac troponin I, creatine kinase (CK)-MB, and myoglobin concentrations were measured in early serum specimens at 30, 60, and 90 minutes after initiation of therapy (0 minutes) in 25 consecutive patients given front-loaded rt-PA during acute myocardial infarction. Angiography, determined at 90 minutes after therapy, was used to classify patients as follows: group I (n = 17) reperfusion (TIMI flow grade 2, 3); and group 2 (n = 8) absence of reperfusion (TIMI flow grade 0, 1). The authors calculated the ratio increase in cTnI (delta cTnI), CK-MB (delta CK-MB), and myoglobin (delta myoglobin) 90 minutes after therapy in group 1 and 2. Serum cTnI, CK-MB and myoglobin concentrations significantly increased at 60 and 90 minutes in group 1, but not in group 2. Delta cTnI, delta CK-MB, and delta myoglobin levels were significantly increased in group 1 versus group 2 at 90 minutes. Further, delta cTnI was significantly greater at 90 minutes within group 1 compared to delta CK-MB and delta myoglobin. The sensitivity for detecting reperfusion at 90 minutes angiography using threshold values of 6.0 for delta cTnI, 7.0 for delta CK-MB, and 5.0 for delta myoglobin were: delta cTnI 82.4%; delta CK-MB 64.7%; delta myoglobin 76.5%; respectively. This study indicates that early serial measurements of cTnI were a more accurate predictor of early coronary artery reperfusion 90 minutes after thrombolytic therapy compared to CK-MB and myoglobin. Larger population studies will be necessary to confirm these finding.

Comparable detection of acute myocardial infarction by creatine kinase MB isoenzyme and cardiac troponin I.

Although measurement of cardiac troponin I (cTnI) is, in some situations, more specific for detection of cardiac injury than is measurement of the MB isoenzyme of creatine kinase (MBCK), its sensitivity and specificity relative to MBCK for detection of myocardial infarction has not been established. Accordingly, we studied prospectively 199 consecutive patients admitted to the coronary care unit. Values of MBCK and cTnI mass were determined in all samples. Of the 188 patients admitted with a suspicion of acute myocardial ischemia, 89 were diagnosed as having an acute myocardial infarction on the basis of the patterns of MBCK values. Eighty-six of these patients also had increased cTnI (concordance, 96.6%); three did not. Of the patients diagnosed as without infarction, five with unstable angina and symptoms in the day(s) prior to admission had increased cTnI, for a cTnI specificity of 94.9%. Receiver operating characteristic curve analysis indicated that cTnI and MBCK had statistically indistinguishable diagnostic accuracies for the detection of acute myocardial infarction.

Development of monoclonal antibodies for an assay of cardiac troponin-I and preliminary results in suspected cases of myocardial infarction.

To improve the specificity of biochemical markers of myocardial infarction (MI), we have developed a double monoclonal "sandwich" enzyme immunoassay to measure cardiac troponin-I (cTnI) in serum. We produced eight IgG monoclonal antibodies against human cardiac troponin-I (cTnI) and tested them against human and animal (canine, bovine, and rabbit) troponins. Five antibodies were cardiac-specific; none of the antibodies were species-specific. Two of the five cTnI-specific monoclonal antibodies were utilized in an immunoassay. Standards were made by adding purified human cTnI to affinity-stripped cTnI-free human sera to cover the range 0-100 micrograms/L for cTnI. The dose-response curve was nonlinear but reproducible. Total assay imprecision (CV) varied between 11% and 21%. The upper limit of the reference range (nonparametric 95% interval) was established as 3.1 micrograms/L by measuring cTnI concentration in sera of 159 hospitalized patients without evidence of cardiac disease. Purified human skeletal TnI up to 10,000 micrograms/L did not affect the assay (calculated cross-reactivity < 0.1%). Diagnostic sensitivities of creatine kinase MB isoenzyme (CK-MB) and cTnI were evaluated retrospectively in 49 consecutive patients with proven MI. In the 30 patients for whom sufficient information was available to establish an accurate time course, CK-MB was more sensitive during the first 4 h after the onset of chest pain, but thereafter the sensitivities were similar up to 48 h. However, cTnI is more cardiac-specific than is CK-MB and remains increased longer than does CK-MB.

Development and application of monoclonal antibodies to human cardiac myoglobin in a rapid fluorescence immunoassay.

Myoglobin (Mb) is considered a useful marker for early detection of myocardial infarction and for monitoring cardiac reperfusion after thrombolytic therapy. We developed eight monoclonal antibodies to human cardiac Mb, characterized their epitopic reactivity, and determined which combinations of the antibodies are useful in two-site immunoassays. We configured two of the monoclonal antibodies in a one-step, two-site particle concentration fluorescence immunoassay (PCFIA) for measurement of Mb. The PCFIA has rapid kinetics of reaction, being complete in 15 min, and has a linear analytical range of 20-675 micrograms/L for human Mb. Although the PCFIA has a high-dose "hook" effect, this is of no analytical importance at concentrations of Mb less than or equal to 148,000 micrograms/L. The assay is not subject to interference from icterus (bilirubin less than or equal to 360 mg/L), has no cross-reaction with hemoglobin (less than or equal to 42 g/L), and may be performed with either plasma or serum in approximately 1 h. The intra- and interassay imprecisions (CV) of the method are less than 10% for concentrations of Mb within the normal range and less than 4% at higher concentrations. A comparison of the PCFIA with a commercial radioimmunoassay showed that results of the two assays correlate well (PCFIA = 0.88 x RIA + 18, r = 0.990, n = 171).

Immunoaffinity purification of creatine kinase-MB from human, dog, and rabbit heart with use of a monoclonal antibody specific for CK-MB.

We describe a simple, rapid immunoaffinity procedure for purifying the MB isoenzyme of creatine kinase. Immunoaffinity gel is prepared by linking a monoclonal antibody ("Conan-MB"), specific for this isoenzyme, to Sepharose 4B. Heart tissue is homogenized and fractionated with 40-70% saturated ammonium sulfate before it is applied to the immunoaffinity gel. CK-MB activity, retained on the gel, is then eluted with a high-pH diethylamine buffer (0.1 mol/L, pH 10.5). The purified CK-MB isoenzyme is stabilized by collection directly into tubes containing glycerol (to prevent dissociation of the enzyme subunits) and pH-neutralizing buffer. This procedure compares favorably in yield, specific activity, and technical ease with a multi-column purification method previously used in our laboratory. We have used the immunoaffinity procedure to purify to homogeneity CK-MB from human, dog, and rabbit heart, with yields of 50.0%, 53.1%, and 49.3% and specific activities of 540, 477, and 377 kU/g, respectively. The preparations are pure as judged by protein staining with silver nitrate after electrophoresis on sodium dodecyl sulfate/polyacrylamide gel.

Quantification of lactate dehydrogenase-1 in serum with use of an M-subunit-specific monoclonal antibody.

We have developed a rapid, one-step assay for measuring lactate dehydrogenase-1 (LD-1) activity in serum after extraction of LD-2, LD-3, LD-4, and LD-5 isoenzymes by an immobilized M-subunit-specific monoclonal antibody (D.8.1). In the assay, 100 microL of serum is mixed with 50 microL of a suspension of 0.8-micron-diameter latex particles coated with 30 micrograms of the monoclonal antibody D.8.1, then incubated at room temperature for 5 min. The latex particles, to which LD-2 through LD-5 are bound, are pelleted by centrifugation for 2 min at 12,000 X g, and the LD-1 activity is measured kinetically in the supernatant fluid. We optimized the assay for antibody immobilization, antibody concentration, and time and temperature of incubation. Serum bilirubin concentrations up to 0.33 g/L (0.56 mmol/L) did not interfere in the assay. Hemolysis interfered solely through LD-1 released from erythrocytes. The within-assay CV for low-concentration quality-control material (LD-1 33 U/L) was 3.5% (n = 9) and for high-concentration material (LD-1 185 U/L) was 1.9% (n = 8); the between-assay CVs for the two materials were 6.1% (n = 9) and 2.5% (n = 10), respectively. The LD-1 activity measured in 98 samples by our assay compared well with a two-step polyclonal antibody-based assay (Isomune-LD, Roche Diagnostic Systems; r = 0.998) and with an electrophoretic method (Paragon, Beckman Instruments; r = 0.956).

Semi-automated direct colorimetric measurement of creatine kinase isoenzyme MB activity after extraction from serum by use of a CK-MB-specific monoclonal antibody.

This semi-automated colorimetric assay for the MB isoenzyme of creatine kinase (EC 2.7.3.2) is based on a monoclonal antibody ("Conan-MB") specific for this isoenzyme and is a modification of a previously published method (Vaidya et al., Clin Chem 1986;32:657-63). A 0.64-cm bead coated with 2 to 3 micrograms of antibody is incubated with 100 microL of serum and 10 microL of 0.2 mol/L beta-mercaptoethanol for 1 h at room temperature, to extract CK-MB. The beads are washed with de-ionized water and incubated with CK substrate for 45 min at 37 degrees C. A solution containing trans-1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid, p-iodonitrotetrazolium violet, and diaphorase is added and the resulting colored product is measured at 492 nm. The standard curve is linear to 200 U of CK-MB per liter, and analytical recovery is 97-113%. Total assay CV for low (9.7 U/L) and high (50.7 U/L) quality-control materials was 14.1% (n = 1878) and 11.6% (n = 1842), respectively. CK-MB activity correlated well (r = 0.978, n = 226) with CK-MB measured by a two-site mass immunoassay, and 99.4% of samples with CK-MB greater than or equal to 12 U/L (n = 347) were verified by electrophoresis on agarose.