Time-resolved fluorometric sandwich immunoassay for human growth hormone in serum and urine

A specific and sensitive time-resolved fluorometric sandwich immunoassay for human growth hormone (hGH) in serum and urine is described. A monoclonal anti-hGH IgG1 (5802)-coated polystyrene ball was incubated with serum or dialyzed urine and subsequently with europium ion-labeled monoclonal anti-hGH IgG1 (5801). Europium ion bound to the polystyrene ball was measured by time-resolved fluorometry. The detection limit of hGH was 0.3 pg/tube, which was 15-fold higher than that by sandwich enzyme immunoassay using horseradish peroxidase as label. The assay range of serum hGH was 15-15,000 ng/liter using 20 microliters of serum. The assay range of urinary hGH was 2-2,000 ng/liter using 150 microliters of dialyzed urine. The detection limits of hGH in serum and urine by this immunoassay were satisfactory for measuring hGH levels in serum and urine of healthy children and in serum of healthy adults and higher levels but not in urine of healthy adults and in serum and urine of patients with hGH deficiency.     

Comparison of a cytosol radioreceptor assay with a radioimmunoassay for 1,25-dihydroxyvitamin D in serum or plasma

Human plasma and serum levels for 1 alpha,25-dihydroxyvitamin D were determined by a cytosol radioreceptor assay (RRA) and a radioimmunoassay (RIA). For both assays, 1.5 ml of human serum or plasma is used. Prior to RRA or RIA, extraction with benzene is performed followed by 'high-performance' liquid chromatography (HPLC) on a silica column (25 X 0.46 cm) with hexane/isopropanol (9/1 by vol), to isolate 1 alpha,25-dihydroxyvitamin D from the other vitamin D metabolites. The cytosol receptor was isolated from the intestine of healthy chickens. The antisera were raised in rabbits to 1 alpha,25-dihydroxyvitamin D3-3-hemisuccinate coupled to bovine serum albumin. The standard curves for RRA and RIA are prepared with 1 alpha,25-dihydroxyvitamin D3. 1 alpha,25-dihydroxy[3H]vitamin D3 of high spec act (158 kCi/mol) is used as tracer. The reactants are incubated for 16 h at 4 degrees C. Then, bound and free ligand are separated after the addition of dextran-coated charcoal. Both assays have a sensitivity of 2 pg/tube. The cytosol receptor and the antibodies have about the same absolute affinity for 1 alpha,25-dihydroxyvitamin D3 but the cytosol receptor has a higher relative affinity for 1 alpha,25-dihydroxyvitamin D3 (compared with other vitamin D metabolites). Reproducibility and precision are better for the RIA. The between- and within-assay CVs are 16.0% (mean = 58.7 ng/l, n = 16) and 11.2% (mean = 52.1 ng/l, n = 15), respectively, for RRA and 12.6% (mean = 61.8 ng/l, n = 27) and 7.4% (mean = 61.8 ng/l, n = 15), respectively using RIA. Reference values obtained by both assays on healthy males and healthy premenopausal females are the same for both sexes; 53.9 +/- 31.0 ng/l (n = 46) using RRA and 51.8 +/- 30.2 ng/l (n = 91) for RIA (mean +/- 2 SD).     

A sensitive sandwich enzyme immunoassay for human myoglobin using Fab'-horseradish peroxidase conjugate: methods and results in normal subjects and patients with various diseases

A sensitive sandwich enzyme immunoassay (EIA) for human myoglobin (Mb) was developed. Serum Mb was assayed by incubation with an anti-Mb rabbit IgG-coated polystyrene ball and then with affinity-purified anti-Mb Fab'-horseradish peroxidase (HRP) conjugate. The HRP activity bound to the polystyrene ball was assayed fluorimetrically. The sensitivity was 3.1 pg/tube and serum Mb levels of 0.4-1000 micrograms/l could be determined. The recoveries of Mb added to human sera at 3 concentrations were 92.8-99.8%. The within- and between-assay coefficients of variations (CV values) were both below 10%. The regression equation of values determined by EIA and radioimmunoassay (RIA) was y(EIA) = 0.964x (RIA)--2.30 and the correlation coefficient was 0.984. The mean normal serum concentration of Mb was 21.5 +/- 6.0 micrograms/l (mean +/- SD) in males and 16.9 +/- 5.8 micrograms/l in females. The significance of serum Mb determination by the EIA in various diseases was confirmed. The present EIA for Mb is more sensitive and economical than RIA and should be useful for determining Mb in biological materials.     

Mitogenic effects of growth hormone in cultured human fibroblasts. Evidence for action via local insulin-like growth factor I production.

We examined human growth hormone's (hGH) effect on mitogenesis in cultured human fibroblasts, and the role of local insulin-like growth factor I (IGF-I). With 0.5% human hypopituitary serum (HPS), hGH increased thymidine incorporation (TI) over serum-free medium dose responsively, with half-maximal effect at 10 ng/ml (0.5 nM) (hGH 127 +/- 8.8%; IGF-I 107 +/- 1.7% [SEM]) (n = 10). Similarly, with 0.5% HPS, hGH and IGF-I increased cell replication by 172 +/- 8.2% and 169 +/- 25%, respectively (n = 4). Specific IGF-I monoclonal antibody (Sm1.2) dose dependently blunted TI stimulated by 10 ng/ml hGH or IGF-I (at 1:1000, 38 +/- 6.5% and 30 +/- 14% reduction, respectively). Sm1.2 also reduced cell replication by both 10 ng/ml hGH and IGF-I, respectively, to 32% and 42% of stimulated values. Dexamethasone (0.1 microM) synergistically enhanced TI by both IGF-I and hGH. A 28-h time course for TI showed that hGH stimulated a similar peak to IGF-I, lagging in its effect by 4-10 h. We have provided further evidence that hGH stimulates growth of cultured human fibroblasts via local IGF-I production, consistent with IGF-I's paracrine-autocrine role.     

Influence of matrix on concentrations of somatotropin measured in serum with commercial immunoradiometric assays

Using immunoradiometric assays (IRMAs) from Hybritech Inc. (H) and Nichols Institute Diagnostics (ND), we measured somatotropin (human growth hormone, hGH) in serum samples obtained every 20 min for 24 h from 10 prepubertal subjects with short stature. Results obtained with the ND reagents were 2.74 times greater than those obtained with the H reagents (P = 0.00001, r = 0.94, SEE = 3.9, n = 720). We therefore compared the IRMAs with the standard hGH RIA from the National Institutes of Health (NIH) National Hormone and Pituitary Program, using the genetically engineered hGH preparations (from Genentech Inc.) 22-kDa hGH and methionated 20-kDa hGH. We also assayed human pituitary hGH (NIH, lot no. AFP-4793B). Each hGH preparation was diluted in three diluent buffer systems: horse serum from H and from ND, and human serum. The RIA and H-IRMA gave superimposable standard curves for all hGH preparations in each diluent. The methionated 20-kDa hGH was not detected in the H-assay. Use of human serum matrix in the ND-IRMA shifted the standard curve as compared with the horse-serum matrix, giving equivalent binding at lower concentrations; i.e., serum hGH was overestimated in samples assayed against standards diluted in horse serum. Quality-control materials (Ciba-Corning) yielded disparate results in all three assays, yet human serum pools containing hGH gave similar results in the H and the NIH assays, and higher values in ND. When a human serum standard was used in the ND assay, both IRMAs gave similar results to the RIA assay for human serum samples. Reference intervals for hGH should be determined by each analytical laboratory, to prevent misdiagnosis of patients. Furthermore, quality-control material should be of human origin, because commercially supplied quality-control material does not react the same as human serum in some hGH assays.     

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.     

Development of a highly sensitive sandwich enzyme immunoassay for human ferritin using affinity-purified anti-ferritin labelled with β-d-galactosidase from Escherichia coli

A highly sensitive sandwich enzyme immunoassay of human ferritin was developed. Polystyrene balls were coated with rabbit anti-human ferritin IgG by physical adsorption, and rabbit anti-human ferritin Fab' was purified by affinity chromatography and labelled with β-d-galactosidase from Escherichia coli. Using the antiferritin-coated polystyrene balls and labelled anti-ferritin, the sensitivity obtained was 23 fg (0.05 amol) of ferritin per tube. The range of serum ferritin levels that could be determined using 0.1 μ1 of serum was 0.23–4500 ng/ml, and even 2.3 pg/ml was measurable by using 10 μl of serum. The coefficients of within-assay (n = 25) and between-assay (n = 10) variations were 5.9–8.8%. The regression equation and coefficient of correlation to a radioimmunoassay were Y(RIA) = 0.92X(EIA) + 3.0 and 0.99 (n = 78), respectively. The corresponding sandwich radioimmunoassay was less sensitive, partly because the specific radioactivity of ¹²⁵I-labelled anti-ferritin IgG used was not sufficiently high.     

Primary hyperparathyroidism with normal serum intact parathyroid hormone levels

To evaluate the features of primary hyperparathyroidism (HPT) with normal serum intact parathyroid hormone (iPTH) levels, we studied 271 consecutive patients undergoing surgery for primary HPT. In 20 patients, serum iPTH levels were within the normal range (10-65 ng/l). In their records, the most common clinical features were fatigue (n=13), polyuria (n=6), renal stone (n=5), and hypertension (n=5). Mean serum calcium and phosphorus were 2.78 and 0.85 mmol/l, respectively: 14 had serum phosphorus within the normal range. Mean serum iPTH was 48.5 ng/l, and was <45 ng/l in nine patients. Cervical ultrasound demonstrated a parathyroid adenoma in nine, and was normal in four. Tc sestamibi parathyroid scintigraphy always demonstrated an adenoma (9/9). In eight patients, normal iPTH values delayed diagnosis. Physicians should be aware of the possibility of HPT in patients with hypercalcaemia, even when serum phosphorus and iPTH levels are within the normal limits. Particularly, HPT cannot be excluded when serum iPTH levels are below the upper part of the normal range. In such cases, cervical imaging, which has the same sensitivity as in other HPT, should be undertaken. These explorations are useful, because many patients are symptomatic and can take advantage of surgery.     

Measurement of serum digitoxin in patients by radioimmunoassay using specific antiserum.

INTRODUCTION: Digitoxin is used to treat patients with heart failure. METHODS: A radioimmunoassay procedure for the specific determination for digitoxin in serum was developed using the antiserum (antiserum (A)) raised against digitoxin 3'-hemisuccinate-BSA conjugate. RESULTS: The intra- and interassay variability were <10% in the range of 5-100 ng/ml. The specificities of antiserum (A) and the commercial anti-digitoxin antiserum (antiserum (B)) were assessed by cross-reactivity studies with various related compounds. Antiserum (A) was highly specific for digitoxin. Mean digitoxin concentrations in serum samples (n=34) from digitalized patients by RIA using these antisera were 10.0 and 12.4 ng/ml, respectively. CONCLUSION: This RIA using antiserum (A) measure unmetabolized digitoxin and may be applicable for pharmacokinetic studies.     

Improved radioimmunoassay of atrial natriuretic peptide in plasma

We describe a radioimmunoassay (RIA) for measurement of atrial natriuretic peptide (ANP), based on one-step incubation and a simplified extraction procedure. The extraction was performed on a "Supelclean LC 18" column, with 2-mL plasma samples. Use of a diiodinated tracer improved the sensitivity of the RIA method. The minimal detectable value was 5 ng/L. Simplification of the extraction procedure and simultaneous incubation of the reagents provide a method more suitable for routine standard assay of ANP than those currently available. Intra- and interassay CVs were 6% (n = 12) and 11% (n = 10), respectively. The mean concentration of ANP in plasma of 32 healthy volunteers was 33 (SEM 4) ng/L. The ANP values for plasma after one-step incubation correlated well with those determined by a commercial RIA kit: r = 0.971, slope = 1.099, intercept = 1.949 ng/L (n = 25).     

A sensitive and specific sandwich enzyme immunoassay for human thyroid-stimulating hormone

A sensitive and specific sandwich enzyme immunoassay (EIA) for human thyroid-stimulating hormone (hTSH) has been developed. hTSH is incubated with anti-hTSH IgG-coated polystyrene balls, and after washing they are further incubated with anti-hTSH Fab'-β-d-galactosidase conjugate. The β-d-galactosidase activity bound to the polystyrene balls is proportional to the amount of hTSH to be assayed. Polystyrene balls are coated with rabbit anti-hTSH IgG which had been affinity-purified and treated with human chorionic gonadotropin-Sepharose 4B to remove antibodies cross-reacting with structurally related hormones. Rabbit anti-hTSH Fab', which had been affinity-purified was conjugated with β-d-galactosidase from Escherichia coli using N,N′-o-phenylenedimaleimide.In the specific sandwich enzyme immunoassay developed, 1 nU (1 × 10^?9 U) of hTSH per tube can be measured and the sensitivity for serum hTSH level is 0.1 μU/ml when 10 μl of serum is used. No significant interference was observed in the presence of 1.3 mU hLH/tube, 0.5 mU hFSH/tube or 0.5 U hCG/tube. Recoveries of hTSH added to human sera were 95.3–104% with a standard deviation of 12.0–14.9%. The coefficients of within-assay and between-assay variations were 6.0–7.5% and 4.9–8.7%, respectively. The regression equation and coefficient for correlation to radioimmunoassay (RIA) were y (RIA) = 0.95x (EIA) + 3.2 and 0.97, respectively.Serum levels of hTSH in normal male and female adults were 2.4 ± 1.0 (SD) (n = 41) and 2.9 ± 1.3 (n = 46) μU/ml, respectively; those in hyperthyroidism and hypothyroidism were 0.28 ± 0.06 (n = 20) and 49.6 ± 24.7 (n = 22) μU/ml, respectively; and those in pregnant and postmenopausal women were 2.5 ± 1.2 (n = 7) and 2.7 ± 1.0 (n = 35) μU/ml, respectively, indicating that high serum levels of hCG or hLH and hFSH under these conditions did not significantly interfere with the present assay of hTSH at normal levels.     

A simple radioimmunoassay for unconjugated estriol in pregnancy plasma.

A simple and specific radioimmunoassay (RIA) for the measurement of plasma or serum unconjugated estriol (E3) in pregnancy is described for general laboratory use. Two different antibodies utilized had low cross-reaction to estrone (E1) and estradiol-17 beta (E2). With either of these antibody reagents, a method was designed that requires only an extraction step using a specific solvent mixture of ethyl acetate and n-hexane (3:2, v/v), one-hour incubation with working antibody, and separation of bound from free E3 by ammonium sulfate. Standard curves useful in the range of 0 to 2,000 pg. were obtained. This assay, requiring only 0.05 ml. of plasma, can be completed within 4 hours. The sensitivity of this RIA was 10 pg. The intra- and interassay coefficients of variation (CV) were 5.0 and 9.4 per cent, respectively. When E3 determinations by this method are compared with RIA values by a more complicated technique using Celite column chromatography, the results are not significantly different. There was a good correlation between our RIA and a standard urine E3 determination (n = 137, r = 0.82, p less than 0.001). Using our simplified method, the mean plasma E3 during pregnancy were 1.0 +/- 0.6 ng. per milliliter at 10 weeks, 2.3 +/- 0.6 ng. per milliliter at 20 weeks, 6.5 +/- 1.4 ng. per milliliter at 30 weeks, and 16.5 +/- 5.1 ng. per milliliter at term. Since the serum RIA reflects changes in free E3 which has a shorter half-life than conjugated E3, this rapid and simple method is attractive as an approach to monitor fetal-placental function.     

The ontogenesis of human fetal hormones. III. Prolactin.

The synthesis and release of human prolactin (hPRL) in the human fetus was assessed by radioimmunoassay analysis of the content and concentration of hPRL in 82 pituitary glands and the concentration of serum hPRL in 47 fetuses of gestational age 68 days to term. Fetal hPRL exhibited parallelism with the reference standard (Lewis 203-1). hPRL was detected by 68 days of gestation (10 wk), the earliest fetal pituitary gland studied; 8 out of 33 pituitaries had a prolactin (PRL) content above 2.0 ng between 10-15 wk gestation. The mean ocntent of PRL in the pituitary gland increased sharply from 14.8 plus or minus 4.6 ng at 15-19 wk to 405 plus or minus 142 ng at 20-24 wk and 542 plus or minus ng at 25-29 wk gestation. By term, the mean content was 2,039 plus or minus 459 (range 493-3,689) and the mean concentration 15.9 plus or minus 2.4 ng/mg (range 7-20). There was a significant positive correlation (P less than 0.001) between the hPRL and human growth hormone (hGH) content of fetal pituitary glands; at term the hPRL/hGH ratio was 1/290. The concentration of serum hPRL between 12 and 24 wk ranged from 2.9 to 67 ng/ml, mean 19.5 plus or minus 2.5 ng/ml )n = 21); by 26 wk fetal serum hPRL increased sharply and attained levels of 300-500 ng/ml in late gestation. At delivery, the mean plasma concentration of hPRL was 167 plus or minus 14.2 ng/ml in 36 umbilical venous specimens and 111.8 plus or minus 12.3 ng/ml in the matched maternal venous specimens. No correlation between serum hPRL and the pituitary content or concentration of hPRL was demonstrable in 12 matched fetal specimens. In five anencephalic infants, umbilical venous hPRL levels were between 65 and 283 ng/ml. In two anencephalic infants, thyrotropin releasing factor (TRF) (200 mug IV) evoked a rise in serum hPRL in one patient from 43 to 156 ng/ml at 30 min, and in the other from 65 to 404 ng/ml at 120 min. In both patients, plasma thyroid-stimulating hormone (TSH) rose from undetectable base-line levels to peak levels of 97 and 380 muU/ml, respectively. The pattern of change in serum hPRL in the human fetus contrasts sharply with that of serum hGH, luteinizing hormone, or follicle-stimulating hormone. These observations in the fetus and in anencephalic infants suggest that the striking elevation of serum PRL in the fetus is neither mediated by a putative PRL releasing factor or by TRF, nor is a consequence of suppression or absence of PRL release inhibiting factor alone, as a functional hypothalamus is not required to attain the high PRL concentration at term. Several lines of evidence support the view that high plasma estrogen levels characteristic of gestation act directly on the fetal anterior hypophysis to stimulate PRL secretion or to sensitize the secretory mechanism of the lactotrope, increasing its responsiveness to other stimuli.     

Radioimmunoassay of growth hormone-releasing hormone (GHRH) with a polyclonal antibody against synthetic GHRH(1-29)-Gly4-Cys-NH2: method and clinical studies.

A radioimmunoassay (RIA) for growth hormone-releasing hormone (GHRH) using a polyclonal antibody against synthetic GHRH(1-29)-Gly4-Cys-NH2 has been developed. The antiserum (RBM105) showed full cross-reactivity with GHRH-(1-44)NH2, GHRH-(1-40)OH, GHRH-(1-37)OH and GHRH-(3-44)NH2, and probably recognized the region of Ala4 to Lys12 of GHRH. Since the sensitivity of the GHRH RIA was 1.5 pg/tube, the lowest detectable plasma level was 5 ng/l when an extract of 0.3 ml of plasma per tube was used. On gelfiltration chromatography, the GHRH immunoreactivity of normal plasma was eluted in the same position as synthetic GHRH. The plasma GHRH concentration in healthy subjects was 20.5 +/- 6.5 ng/l (mean +/- SD), and in patients with hypothalamic disorders was 17.4 +/- 2.0 ng/l. In contrast, the plasma GHRH level in hemodialysis-dependent, chronic renal failure (CRF-HD) patients (38.7 +/- 13.1 ng/l) was significantly higher than normal. The acromegalic patients were 24.3 +/- 11.9 ng/l, except for one patient with ectopic GHRH syndrome (990 ng/l): his plasma GHRH level reached 7,100 ng/l during operation, and then decreased logarithmically to 70 ng/l after 6 h. Somatostatin at concentrations of 10 and 1,000 nmol/l significantly suppressed (GHRH release) from primary culture cells of the GHRH-producing tumor from 17.3 +/- 0.92 ng/2 x 10(5) cells to 9.98 +/- 3.61 and 4.32 +/- 1.01 ng/2 x 10(5) cells, respectively after 48 h. These data indicate that this GHRH RIA is useful for determining the plasma GHRH concentration in normal and diseased states and also for in vitro studies of GHRH release.     

Radioimmunoassay of human sex hormone binding globulin: improved radioiodination procedure

Sex hormone binding globulin (SHBG), purified by affinity chromatography from retroplacental blood plasma, was reacted with 3-(p-hydroxyphenyl) propionic acid N-hydroxysuccinimidyl ester (PHPPS, Bolton-Hunter reagent). The derivative of SHBG obtained (parahydroxyphenylpropionyl-SHBG; PHPP-SHBG) was stable and could, in contrast to underived SHBG, be efficiently 125I-iodinated with a lactoperoxidase technique. The PHPP-SHBG labelled with 125I had good antiserum binding and stability properties and was used for radioimmunoassay (RIA) of SHBG in serum. The RIA requires a total incubation time of 3 h. It has been standardized with purified SHBG and has a sensitivity of 5 micrograms/l, giving a lowest detectable concentration in the routine procedure (samples diluted 1:40) of about 0.2 mg/l. Variation within and between assay was 4.1% and 7.2%, respectively, for samples with values within the normal range. Values obtained by this RIA procedure correlate well with those obtained by a dihydrotestosterone binding method and by an electroimmunoassay technique. The mean serum concentration of SHBG in healthy, regularly menstruating women (n = 42) was 3.7 +/- 1.0 (SD, standard deviation) mg/l and in healthy men (n = 100) 2.0 +/- 0.9 mg/l.     

Radioimmunoassay of glandular kallikrein in human plasma after partial purification by immunoaffinity column

Glandular kallikrein in human plasma was partially purified by immunoaffinity column (1.0 X 2.0 cm) and was measured by a radioimmunoassay (RIA). Plasma (5-10 ml) was diluted with an equal volume of 10 mmol/l sodium phosphate buffer, pH 7.4, containing 0.9% NaCl (PBS) and was applied to an immunoaffinity column from which glandular kallikrein was eluted with 3 mol/l NaSCN (20 ml). The enzymic fraction was concentrated with an Amicon PM 10 filter and dialyzed against PBS. The final recovery of the enzyme was 51.6 +/- 1.6% (mean +/- SD), determined by using [125I]kallikrein. The usable range of the standard curve covered 2.5-100 ng/tube. The coefficient of variation within the series was 5.9%, and the coefficient of variation between the series was 7.6%. In healthy controls (n = 25), the plasma content of glandular kallikrein was 1.36 +/- 0.39 ng/ml. In patients with acute pancreatitis (n = 6), the plasma concentration was 8.02 +/- 6.15 ng/ml, significantly different from the control group (p less than 0.01).     

A sensitive two-site enzyme immunoassay for human pancreatic secretory trypsin inhibitor (PSTI) using monoclonal antibodies

By using monoclonal antibody against human pancreatic secretory trypsin inhibitor (PSTI), we developed a highly sensitive, simple, and reliable two-site enzyme immunoassay system. The minimum amount of PSTI detected by this EIA is approximately 10 pg/ml when a 100 microliter aliquot of the sample is used. Good reproducibilities of within- and between-assay series and excellent recovery of exogenous PSTI from serum were observed. The correlation between the values obtained by the EIA and RIA methods was given by the linear regression equation, y = 1.09x + 4.6, for which the correlation coefficient (r) was 0.980 (n = 20). Antigenicity of the trypsin-PSTI complexes was found to be approximately 10% of that of PSTI. From these results, it seems that our recently developed EIA system for PSTI is useful in clinical testing for quantitation of PSTI in body fluids, for biochemical studies on synthesis and secretion of PSTI, and also for study of pathophysiological mechanisms involved in the development of acute pancreatitis and certain malignant neoplasms.     

Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers.

We developed a chemiluminescent enzyme immunoassay (CLEIA) to quantify such tumor markers as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA19-9, and CA125. We used a novel chemiluminescent substrate, a derivative of 1,2-dioxetane phosphate (AMPPD), to measure alkaline phosphatase as a labeling enzyme to Fab' fragments of antibody. Regardless of the solid phase, i.e., polystyrene beads (6 mm diameter) or ferrite-coated particles (0.3 microns diameter), the standard curves within the dynamic ranges of the conventional RIA or enzyme immunoassay (EIA) were linear in all cases except for those with AFP. Use of the ferrite particles further shortens the immunoreaction, so the assay can be performed in 30 min. In addition, the relationships between concentrations of the marker and chemiluminescent signals for CA19-9, CA125, and CEA were linear up to concentrations about 10-fold greater than the ordinary dynamic ranges. Intra- and interassay CVs (averages for individual analyte) were 2.2%-4.9% and 2.0%-5.8%, respectively. In an analysis of serum samples, results of the CLEIA correlated reasonably well with those of RIA or EIA. The lower limit of detection by CLEIA with ferrite particles was 390 arb. units/L for CA19-9, 990 arb. units/L for CA125, 0.06 micrograms/L for CEA, and 0.03 micrograms/L for AFP. Thus, the sensitivity increased to between two- and 10-fold that of RIA or colorimetric EIA, depending on the analytes.     

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.     

Thyrotropin enzyme-immunoassay in dried blood spots: a spectrophotometric method for neonatal thyroid screening

We describe an enzyme-immunoassay with photometric endpoint determination, suitable for the measurement of thyrotropin (TSH) in dried blood spotted on filter paper. Using reagents of a commercially available test kit provided for the measurement of thyrotropin in 200 microliter serum, we have adapted the method to the determination of thyrotropin in blood spots containing ca. 10 microliter blood. This was achieved by prolongation of the assay time from 3 to 20 hours, and by increasing the amount of enzyme-antibody complex. Precision and sensitivity of the blood spot assay are comparable to those of our in-house thyrotropin RIA, and the RIA/EIA correlation coefficient is 0.987 (n = 150). The advantages of EIA are the simplicity of the photometric end point determination (although strict time control has to be observed in order to avoid drifts in results), the long stability of reagents, and the non-isotopic label. The method therefore appears to be a suitable alternative to thyrotropin RIA for the determination of thyrotropin in neonatal thyroid screening.