Immunochemical determination of CK-MB isoenzyme in human serum. II. An enzymic approach.

A novel immunochemical technique for a specific enzymic determination of the myocardial isoenzyme of creatine kinase, CK-MB, involves determination of B-subunit activity of a specimen in which the M-subunit activity has been inhibited by specific antibodies to the M-subunit. Interfering activities from CK-BB isoenzyme, atypical forms of creatine kinase, and adenylate kinase are eliminated by using a blank tube in which all the M-subunit-containing isoenzymes have been removed by a specific immunoprecipitation step. The assay is convenient, linear, and reproducible, and results compare well with those by agarose electrophoresis.

Immunochemical determination of CK-MB isoenzyme in human serum: a radiometric approach.

The presence of creatine kinase isoenzyme MB (CK-MB) in human serum is an indicator of myocardial injury. In this assay for CK-MB, reagent containing both rabbit antibodies to CK-MM and 125I-labeled sheep antibodies to CK-BB is added to the patient's serum and incubated for 1 h at ambient temperature. Goat anti-rabbit immunoglobulin, conjugated to a mixture of amylose and polyvinylidene fluoride floccules, is then added. After a 15-min incubation, the mixture is centrifuged, isolating the two M-subunit-containing isoenzymes as insoluble complexes. Because the B-subunit portion of MB is labeled with 125I, the radio-activity in the pellet will be proportional to the amount of MB present. The discarded supernate contains excess sheep 125I-labeled BB antibodies, free or bound to BB isoenzyme if it is present. The preparation and characterization of the isoenzymes and antibodies is explained. Concentrations of CK-MB is sera of patients with and without an acute myocardial infarction were assayed in serial specimens obtained in 103 consecutive admissions to a coronary care unit. The performance of this radiometric procedure compared well with CK-MB electrophoresis, giving a sensitivity of 100% and a specificity of 92%.

Immunochemical determination of LDH-1.

An evaluation of a new immunochemical method for the heart isoenzyme of lactic dehydrogenase (LDH-1) is described. In this assay, a goat antibody against the M subunit of LDH is added to a patient's serum. Donkey antigoat gamma globulin is then added, which precipitates the M-anti-M complex. LDH activity in the supernatant is thus a measure of the heart isoenzyme, since this isoenzyme is not precipitable by the anti-M antibody. Assays for total CK, CK-MB, and total LDH, and LDH electrophoresis were performed along with an immunochemical LDH-1 assay on 261 serum samples from 49 patients clinically suspected to have acute myocardial infarctions. These were performed on admission and 12, 24, 48, 72, and 96 hours later. The diagnosis of acute infarction was confirmed for 25 of the 49 patients and ruled out for 19. Five patients were classified clinically as borderline cases for the diagnosis of acute myocardial infarction. All 25 patients with the clinical diagnosis of myocardial infarction had positive results by both electrophoresis and immunochemistry; four of the five borderline patients had positive results by electrophoresis. All five borderline patients had positive results by immunochemistry. None of the 19 patients without apparent myocardial injury had positive results by either electrophoresis or immunochemistry. Ten (40%) of the 25 samples collected at the time of admission from 25 patients who had myocardial infarctions had elevated LDH-1 activity by electrophoresis, while 14 patients (56%) had increased LDH-1 by immunochemistry. The immunochemical method for LDH-1 is more sensitive than electrophoresis for the detection of myocardial infarction and offers the further advantages of greater simplicity, precision, and accuracy, while being no less specific.

Plasma human placental lactogen, oxytocinase, and placental phosphatase in normal and toxemic pregnancies.

A total of 625 serum samples were drawn from 400 normal and 225 hypertensive toxemic pregnant women. Each sample was simultaneously assayed for its human placental lactogen (HPL), oxytocinase (O), and placental phosphatase (PP) concentration. In addition, accurate placental and infant birth weights were determined in those cases where the serum sample was obtained within 14 days of delivery. The results showed a significant rise and correlation of each of the three proteins with increasing weeks of gestation. Although the infant birth weight was unrelated to the serum level of the three proteins, both the HPL and O concentrations were significantly correlated with the placental weight in the normal pregnancies. In both types of pregnancies, the concentration of O was significantly related to that of PP and this was also true for HPL and O and HPL and PP. In all instances O was more strongly related than PP. In the toxemic pregnancies there was a higher O and lower PP level than in normal gestations. These data suggest that placental enzyme measurements, especially O, could be clinically helpful in monitoring high-risk pregnancies.

Detection of drugs of abuse by radioimmunoassay: a summary of published data and some new information.

We review the status of radioimmunoassays for detection of abused drugs. Individual assays with use of 125I-labeled antigens are all performed in an identical manner and can be completed in 30 min to 1 h. Combined assays for simultaneous detection of two or more such drugs or assays in which a tritium-labeled antigen is used require 1-2 h for completion. All tests can be performed with 0.1 ml or less of specimen. The assays involving 125I reliably detect urinary concentrations of, per liter, 40-100 mug of morphine, 100 mug of barbiturates, methadone, methaqualone, or benzoylecgonine, and 1000 mug of amphetamine. The assay for morphine involving 3H detects 60 mug/liter. Each assay is capable of providing a qualitative and quantitative estimate of the drugs sought. The 125I-labeled antigens have a usable shelf life of at least two to four months after the antigen is iodinated; the tritium assay is stable for six months. The assays can be performed with use of paper discs that have been suspended in urine and then dried, in place of the liquid specimen. The assays appear to be equally applicable to detection of drugs in urine, blood, saliva, and tissues. All of them are done at ambient temperature and can be used equally well for emergency (stat) tests or mass screening. Except for the benzoylecgonine assay, the clinical reliability of these tests has been demonstrated.

The isolation of carcinoembryonic antigen from tumor tissue at neutral pH.

Carcinoembryonic antigen (CEA) was purified from tumor tissue under mild conditions at neutral pH by a procedure that utilized affinity chromatography on concanvalin A. Further purification by gel filtration provided CEA in 10 to 20% yield and 10% purity. Antibody to this preparation was rendered specific for CEA by adsorption on a column of normal liver proteins bound to Sepharose. On reaction by immunodiffusion against a crude tumor extract, the adsorbed antibody produced two precipitin lines, of which one was relatively weak. These two precipitin lines fused completely with the two respective lines produced by antibody to perchloric acid-treated CEA. The major antigen found in crude tumor extracts and in CEA preparations purified at neutral pH was nearly undetectable in perchloric acid extracts of tumor homogenates. Further investigations showed that 60 to 70% of the CEA in crude tumor extracts and in preparations isolated at netural pH is destroyed and/or becomes insoluble acidic conditions.

Simultaneous detection of morphine and barbiturates in urine by radioimmunoassay.

This report describes a radioimmunoassay for the simultaneous detection of morphine and barbiturates. Morphine and barbiturate antibodies, obtained from goats, were mixed with 125l-labeled antigens. By adjusting concentrations of the morphine and barbiturate antibodies and radiolabeled antigens, closely superimposed standard curves for the two drugs would be obtained. As a consequence, similar response curves were obtained for urine specimens containing morphine or barbiturates. Although concentrations as low as 25 mug/liter could be measured, to ensure against false positive reactions the test should be performed at the 100 mug/liter concentration. Unknown samples positive by the dual assay were confirmed by separately testing the specimens with the individual radioimmunoassay specific for morphine or barbiturate. Equivalency tests of urines positive for morphine, positive for barbiturates, or negative for both demonstrated complete correlation between the single and dual assays. The mixed reagent retained its sensitivity and specificity for at least three months when stored at 4 or 25 degrees C. The dual radioimmunoassay is a rapid, simple procedure that can be adapted to automated processes and that is suitable for large- and small-scale screening.

Amniotic fluid components as determinants of fetal maturity.

In order to evaluate the most reliable means of determining fetal maturity, six amniotic fluid components were analyzed from 99 women who were 22 to 40 weeks pregnant. The six parameters investigated were: amylase activity the lecithin to sphingomyelin (L/S) ratio, percentage of fat cells, and the concentrations of protein, creatinine, and bilrubin. In general, single components and combinations of two or three of the tests did not significantly enhance the predictive accuracy or reduce the error from that obtained with the L/S ratio used alone.

Rh-D antibody titer in amniotic fluids. Its use as an index of the severity of erythroblastosis fetalis.

PIP: Report of a study of Rh antibody titers in the amniotic fluids from 42 Rh-negative sensitized pregnancies is presented. Amniocentesis was performed in the pregnant Rh-negative women having serum Rh antibody titers (indirect Coombs) ranging from 1/64 to 1/4,096. Spectrophotometric analysis was done on the antiRh-D titers. In order to predict the outcome of pregnancy based on the amniotic fluid Rh antibody content, all fetuses were divided into 3 categories. Those with titers of 1/4 or below should correspond to Category I; unaffected or mildly affected babies; those with titers of 1/8 to 1/16 should be in Category II or have moderate to severe disease; while fetuses with titers of 1/32 or above would be in Category III indicating very severe disease. Out of 18 cases with titers of 1/4 or below, 17 were unaffected or mildly affected. In 10 patients titers of 1/8 or 1/16 were observed as the highest titer. 8 infants in this group were moderately to severely affected. 14 cases were observed with titers of 1/32 or above and in 11 of them, the pregnancy outcome was a severely ill or dead infant. The other 3 cases in this group were babies with severe anemia who required 2 or 3 exchange transfusions. It was learned that the antibody titer of amniotic fluid of Rh-sensitized pregnancies can be used as a reliable index of the severity of hemolytic disease in the fetus.^ieng