Semi-quantitative estimation of serum myoglobin by a rapid latex agglutination method: an emergency screening test for acute myocardial infarction

The present study reports the evaluation of a new latex agglutination test for serum myoglobin (SMb). The time of agglutination of the latex particles coated with antibodies to myoglobin was measured in 172 serum specimens with known concentration of myoglobin quantitated by a radioimmunoassay (RIA), collected from myocardial infarction (MI) patients, subjects suffering from various diseases, and normal controls. Myoglobin levels in the samples were found to decrease exponentially with time of agglutination. Agglutination occurring within 1 min (result coded as + + + +) corresponded to 761 +/- 366 micrograms/l of myoglobin; between 1 and 2 min (+ + +), to 285 +/- 101 micrograms/l; between 2 and 3 min (+ +), to 85 +/- 47 micrograms/l; between 3 and 4 min (+), to 51 +/- 38 micrograms/l; and after more than 4 min (-), to 31 +/- 16 micrograms/l. Blood samples were serially drawn from 24 MI patients with short hospitalization delays; the rapid agglutination which was obtained in the specimens taken upon admission (20 results coded as + + + + and four as + + +) actually corresponded to markedly increased SMb levels. In contrast, serum creatine kinase (CK) activities were still less than 150 U/l in four patients (16.6%); CK-MB was less than 5 U/l in five cases (20.8%). Positive agglutinations for SMb were also obtained 4 and 8 h following admission in all subjects, confirming that the latex test is an early and very sensitive indicator for MI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and sensitive radioimmunoassays for human myoglobin

A radioimmunoassay for quantitation of serum myoglobin in healthy individuals and patients with different diseases is described. Purified myoglobin was labelled by an 125I-labelled ester (N-succinimidyl 3-(-4 hydroxy, 5-[125I]iodophenyl) propionate), a commercially available antiserum was used, and the antigen-antibody complex was precipitated with polyethylene glycol 6000. The rapid assay can be performed within 1 h at 37 degrees C with a detection limit of 45 micrograms/l. Prolonged incubation at 4 degrees C for 18 or 72 h gives a detection limit of 6 and 2 micrograms/l, respectively. The mean coefficient of variation of the routine assay was 11%. In healthy human subjects a significant difference in mean serum myoglobin concentration was found between 43 women (34 +/- 17 micrograms/l) and 51 mean 47 +/- 15 micrograms/l). In twenty patients admitted to hospital with the clinical diagnosis acute myocardial infarction, the serum myoglobin concentration profiles were in close agreement with the final diagnosis. In three patients with myocardial infarction serum samples were taken every 2 h after the acute episode, and serum myoglobin levels were compared with the levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase and creatine kinase isoenzyme-MB.     

Automated quantitative nephelometric latex immunoassay for determining ferritin in human serum.

We describe a rapid and sensitive latex nephelometric immunoassay for quantifying ferritin in human serum. This latex immunoassay procedure uses commercially available ready-for-use reagents [Tina-Quant (a) Ferritin, Boehringer Mannheim] that have a long shelf life. The assay consists of incubating the diluted serum sample (5-fold) for 12 min at room temperature with latex particles covalently coated with anti-ferritin antibodies, and then quantifying the change of light-scatter produced. The assay is fully automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. The method has an analytical range of 3 to 260 micrograms/l. Maximal intra- and inter-assay CVs were 4.0 and 6.2%, respectively. Analytical recoveries ranged from 91.3 to 103.6%. Assay detection limit was less than 3 micrograms/l. Linearity of the test is given throughout the measuring range. There was no interference from bilirubin (up to 340 mumol/l), haemoglobin (up to 7 g/l), or rheumatoid factor (up to 1,100 IU/ml). Turbid and lipemic samples interfere. This interference may be avoided by pretreating these samples prior to assay. Results correlated well with those obtained by an automated ELISA test (r = 0.995) and with those of two commercial RIA methods (r greater than 0.97). This latex nephelometric procedure is a convenient method and represents an interesting alternative to other immunoassays for measuring ferritin in human serum.     

Automated latex nephelometric immunoassay of theophylline in human serum.

A latex test was adapted for the nephelometric quantitation of theophylline in human serum. The assay is fully automated on the Behring Nephelometer Analyser with a sampling rate of 150 samples per hour. There is no interference from bilirubin (up to 340 mumol/l), haemoglobin (up to 7000 mg/l), Intralipid (up to 5 g/l), or rheumatoid factor (up to 1100 x 10(3) IU/l). The theophylline standard curve extends from 1.25 to 40 mg/l. The coefficient of variation ranged from 2.4 to 7.4%. The correlation coefficient between the latex immunoassay and the TDx theophylline procedure was 0.988, calculated from the assay of 74 samples.     

Study of a myoglobin test in patients hospitalized for suspected myocardial infarction

An immunoagglutination latex test was studied in comparison with a plasma myoglobin radioimmunoassay in 103 subjects with suspected myocardial infarction. The test provided an early and reliable indication of raised plasma myoglobin (greater than 85 micrograms/l), a biochemical marker for the early phase (12 h) of myocardial infarction. The diagnostic values (sensitivity and specificity) studied over a 36 h period were the same as those for the plasma myoglobin assay. The sensitivity was similar to that of creatine kinase activity and better than that of the creatine kinase MB/creatine kinase ratio; the lower specificity was due to false-positive results in some subjects with angina. The myoglobin test, which provides rapid results, may be substituted in early diagnosis of myocardial infarction for the plasma myoglobin assay which is unsuitable for emergency analysis.     

A rapid latex agglutination test for detection of elevated levels of myoglobin in serum and its value in the early diagnosis of acute myocardial infarction

A latex agglutination test for detection of elevated levels of myoglobin in serum has been studied, and its clinical value in diagnosis of acute myocardial infarction (AMI) has been evaluated on a retrospective material of fifty-five patients consecutively admitted to hospital on suspicion for AMI within 4 h after onset of symptoms. The analysis time was less than 10 min. There was no evidence of interference with other related proteins, as judged from analysis of sera with high content of aspartate and alanine aminotransferase, lactate dehydrogenase and creatine kinase, nor was the test sensitive to haemolysis. However, unspecific agglutination was seen with some sera containing a high concentration of rheumatoid factors. In sera with known concentrations of myoglobin, quantitated by a radioimmunoassay, the test was negative in all sera below 80 micrograms/l (n = 187), and positive in all sera above 140 micrograms/l (n = 209). In the range 80-140 micrograms/l the latex test could be either positive or negative (n = 105). The day-to-day reproducibility was high in the ranges below 80 micrograms/l and above 140 micrograms/l, but range 80-140 micrograms/l. In the diagnosis of AMI the predictive value of a positive result was 0.64, and the predictive value of a negative result was 1.0. The latex agglutination test is therefore clinically useful as an emergency test, and as a very early and sensitive indicator for AMI. Patients with a negative test result during the first 12 h after debut of symptoms can with great certainty be identified as not suffering from AMI, whereas patients with a positive test result should be monitored by further laboratory analyses.     

Evaluation of a quantitative photometric latex agglutination immunoassay for alpha-foetoprotein

A simple quantitative photometric method is described for the determination of serum alpha-foetoprotein using latex particle agglutination in an immunochemical system. This method is based on the quantitative photometric measurement of agglutination of latex particles coated with antibodies against alpha-foetoprotein. The turbidity is measured at a wavelength of 340 nm. Agglutination causes a decrease in absorbance. Interference by serum constituents, e.g. rheumatoid factors, are avoided by pretreating the serum samples with buffered polyethylene glycol. Concentrations of 0 to 640 micrograms/l were used for the standard curve. Analytical recoveries were 99.5 to 105.2%. Maximum within and between runs coefficients of variation were 6.2 and 11.6%. The correlation coefficient of the method with radioimmunoassay (RIA), calculated from results on 117 serum samples, was 0.997, and the regression equation y = 0.99x (RIA) - 7.23.     

An enzyme linked immunosorbent assay (ELISA) for measurement of human serum thyroglobulin. Evaluation of the influence of thyroglobulin auto-antibodies

The aim of the study was to develop an enzyme linked immunosorbent assay (ELISA) for measuring thyroglobulin (Tg) in human serum and to evaluate the influence of serum thyroglobulin auto-antibodies (TgAb) on the ELISA. The sensitivity of the ELISA was 2.1 micrograms/l. Serum Tg levels in healthy controls were from less than 2.1 to 55.5 micrograms/l (n = 46) (95% reference range). With serum Tg concentrations between 19.6 to 90 micrograms/l the within-assay coefficient of variation (CV) was from 4.5 to 6.6% (n = 12) and the between-assay CV from 8.5 to 10.5% (n = 6). The recovery from 20 to 89 micrograms Tg/l serum was from 93 to 101%. There was significant correlation between serum Tg concentrations measured by the ELISA and a RIA method in healthy controls (r = 0.85, n = 46, p less than 0.001) and in patients with differentiated thyroid carcinoma (r = 0.97, n = 28, p less than 0.001). The TgAb interfered with the serum Tg determination both in the ELISA and in the RIA method. The assay is simple and easy to perform, and the equipment is inexpensive and useful for large-scale serum Tg measurements as an alternative to RIA.     

One-step sandwich enzyme immunoassay for human laminin using monoclonal antibodies

A one-step sandwich enzyme immunoassay (one step sandwich EIA) for human serum immunoreactive laminin was set up with a pair of monoclonal antibodies prepared against human placental laminin P1 fragment. The assay was characterized by carrying out two immunoreactions simultaneously, laminin P1 fragment reacting with both a monoclonal antibody as a solid phase and a horseradish peroxidase-labeled monoclonal antibody (Fab') against human laminin P1 fragment as conjugate. Sensitivity of the immunoassay was 0.01 ng/well (0.5 microgram/l), and linearity was obtained between 0.01-20 ng/well (0.5-1,000 micrograms/l). The levels of laminin in sera from normal individuals and patients with liver cirrhosis, hepatocellular carcinoma and primary biliary cirrhosis were 103 +/- 15 micrograms/l, 228 +/- 70 micrograms/l, 341 +/- 163 micrograms/l and 232 +/- 93 micrograms/l, respectively. Protein immunoblotting showed that the serum immunoreactive laminin measured by the assay was a fragment with rel mol mass of 200 kDa.     

Quantitative nephelometric assay for determining myoglobin evaluated

A recently introduced automated nephelometric immunoassay involving shell/core particles for determination of myoglobin (Behringwerke) was evaluated with the BNA Nephelometer. Method precision was good: the intra-assay CV varied between 1.5% and 6.1%; with daily calibration, the interassay CV ranged between 1.5% and 7.5%. For usual sample dilutions, the assay response varied linearly with myoglobin concentrations up to 23.1 nmol/L. After automatic dilution by the instrument, concentrations up to 2310 nmol/L could be measured without high-dose "hook" effect. Further manual dilution allowed measurement of myoglobin concentrations up to 26,000 nmol/L. Calibration was stable for at least seven days. We detected no significant interferences from hemoglobin, haptoglobin, bilirubin, iodine-containing contrast media, and rheumatoid factors. Treating lipemic samples with Lipoclean (Behringwerke) decreased test results. Simultaneously drawn serum and plasma samples from the same subject showed no consistent differences in myoglobin concentrations. The mean reference myoglobin concentration was 1.380 (SD 0.82) nmol/L for men and 0.878 (SD 0.45) nmol/L for women. In patients with renal insufficiency, serum creatinine values were moderately related to serum myoglobin values (r = 0.465). Although a commercial radioimmunoassay (Byk-Sangtec) and the nephelometric assay intercorrelated well (r = 0.929), values obtained by nephelometry were significantly lower (P less than 0.05). By both assays, results for heart and skeletal muscle tissue extracts showed no correlation, a finding that suggests the existence of multiple forms of myoglobin in human tissues. We conclude that immunonephelometry is a rapid, practical, and reliable method for measuring myoglobin in serum.     

Comparison of high performance liquid chromatographic and microbiological methods for determination of itraconazole

A reversed-phase high-performance liquid chromatographic (HPLC) method, with internal standard quantification, is described for the analysis of itraconazole in human serum. No interference was encountered from over 60 drugs tested. The standard curve was linear from 10 to 10,000 micrograms/l. The detection limit of the method was 10 micrograms/l, with coefficients of variation from 2.2 to 7.8% over a range of itraconazole concentrations from 20 to 1600 micrograms/l. An agar diffusion method is also described with a lowest reproducible limit of 100 micrograms/l. This method had coefficients of variation from 11.0 to 17.1% over a range of itraconazole concentrations from 100 to 1600 micrograms/l. Comparison of the methods showed that HPLC gave much lower values of itraconazole concentrations in patient serum samples than did the microbiological method.     

Quantitation of serum lactate dehydrogenase-5 with monoclonal antibodies

Spleen cells from BALB/cJ mice which had been immunized with human lactate dehydrogenase-1 (LDH-1) were fused with SP2/0-Ag14. Two hybridomas were produced which recognized the antigen. Competitive RIA revealed that one antibody ('Smit-LDH') recognized the H subunit of LDH while the other ('Hem-LDH') recognized both H and M subunits of LDH. With the use of these antibodies we developed an assay for LDH-5 activity in which serum is incubated for 30 min at room temperature with the two antibodies ('Smit-LDH' and 'Hem-LDH' in the ratio 64:1.3, micrograms/ml) immobilized on latex beads to extract LDH-1 through LDH-4. After centrifugation, the LDH activity of the supernatant is measured and represents LDH-5 activity. Latex beads coated with bovine serum albumin were used as control. The LDH-5 activity as determined by our assay correlated well (r = 0.98) with the values obtained by an electrophoresis method. There was no interference due to LDH-1 through LDH-3 up to 3,000 U/l and LDH-4 up to 350 U/l. Serum samples with total LDH activity above 1,000 U/l were appropriately diluted in order to avoid interference by LDH-4. Use of these monoclonal antibodies allows precise, rapid and direct measurement of LDH-5 activity in serum.     

Radioimmunoassay of human platelet-derived growth factor using monoclonal antibody toward a synthetic 73-97 fragment of its B-chain

A mouse monoclonal antibody toward a 73-97 fragment of human platelet-derived growth factor (hPDGF) B-chain was used to develop a radioimmunoassay (RIA) for serum hPDGF. By the single step procedure of the double antibody technique, the measurable range was 10-1,000 micrograms/l. The coefficients of variation within and between series were 10.2% and 12.1% respectively, and satisfactory dilution curves were obtained for sera from healthy subjects. The hPDGF levels in all plasma samples from 15 healthy subjects examined were below the detection limit (10 micrograms/l), whereas the mean hPDGF concentration (+/- SD) in serum samples of 60 healthy subjects was 31.9 +/- 20.4 micrograms/l. This value was significantly (p less than 0.01) higher than the mean for 21 patients with idiopathic thrombocytopenic purpura (12.6 +/- 4.5 micrograms/l). There was a significant positive (r = 0.481, p less than 0.01) but not a strong (r2 = 0.23) correlation between the peripheral blood platelet counts and serum hPDGF levels of all subjects. This RIA system should be useful clinically for measurement of serum hPDGF.     

Determination of beta 2-microglobulin in human urine and serum by latex immunoassay

This highly sensitive method for determination of beta 2-microglobulin (beta 2-m) in human urine or serum is based on direct agglutination by beta 2-m of latex particles on which an antibody against beta 2-m is adsorbed. The agglutination is quantified by counting the remaining unagglutinated particles, or by turbidimetry. A novel aspect of this method is the capability to prevent nonspecific agglutination of the antibody-coated particles by diluting them with an albumin solution of well-defined characteristics (pH, freshness, concentration) just before the assay. The assayable concentration range is 1--32 micrograms/L, the detection limit 0.5 micrograms/L. Within-assay CV, based on 10 determinations of beta 2-m in urine and serum at two different dilutions, ranged from 4.6 to 8.7%. Between-assay CV, calculated from 10 determinations of beta 2-m in urine and serum, was 10 and 8.4%, respectively. Analytical recovery of beta 2-m in urine averaged 97% and in serum 104% (n = 10). No component of urine or serum interfered. Coefficients of correlation for beta 2-m in urine or serum as measured by radioimmunoassay and latex immunoassay were 0.97 and 0.93, respectively. Concentrations of beta 2-m in serum and urine from 33 healthy men (ages 20 to 67 years) averaged 1.5 mg/l and 54 micrograms/g of creatine, respectively.     

Evaluation of a sensitive immunoluminometric assay for the determination of C-reactive protein (CRP) in serum and plasma and the establishment of reference ranges for different groups of subjects

A sensitive immunoluminometric assay originally designed to measure C-reactive protein (CRP) in neonates and minimal serum volumes was adapted to measure this protein in a routine method without prior sample dilution. The concentration range covered without prior dilution was 10 micrograms/l to 20 mg/l using a sample volume of 5 microliters serum and a total assay time of less than 2 h. Serum samples were assayed from participants in a community medicine programme (SHIP--Study of Health in Pomerania) of the University of Greifswald, Germany (n = 414), as well as from mother-child pairs at birth (n = 30) and women attending the infertility clinic (n = 36). The validation of the assay was compared with a commercial latex-enhanced turbidimetric immunoassay (Roche Diagnostics--Integra 700) using routine serum samples (n = 60) from hospital patients. Comparison was made with the routine assay used in the SHIP study (Roche Diagnostics--Hitachi 717/Tina Quant). From 414 SHIP samples measured in the immunoluminometric assay, 289 were below the detection level in the turbidimetric (Tina Quant) assay. A significant positive correlation (p < 0.01) between log C-reactive protein concentration with age was found, both in the non-screened (all CRP values) (n = 414, r = 0.222) and selected (CRP < 5.00 mg/l = 90th percentile) (n = 370, r = 0.242) SHIP participants. Women were found to have significantly higher CRP levels than men (women: median age 47 a, median CRP 1.29 mg/l; men: median age 55 a, median CRP 1.00 mg/l--p = 0.016) in the non-selected SHIP participants. The situation was different in the selected group, (median age: men 54 a, women 48 a) where no significant difference in median CRP values between the sexes was seen (men: 0.874 mg/l, women 0.951 mg/l, p = 0.206). The distribution of CRP values in a "Normal Healthy Population" is skewed (mean/median--SHIP: all--2.08; selected--1.49). From the 414 SHIP samples measured in the immunoluminometric assay, 289 were below the detection level (2.5 mg/l) in the turbidimetric (Tina Quant) assay. From the 125 remaining samples the correlation between both methods was acceptable (r = 0.813), the regression line y = a + bx being: CRP (ILMA) = 1.83 + 0.842*CRP (Tina Quant). The Tina Quant assay gave values significantly higher than the ILMA in the range 2.5-25 mg/l CRP (p < 0.001). The total information loss in 289/414 subjects with a CRP < 2.5 mg/l with the Tina Quant assay makes it no longer suitable for epidemiological studies in which CRP is to be studied as a risk factor for cardiovascular events. The comparison between the immunoluminometric assay and the latex-enhanced immunoturbidimetric assay (Roche Integra) was much better. The latter measured down to less than 0.3 mg/l, thus being more suitable for epidemiological studies than the Tina Quant assay from the same producer. The correlation and regression data between the ILMA (x) and the Roche Integra assay (y) were: r = 0.971; CRP (Roche Integra) = 0.635 + 0.984*CRP (ILMA); n = 50.10 sera with CRP levels between 25 and 460 mg/l showed no high-dose hook effect in either assay. The remaining 50 sera were measurable in both assays. The turbidimetric assay gave rise to marginally but significantly higher values than the immunoluminometric assay (p = 0.004). The mothers at birth had a median CRP of 3.64 mg/l (range 1.49-12.6 mg/l), the neonates a median CRP of 34 micrograms/l (range 4-288 micrograms/l). All births were without complications, with gestational periods between 38 and 42 weeks. There was no correlation between maternal and neonatal CRP at birth. Mothers at birth had significantly higher CRP levels than healthy non-pregnant women (p < 0.001). Women attending the infertility clinic had CRP-values similar to age-matched healthy non-pregnant women (median 0.698 mg/l, range 0.05-9.97 mg/l). Interassay coefficients of variation at CRP concentrations of 0.85 and 7.9 mg/l were 8.99 and 7.93%, respectively, for the immunoluminometric     

Measurement of serum myoglobin by a turbidimetric latex agglutination method.

We evaluated an immunoturbidimetric quantitation for serum myoglobin by the latex agglutination method using an automated biochemical analyzer. This method is rapid, specific, accurate, precise, and has wide dynamic range. The total assay time is 10 min and is performed at 37 degrees C with continuous monitoring at 570 nm. The assay results were compared with radioisotopic immunoassay results and showed a good correlation coefficient, r = 0.99; Y = 0.98 x + 9.3; N = 79. Sera from healthy adults has a myoglobin concentration in the range of 15-80 ng/ml(N = 362). Sex- and age-related differences were observed. The serum myoglobin levels in males and elderly people showed higher concentration than in females and younger people. The peak elevation of serum myoglobin compared with other cardiac markers was observed within 6 hours after onset of chest pain as well as the CK-isoform ratio (MM3/MM1). All of the serum from 21 patients with definite acute myocardial infarction showed increased serum myoglobin levels (100-1200 ng/ml) upon admission and within 6 hours. The results suggest that assays for serum myoglobin levels are helpful in the early diagnosis of acute myocardial necrosis.     

Application of an enzyme-linked immunoassay for the measurement of pregnancy zone protein (PZP) in cell culture supernatants and sera

A simple and sensitive enzyme-linked immunosorbent assay (ELISA) measuring specifically the pregnancy zone protein (PZP) was constructed. The assay range was 2.0-500 micrograms/l. The intra-assay coefficient of variation (CV%) was 5.9% at the level of 100 micrograms/l and 3.5% at 10 micrograms/l. The imprecision between runs was 4.5% at 100 micrograms/l and 7.6% at 10 micrograms/l. Recovery of the native PZP standard added to serum-free cell culture medium was 98.1 +/- 3.7% (mean +/- SD), and recovery from serum of women in late pregnancy was 96.0 +/- 9.3%. Recovery from PZP-chymotrypsin (PZP-CT) complexes added to serum-free medium was 141 +/- 4.3%. There was no detectable cross-reactivity between the anti-human PZP antibody and human alpha 2-macroglobulin (alpha 2-M). The dose-response of two PZP standards and the PZP serum concentrations of 100 blood donors were determined. Furthermore, the serum level of PZP from 11 patients suffering from IgA myeloma was quantitated and found within the normal range when compared to serum levels of healthy blood donors of the same age and sex. Finally, supernatants from serum-free cultures of different human peripheral blood mononuclear cell (PBM) subpopulations were assayed. Neither of them were found to exhibit any detectable increase in PZP concentration during culture, but cultures of monocytes were found to produce alpha 2-M.     

Time-resolved immunofluorometric assay for placental protein 14

A sandwich-type solid phase time-resolved immunofluorometric assay (IFMA) was developed for endometrial protein PP14 (placental protein 14). The assay utilizes affinity-purified polyclonal antibodies for coating the microtiter wells and for labelling with europium (III) chelate. Maintaining specificity, the 0.6 micrograms/l sensitivity of IFMA is over 25 times higher than that of RIA. The immunofluorometric method enables detection and accurate quantitation of PP14 in all those samples in which PP14 is undetectable by RIA. The method is suitable for quantitative measurement of low PP14 levels in serum of postmenopausal and fertile-aged women and men, as well as in follicular fluid. At 14-16 micrograms/l, which is the sensitivity of radioimmunoassay, the intra-assay variation of IFMA is 6.6% and inter-assay variation 11.4%. In postmenopausal women the PP14 levels are 12.7-56.7 micrograms/l, in fertile-aged women 13.7-113.4 micrograms/l, and in men 3.1-53.1 micrograms/l. The levels in preovulatory follicular fluid are 1.2-20.5 micrograms/l. It is concluded that PP14 IFMA is highly sensitive, accurate and suitable for measurement of protein levels undetectable by other currently available methods.     

Radioimmunoassays of human myoglobin in serum and urine.

Two solid-phase radioimmunoassays have been developed for the detection of myoglobin in serum and urine. The sensitivity of the methods is 0.1 and 0.5 microgram/l, respectively, with a coefficient of variation of the respective method of 7-8%. The mean serum concentration of myoglobin in ninety-nine healthy blood donors was 44.3 microgram/l +/- 18.0 microgram/l (SD) with a significant difference (P less than 0.001) between men (50.6 +/- 19.8) and women (35.7 +/- 10.4). Serum myoglobin was positively correlated to age (P less than 0.05), body weight (P less than 0.02), serum creatine kinase (P less than 0.001), and serum creatinine (P less than 0.001) to galactose elimination rate. Serum myoglobin levels were not influenced by exhaustive short time dynamic exercise. The mean urinary excretion of myoglobin in twenty-four healthy students was 1.2 microgram/24 h (range 0.1-4 microgram/24 h). Myoglobin excretion was correlated to excretion of beta 2-microglobulin (P less than 0.02) but not to serum levels of myoglobin. No indications of circulating antibodies to myoglobin were obtained when assaying sixty-seven rheumatoid arthritis and thirteen myastenia gravis sera. Presence of other myoglobin binding substances in serum, which would interfere with the assays also seemed unlikely. Determination of myoglobin in serum by sensitive and specific method might be of clinical value in the diagnosis of diseases involving muscle tissues.     

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).