Intra-assay CV Research Articles

GROWTH HORMONE RESPONSES TO GROWTH HORMONE RELEASING PEPTIDE (GRP) AND TO GROWTH HORMONE RELEASING HORMONE (GRF) IN GROWTH HORMONE DEFICIENT CHILDREN (GHD)

GH-releasing peptide is a potent and specific stimulator of GH secretion. It is a 6 aminoacid peptide with lower m.w. and loger half life than GRF. To clarify how this peptide acts, we administered separately 1 ug/kg bolus doses of GRP and GRF iv to 29 children (15 M, 14 F), ages 3-16 years, with previously documented GHD. Tests were separated by at least 1 week. Bone ages ranged from 1-10 years and growth velocities were less than 3 cm/year. GH responses to 2 indirect GH stimulation tests were less than 7 ng/ml. GH was meassured by RIA with an intraassay cv of 5%. A positive response was defined as a GH increase greater than 4 cv's. We observed 14 (48%) positive responses to GRP, and 18 positive responses to GRF (62%), 9 (31%) patients responded to both peptides. Out of the 14 GRP responders only 9 patients (64%) had a positive response to GRF, and out of the 18 GRF responders only 9 (50%) had a positive response to GRO. GH peak levels were observed between 5 and 60 min (mean 30 min)after GRF, and between 10-45 min (mean 24 min) after GRP. We conclude that GRP is a potent secretagogue of growth hormone in a substantial proportion of GH deficient children. The dissociated response to GRP and GRF suggests that they stimulate GH release through different mechanisms.

Automated spectrophotometric method for determining oxidized and reduced glutathione in liver

An enzymatic recycling method has been applied to the measurement of total and oxidized glutathione with a centrifugal analyzer. When the reduced form of glutathione (GSH) was masked with 2-vinylpyridine to measure the oxidized glutathione (GSSG), the time to ensure full derivatization was three times longer than has been reported. The method is quick, simple, accurate, and precise (1.27% for GSH, 3.3% for GSSG intraassay CV; 2.15% for GSH, 5% for GSSG interassay CV), and the automation allows large numbers of samples to be conveniently assayed.

Rapid determination of erythrocyte pyruvate kinase activity

We report a new potentiometric method for determining pyruvate kinase (PK). Enzymatic activity is measured by monitoring the change in pH produced in the reaction buffer under International Committee for Standardization in Haematology (ICSH) standardized assay conditions, and the lactate dehydrogenase reaction is automatically subtracted in each measuring cycle. The analysis, performed at 37 degrees C, requires a 10-microL sample (isolated erythrocytes or whole blood) and is completed in 2.5 min. The intra-assay CV is < 4% (PK between 3 and 35 U/g Hb); the interassay CV is 4.0% (PK 15 U/g Hb); results are linear from 3 to 30 U/g Hb. A good correlation with the ICSH reference method (x) was found: y = 1.011x - 0.05; n = 32; r = 0.9939; Sylx = 0.75 (units: U/g Hb). The reference intervals of the PK activity in isolated erythrocytes (RBC-PK) were estimated in 89 normal subjects. We found that women possess a higher RBC-PK than do men (P < 0.0001) and that the biological variability (CVb) of RBC-PK is 13.5%. Applications of the proposed method to the hematological routine are reported.

Plasma and urinary oxalate and glycolate in healthy subjects

High-performance ion chromatography (HPIC) has been widely used for oxalate analysis and, more recently, for glycolate analysis. We describe a procedure for sample preparation in which the plasma ultrafiltrate is acidified during harvesting with a cation-exchange resin, and the chloride is removed before the ion chromatography, which is performed with a newly developed AS10 column. The same ultrafiltrate sample is analyzed for glycolate. For plasma oxalate, the mean recovery of sample in eluted fractions was 95-96%, and intraassay CV was 6.2-8.1%. The reference interval (mean +/- 2 SD) for men was 0.8-3.2 mumol/L and for women, 1.0-2.6 mumol/L. For urinary oxalate, the reference interval for men was 175-560 mumol/day and for women, 107-432 mumol/day. For plasma glycolate, the mean analytical recovery was 96-98%, and the intra-assay CV was 2.4-6.2%. The reference interval for men was 1.9-7.5 mumol/L and for women, 1.4-7.4 mumol/L. For urinary glycolate, the reference interval for men was 0-1400 mumol/day and for women, 91-1001 mumol/day.

Application of an Improved HPLC Perhexiline Assay to Human Plasma Specimens

Abstract The analysis of perhexiline in plasma offers an important contribution to the management of patients prescribed such therapy for refractory angina pectoris. Perhexiline has properties and complexities of non-linear kinetics and is subject to genetically predetermined metabolic variants in hydroxylation. The present communication describes a refinement of a previous high performance liquid chromatographic fluorescence method and the application of this method in the therapeutic monitoring of 100 patients at steady-state. The method described is sensitive, accurate and precise, with intra-assay CV's of 2. 1%, 1.4% and 3.7% at concentrations of 150, 750 and 1500 μg/L, and between-assav CV's of 5.7%, 4.7% and 5.8% at 50, 1000 and 3000 μg/L, respectively. The review of patient specimens received in our therapeutic drug monitoring laboratory, suggested that approximately 6% appear to belong to the “poor-hydroxvlation” metabolic sub-population, with a further 16% attaining steady-state plasma perhexiline concentrations above the “therapeutic range” of 150 to 600 μg/L following standard dosage schedules of 100 to 200 mg/day.

Development of Time-Resolved Immunofluorometric Assay of Vascular Permeability Factor

We describe a two-site time-resolved immunofluorometric assay for guinea pig vascular permeability factor (VPF) for quantifying VPF in different biological fluids. Antibody against the carboxy terminus (C-IgG) is immobilized on microtiter wells, and antibody against the amino terminus (N-IgG) is labeled with Eu(3+)-chelate. Line 10 tumor culture medium, known to be rich in VPF, is assayed in a two-step incubation. Bound Eu3+ is then quantified by dissociation into a fluorescent enhancement solution, with measurement of the time-resolved fluorescence. The analytical sensitivity is 0.35 VPF unit, and the intra-assay CV is about 20%. The assay is specific for VPF, because pre-treatment with the appropriate C- or N-peptide, or pre-extraction of VPF, greatly decreases fluorescence. The VPF immunoassay is highly correlated (r2 = 0.94) with the Miles permeability assay, the classical bioassay of VPF. In addition, the immunofluorometric assay is about 30-fold more sensitive than the Miles assay.

Determination of prostaglandin metabolites in biological samples by competitive time-resolved fluoroimmunoassay

We describe a time-resolved fluoroimmunoassay method for the determination of 13,14-dihydro-15-ketoprostaglandin E 2 (PGEM) and 13,14-dihydro-15-ketoprostaglandin F 2α (PGFM) in femtomolar ranges. Polyclonal antibodies were raised in rabbits and the antigen was labelled with europium by the avidin-biotin technique. A second antibody, directed to rabbit IgG, was coated onto the solid phase. The IgG-PG-europium complex was bound by the second antibody, giving a rapid and complete separation of antibody-bound and free antigen. The detection limit was 0.2 pg/assay for PGFM and 1.2 pg/assay for PGEM. The intra-assay CV ranged from 4.6% to 9.3% and the interassay CV from 6.7% to 11.4%. A good correlation was obtained between the results from TR-FIA and RIA when the method was applied to the investigation of tissues from breast cancer. We conclude that the TR-FIA is more sensitive and much faster than the RIA and avoids the use of radioactive material.

Ultrasensitive, Specific, Two-Antibody Immunoradiometric Assay That Detects Free Alpha Subunits of Glycoprotein Hormones in Blood of Nonpregnant Humans

This noncompetitive, sensitive, immunoradiometric assay of the free alpha subunit of human pituitary glycoprotein hormones is based on two monoclonal antibodies and an avidin-biotin separation system. The affinity of the first antibody, mouse anti-alpha subunit covalently conjugated to biotin, is 3.8 x 10(11) L/mol. The second antibody, radiolabeled with 125I, has an affinity of 5.4 x 10(11) L/mol. A polystyrene ball coated with avidin serves as the separation system. Tests of "purified" immunochemical-grade intact human glycoprotein hormones yielded cross-reactions of approximately 2% in the assay. Sephadex G-100 column chromatography showed that this "cross-reaction" was caused by contamination of the various hormone preparations with free alpha subunit. When the intact glycoprotein hormones were further purified with specific anti-alpha monoclonal antibody, their reaction in the alpha subunit assay was undetectable (less than 0.01%). Interassay CV averaged 3.5%, and intra-assay CV averaged 7.5% at low concentrations of subunit. The detection limit of the assay (0.01 micrograms/L) is adequate to detect free alpha subunit in the blood of normal humans. Mean (SD) concentrations of free alpha subunit in normal humans were as follows: eugonadal men = 437 (35) ng/L; postmenopausal women = 1231 (40) ng/L; eugonadal women, follicular phase = 1061 (40) ng/L; eugonadal luteal phase = 780 (45) ng/L.

Pentobarbital quantification in the presence of phenobarbital by fluorescence polarization immunoassay

We developed a method for measuring pentobarbital in samples that also contain phenobarbital. The phenobarbital is destroyed by adding sodium hydroxide and then heating at 95 degrees C for 60 min. Pentobarbital is not affected by this pretreatment and can then be measured with an Abbott TDx barbiturates kit. Using this method, we obtained an average analytical recovery of 98% of added pentobarbital and a correlation of y = 0.953x + 3.4 vs HPLC. The intra-assay CV was 3.7% at 25.8 mg/L; the interassay CV was 6.2% at 16.1 mg/L. Other long-acting barbiturates, e.g., hexobarbital, are also effectively destroyed by this alkali pretreatment. Other short-acting barbiturates, e.g., secobarbital, are not removed and would produce an interference.

Quantitative Turbidimetric Assay for Determining Myoglobin Evaluated

A recently introduced turbidimetric immunoassay using shell/core particles for determination of myoglobin (Turbiquant Myoglobin, Behringwerke, Marburg, Germany) was evaluated on the Behring Turbitimer analyzer. Intra-assay CV varied between 1.0% and 3.2%; interassay CV was between 2.0% and 3.6%. Linear results were obtained between 2.8 nmol/L and 36.7 nmol/L. Manual dilution in saline solution allowed measurement of myoglobin concentrations up to 25,400 nmol/L. In the tested range, no high-dose 'hook' effect was observed. Haemolysis interfered with the assay when haemoglobin concentrations exceeded 2 g/L. The occurrence of error codes due to the presence of triglycerides was shown to depend on particle size. Repeat assays on diluted samples were necessary at triglyceride concentrations of 2.3 mmol/L for post-prandial chylomicrons (200-1000 nm), and at 11-31 mmol/L for very-low density lipoprotein-triglycerides (80-200 nm). No significant interferences of haptoglobin, bilirubin, iodine containing contrast media, and rheumatoid factors were detected for the assay. Treatment of lipaemic samples with Lipoclean (Behringwerke) resulted in lower myoglobin values. Simultaneously drawn serum and heparin-plasma samples gave comparable myoglobin results. However, values obtained in EDTA- or citrate-treated plasma samples occasionally differed significantly from serum values. The upper reference limit for myoglobin concentration was 4.2 nmol/L. The turbidimetric assay correlated well with a radioimmunoassay (Byk-Sangtec; r = 0.892) and with the automated nephelometric assay (Behringwerke; r = 0.944). Values obtained by turbidimetry were comparable to those obtained by the latex agglutination test (Behringwerke).

Radioimmunoassay of epidermal growth factor in human saliva and gastric juice

A sensitive radioimmunoassay was developed for human epidermal growth factor (hEGF) in saliva and gastric juice. This method was sufficiently sensitive for an accurate measurement of hEGF in these biological fluids. The minimal detectable concentration of EGF was 30 ng/L. The imprecision profile of EGF standard curve had a CV less than 10% in the range of 0.1-3.0 micrograms/L. Serial dilution curves of saliva and gastric juice paralleled that of standard EGF. The antibody to hEGF showed no cross-reactivity with a large excess of growth factors, such as human transforming growth factor alpha, human insulin-like growth factor I, and platelet-derived growth factor (c-sis). No detectable cross-reactivity was observed with some biological gut peptides: somatostatin, gastrin, secretin or pancreatic polypeptide. The intra-assay CV for saliva and gastric juice was less than 10%, and the recoveries were 93.9 +/- 8.7% and 93.7 +/- 11.3%, respectively for saliva and gastric juice. Gel exclusion chromatography revealed hEGF-like substances, heterogeneous in size in saliva and gastric juice, the origins and physiological functions of which are unknown.

A Two-Site Immunoradiometric Assay for Pth(1–84) Using N and C Terminal Specific Monoclonal Antibodies

We report the development of a two-site immunoradiometric assay for PTH(1-84) based on two site-specific monoclonal antibodies--3B3 (radiolabelled antibody) specific for PTH(1-34) and ESQ1 (on solid phase) specific for PTH(74-84). Antibody 3B3 is sensitive to the oxidation of the methionine residues in PTH(1-34) therefore hydrogen peroxide (0.1 M) is added to the incubation mixture. Validation studies confirm quantitative recovery of both oxidized and reduced PTH(1-84). The assay has a minimum detection limit of 0.5 pmol/L and a range of 1.5-250 pmol/L with an intra-assay CV of less than 10% (2.8-250 pmol/L less than 5% CV). Studies on clinical samples indicate good discrimination between normal subjects (mean 2.21; range 1.0-5.0 pmol/L) and patients with primary hyperparathyroidism (mean 21.0; range 5.8-100 pmol/L) who in turn are well separated from patients with hypercalcaemia of malignancy (14/18 less than 0.5 pmol/L).

New enzyme-linked immunosorbent assay for glycocalicin in plasma

A new sandwich-type enzyme-linked immunosorbent assay for quantifying glycocalicin, a proteolytic fragment of platelet membrane glycoprotein Ib, is described. The assay is based on the use of two monoclonal antibodies raised against glycoprotein Ib and involves the avidin-biotin technique. The detection limit is 7 micrograms/L and the range of glycocalicin determined in plasma is 0.01 to 1 mg/L. Assay time is 2 h. The intra-assay CV ranged from 3.6% to 5.2%, the interassay CV from 5.4% to 8.0%. Analytical recovery of purified glycocalicin added to a plasma pool averaged 96%. In 36 healthy subjects, the mean glycocalicin concentration in plasma was 0.36 (SD 0.07) mg/L (2.7 nmol/L). We conclude that this assay is suitable for measuring glycocalicin in plasma and is also more sensitive and precise than the previously published immunoassays based on competitive binding assay.

Within‐clinic Glycosylated Haemoglobin Measurement

The performance of the Diamat HPLC analyser (Bio Rad Instruments) was assessed, and the effect of this on-site HbA1 assay on the therapeutic decisions made at the diabetic clinic evaluated. The intra-assay CV for HbA1 at concentrations of 8.3 and 13.4% was 3.8 and 0.4%, respectively, with inter-assay CV of 5.0 and 3.0%. On a single day 82 HbA1 tests on consecutive patients were performed at the clinic. In 43 insulin-treated patients and 79 non-insulin-treated diabetic patients the HbA1 result changed the management decision in 25 and 18% of patients, respectively. The relationship between HbA1 and self blood glucose monitoring (SBGM) results in the previous 6-week period were also evaluated. In 41% of patients with insulin-treated diabetes who produced SBGM diaries there was a discrepancy between categories of blood glucose control, all of these patients having better SBGM than HbA1 values. This study highlights the feasibility and value of a within-clinic HbA1 assay for clinical decision-making and its usefulness in identifying problems of agreement with self-monitored tests.

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.

Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in plasma

We present a new functional assay for the first complement component (C1) in plasma, based on its activation by inhibition of the C1-esterase inhibitor (C1-inh) when monospecific antiserum to C1-inh is added to the plasma. After maximal activation, we can determine the concentration of activated C1 by using an amidolytic rate assay with a chromogenic substrate. We have optimized the assay conditions with respect to incubation time, concentration of antiserum to C1-inh, ionic strength, and pH. Our method determines specifically the concentration in plasma of free activated C1, not complexes of activated C1 with C1-inh, and is not influenced by the concentration of C1-inh in the test sample. Concentrations of C1 correlated significantly with activities determined by a hemolytic assay (r = 0.55, t = 4.09, P less than 0.001). The estimated interassay CV was 5% and the intra-assay CV was 1%. The sensitivity, imprecision, and practical test performance of our assay are superior to those of conventionally used hemolytic assays.

Effects of methylamine and heparin on a rapid chromogenic assay of C1-esterase inhibitor in plasma

We describe a rapid assay of C1-esterase inhibitor (C1-inh) activity in plasma. After adding purified C1s serine protease (EC 3.4.21.42) in excess to plasma, we determine the residual C1s activity towards a new chromogenic tripeptide, CH3CO-Lys(CbO)-Gly-Arg-pNA. Optimal conditions include the addition of methylamine (final concentration 0.12 mol/L) to reduce the potential inhibitory capacity of alpha 2-macroglobulin towards C1s and the addition of heparin (final concentration 3000 int. units/L) to enhance the reaction of C1s with C1-inh. The correlation with C1-inh antigen concentrations in plasma was excellent. The estimated interassay CV was 4.3%, whereas the intra-assay CV was 2.0% for activity concentrations within the range of normal individuals (means +/- 2 SD: 70-124%), 1.3% at lower concentrations. The method is more convenient, rapid, and precise than previous methods, and C1-inh activity in plasma can be assessed within 30 min. We found that concentrations of C1-inh in plasma were low during open-heart bypass surgery.

A sensitive and rapid luminescence sandwich assay for antibodies to hepatitis‐B surface antigen (Anti‐HBsAg)

A chemiluminescent assay for hepatitis-B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra-assay CV, 1.96-2.45%; inter-assay CV, 5.26-8.11% and has a lower detection limit for hepatitis-B surface antigen of 1.3 U/l.

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

Quantification of Apolipoprotein A-1 in Cow Serum by Single Radial Immunodiffusion

A single radial immunodiffusion assay was developed to determine the concentration of the major apolipoprotein A-I in bovine serum. Assay was performed using agarose gel plates with monospecific rabbit antiserum. Standards made with bovine high density lipoproteins or serum 10 times diluted were applied in 3-μl amounts to each well in the gel. The assay working range was 2 to 22mg of apolipoprotein A-I. The intraassay and interassay CV were 3.7 and 4.9%, respectively. Using protein A-I. The intraassay and interassay CY were 3.7 and 4.9%, respectively. Using radial immunodiffusion assay, 23 serum samples from cows at various stages of lactation were analyzed. Apolipoprotein A-I was significantly increased in mid lactation compared with concentrations in early lactation or dry period.