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.     

An enzyme-linked immunoassay for ferritin in human serum and rat plasma and the influence of the iron in serum ferritin on serum iron measurement, during acute hepatitis

To measure human serum ferritin and rat plasma ferritin a non-competitive enzyme-linked immunoassay has been developed using horseradish peroxidase as the enzyme. In this assay it proved necessary to use heated rat plasma to obtain reproducible ferritin values. The heating procedure caused a loss of 38% of the plasma ferritin. Rat plasma ferritin values have been corrected for this loss. The standard deviation, from duplicate normal human and rat samples is 10 ng ferritin/ml serum and 69 ng/ml plasma, respectively. (The mean ferritin concentrations are: in human sera, 82 ng/ml and in rat plasma 762 ng/ml.) Mean recovery of added liver ferritin in the human serum is 104% +/- 4% (+/-S.E.M') and in the rat plasma 101% +/- 3% (+/- S.E.M.). Normal ferritin concentrations varied in the human material between 30 ng/ml and 300 ng/ml serum, and in the rat plasma between 500 ng/ml and 1300 ng/ml. During increased body iron and acute hepatitis the ferritin concentrations, in patients as well as in rats, exceeded the upper limit of the normal values in most cases. During human hepatitis high serum ferritin levels combined with high serum iron levels were measured. The high serum iron concentrations could not be explained by the high serum ferritin concentrations, even if the iron content of the ferritin is supposed to be high.     

Radioimmunoassay of serum IGF-I and IGF-II in patients with chronic liver diseases and hepatocellular carcinoma with or without hypoglycemia

Insulin-like growth factor (IGF) I and IGF-II were measured by radioimmunoassay in the sera of seven patients with acromegaly, 36 normal control subjects, 15 patients with chronic hepatitis, 15 patients with cirrhosis, 25 patients with hepatocellular carcinomas (HCCs) who did not have hypoglycemia, 20 patients with HCCs who did have hypoglycemia, and 10 patients with metastatic liver tumors. Both IGF-I and IGF-II levels decreased as liver disease progressed from the normal control stage to chronic hepatitis and cirrhosis, and both levels reflected the severity of liver disease. Patients with HCCs who had hypoglycemia had relatively higher IGF-II levels in their sera in comparison with those who did not have hypoglycemia (272 +/- 167.5 ng/ml vs 110.4 +/- 85.9 ng/ml [mean +/- SD], p less than 0.0005), despite the fact that those with hypoglycemia had more advanced liver cancer and had lower IGF-I levels in sera (16.7 +/- 14.1 ng/ml vs 46.8 +/- 47.9 ng/ml, p less than 0.002). It is possible that a labile IGF-II material is produced by the cancer cells of patients with hypoglycemia. This factor is reactive to the IGF-II receptor and partially cross-reacts with an antibody to IGF-II; it accounted for the mildly elevated levels of serum IGF-II. Hypoglycemia may be an integral effect of relatively elevated IGF-II like material and an advanced liver cancer. Also, higher serum alpha-fetoprotein (AFP) levels were more frequently found in patients with hypoglycemia who had relatively elevated IGF-II levels and short survivals.     

A modified radioimmunoassay for arylsulfatase A in human serum and urine

A radioimmunoassay was developed for the determination of arylsulfatase A (EC 3.1.6.1) in human serum and urine. An isoenzyme of arylsulfatase A purified from human urine was used as a standard antigen. The enzyme was radioiodinated with 125I using the Chloramine T method and was stable for about 4 wk. Antibody-bound enzyme was separated from free enzyme by means of a double antibody technique in the presence of polyethylene glycol (PEG). The working range of the method was 0.15-5.0 ng of arylsulfatase A per assay. The within-assay CV was about 8% for both biological fluids and the between-assay CV for serum was 14.1%. Analytical recoveries were 93.2 +/- 9.1% and 97.8 +/- 5.5% for serum and urine, respectively, and the sensitivity was 0.040 ng of arylsulfatase per assay. Serum samples of 50 healthy blood donors were assayed to establish the normal serum level of immunoreactive enzyme, which was found to be 8.3 ng/ml +/- 1.8 ng/ml of serum. Storage of frozen serum was shown to have no significant effect on results obtained using this RIA.     

Sensitive enzyme immunoassay for human Mn superoxide dismutase

A sensitive sandwich-type enzyme immunoassay for measurement of human Mn superoxide dismutase (Mn SOD) was developed using purified antibodies specific to Mn SOD. The antisera were raised in rabbits by injecting Mn SOD purified from human liver. The antibody IgG, purified by the use of Mn SOD-coupled Sepharose, showed a single band on the immunoblotting test with a crude liver extract. The assay system consisted of polystyrene balls with immobilized monospecific antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg human Mn SOD/assay tube. Serum Mn SOD concentrations of healthy adults (77.5 +/- 18.0 ng/ml (1 SD), n = 120, 16-64 yr old) were not related to age or sex. Immunoreactive Mn SOD was detectable in most tissues examined except for erythrocytes. The concentrations of immunoreactive Mn SOD and Cu/Zn SOD in the cerebral cortex were not different among the patients with Alzheimer's disease, and the age matched and young patients without neurological disorders.     

Immunoreactive tissue kallikrein in human serum

Human urinary kallikrein and an antiserum to it raised in the rabbit were used to detect and quantitate immunoreactive tissue kallikrein in human serum. Both 125I-labeled kallikrein and the unlabeled purified enzyme appear complexed to higher molecular weight entities in serum, but specific binding between radiolabeled enzyme and antiserum was unaffected by the presence of serum or plasma. Parallelism to standard displacement curves was always seen with radioimmunoassay of normal sera as well as with human mixed saliva or pancreatic extracts. Assay sensitivity is 160 pg/ml of serum, or 16 pg per tube. Purified plasma kallikrein or prekallikrein in concentrations up to 10 micrograms/ml showed no displacement. Acetone-kaolin activation of plasma produced the expected 30-fold increase in Tos-Arg-OMe esterase activity but no change in immunoreactive tissue kallikrein levels. Serum concentrations were 3.8 +/- 0.7 (mean +/- SE) ng/ml in 21 normal volunteers, and were similar in patients with Fletcher trait or Hageman factor deficiency. Significantly increased serum concentrations were seen with long-term low dietary sodium intake or acute forms of pancreatitis. Although the relation of this immunoreactive material to any active tissue kallikrein within the circulation remains to be determined, our studies provide a new parameter for the assessment of a system repeatedly suggested to have some role in regulation of vascular resistance.     

Characterization and determination of the activity of biliary beta-glucuronidase in rats.

beta-Glucuronidase activity determined in 100 diluted bile samples from 12 rats with bile duct fistula by using phenolphthalein glucuronide as substrate incubated at 56 degrees C and pH 6 was 636 +/- 650 (mean +/- S.D.) modified Sigma units/ml. The enzyme had an optimal pH of 6.0 and was inhibited slightly by cholate by markedly by chenodeoxycholate and deoxycholate. The biliary beta-glucuronidase had, thus, low activity under normal physiologic condition because of the high pH (8.1) and high bile salt content (20 mumoles/ml) of the bile. The enzyme kinetic studies revealed that the direct bilirubin was a competitive inhibitor to phonolphthalein glucuronide for the enzyme. The affinity of the former to the enzyme was 163 times that of the latter. The studies provided a method for measuring the true activity of biliary beta-glucuronidase (Vmax) devoid of interfering factors by measuring the enzyme velocity (v) in the diluted bile with at least five different concentrations of substrate (s). The plotting of (1/v) vs. (1/s) should yield the y intercept or (1/Vmax).     

Elevated level of serum Mn-superoxide dismutase in patients with primary biliary cirrhosis: possible involvement of free radicals in the pathogenesis in primary biliary cirrhosis.

Serum Mn-superoxide dismutase (Mn-SOD) was determined in patients with various liver diseases including 31 patients with primary biliary cirrhosis (PBC), 46 with hepatocellular carcinoma (HCC), 17 with liver cirrhosis (LC), 23 with chronic hepatitis (CH) and 12 patients with obstructive jaundice with an enzyme-linked immunosorbent assay using a specific monoclonal antibody. The serum level in patients with PBC (407 +/- 35 ng/ml, mean +/- SEM; n = 31) was significantly increased (p less than 0.01) compared with those of other liver diseases. Mn-SOD level did not correlate with total bilirubin level, gamma-glutamyl transpeptidase activity, alkaline phosphatase activity, alanine aminotransferase activity, IgM, or with ceruloplasmin level in the sera of the patients. When the patients with PBC were histologically subdivided into four groups according to Scheuer's classification (Scheuer PJ. Primary biliary cirrhosis. In: Scheuer PJ, ed. Liver biopsy interpretation. 3rd ed. London: Bailliere Tindall, 1980:47-56), a high level of serum Mn-SOD was noticed in the early stage as well as in the advanced stage of the disease. Immunoblot analysis confirmed the reactivity and specificity of the monoclonal antibody to the enzyme protein in the patients' sera. Immunostaining of a liver biopsy specimen from the patients with PBC revealed increased expression of the enzyme protein in damaged epithelial cells of interlobular bile ducts, bile ductules, and degenerated hepatocytes. These data suggested that free radicals including superoxide anion are possibly involved in the pathogenesis of the disease and Mn-SOD may play some role in a protection against the superoxide anion.     

Clinical significance of serum 7S collagen in various liver diseases

Serum type IV collagen fragment (7S collagen domain) was measured in 30 controls and 152 liver disease patients with a radioimmunoassay using a polyclonal antibody to human placenta 7S collagen. The serum concentrations of 7S collagen (mean +/- SD) were 4.2 +/- 0.9 ng/mL in controls, 5.1 +/- 2.0 ng/mL in acute hepatitis, 6.5 +/- 2.5 ng/mL in chronic inactive hepatitis, 9.5 +/- 3.8 ng/mL in chronic active hepatitis, 14.4 +/- 7.5 ng/mL in liver cirrhosis, and 14.4 +/- 6.9 ng/mL in hepatocellular carcinoma. In acute hepatitis, 7S collagen was slightly increased, whereas type III procollagen N-peptide and prolyl hydroxylase were markedly increased. In chronic liver disease, 7S collagen concentrations increased with the severity of the disease, and also reflected the degree of fibrosis. The serum 7S collagen concentrations were significantly correlated with those of type III procollagen N-peptide and prolyl hydroxylase in all subjects. These results suggest that serum 7S collagen concentration is a useful diagnostic aid for determining hepatic collagen metabolism in liver diseases.     

Enzyme immunoassay and immunochemical characterization of pancreatic stone protein in human serum.

Monoclonal antibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5 was employed as the standard in the assay. The assay's measurable range was 25-1,500 ng/ml and within run coefficient of variation was 3.7-6.4%. Analytical recovery of the assay was 101.5 +/- 5.65% (mean +/- SD). The results of experiments in which serum was fractionated by Mono S (cation exchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5. The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis.     

Radioimmunoassay for Measurement of Thyroglobulin in Human Serum

A specific and reproducible double antibody radioimmunoassay for the measurement of thyroglobulin (HTg) in human serum has been developed. Since antithyroglobulin autoantibodies combine with the [(131)I] HTg tracer, antibody-positive sera were rejected for measurement. Specificity is demonstrated in that thyroid analogous such as thyroxine (T(4)), triiodothyronine (T(2)) monoiodotyrosine (MIT) and diiodotyrosine (DIT) did not crossreact. Sera previously reacted with anti-HTg-Sepharose contained no immunoassayable HTg. Finally, sera obtained from patients after total thyroid ablation for thyroid carcinoma did not contain demonstrable HTg. The sensitivity of the assay is 1.6 ng/ml, and HTg was detectable in 74% of 95 normal subjects. The mean concentration was 5.1 ng/ml +/-0.49 SEM (range <1.6-20.7 ng/ml). Day to day variation in HTg levels is large in some euthyroid subjects and nearly absent in others. HTg was detectable in 90% of the sera obtained in 23 pregnant women at delivery in whom a mean concentration of 10.1 ng/ml +/-1.3 SEM was observed. The mean level for the corresponding newborn infants at birth was 29.3 ng/ml +/-4.7 SEM a value significantly higher than the mean maternal HTg concentration (P <0.01). A group of 17 thyrotoxic individuals all had elevated HTg levels; the mean for this group was 344.8 ng/ml +/-90.7 SEM. In the acute phase of subacute thyroiditis HTg was also elevated in all of 12 patients, and the mean for this group was 136.8 ng/ml +/-74.6 SEM.     

Radioimmunological determination of insulinlike growth factors I and II in normal subjects and in patients with growth disorders and extrapancreatic tumor hypoglycemia.

Serum levels of immunoreactive insulinlike growth factors (IGF) I and II were determined by a modified IGF I and a new IGF II radioimmunoassay in normal children and adults, and in patients with acromegaly, isolated growth hormone deficiency, and extrapancreatic tumor hypoglycemia. Serum samples were gel filtered by a simple routine procedure at acidic pH to dissociate and separate IGF from the IGF carrier protein. Mean immunoreactive IGF I levels (+/- SD; corrected for crossreactivity of IGF II) were 193 +/- 58 ng/ml in normal adult subjects, 712 +/- 245 ng/ml in acromegalic patients and 24 +/- 14 ng/ml in patients with isolated growth hormone deficiency. The lack of growth hormone alone, irrespective of an otherwise normal hormonal status, appears to be responsible for the drastic decrease of IGF I levels. Oversecretion of growth hormone does not increase the levels of immunoreactive IGF II: mean levels (+/- SD; corrected for crossreactivity of IGF I) in normal and acromegalic subjects are virtually identical (647 +/- 126 and 641 +/- 189 ng/ml, respectively). Apparently, normal growth hormone levels stimulate IGF II production already maximally. However in growth hormone deficiency immunoreactive IGF II is significantly decreased (252 +/- 99 ng/ml). Thus, IGF II, like IGF I, is growth hormone dependent. But in contrast to IGF I, the growth hormone dependence of IGF II seems to become apparent only at subnormal growth hormone levels. In normal children IGF I is age dependent: it is low in newborn cord sera (51 +/- 20 ng/ml) and gradually rises into the adult range with increasing age. At the onset of and during puberty mean IGF I levels lie above prepubertal values. In contrast, IGF II levels in normal children are independent of age and pubertal stage beyond the first year of life, whereas newborns have significantly lower IGF II values. Hypoglycemia resulting from extrapancreatic tumors is not associated with increased immunoreactive IGF I or II levels. IGF I is decreased in most of the sera (mean level +/- SD:56 +/- 39 ng/ml) whereas IGF II lies in the normal range (556 +/- 195 ng/ml).     

Peroxisome localized human hepatic alanine-glyoxylate aminotransferase and its application to clinical diagnosis

The subcellular localization of alanine-glyoxylate aminotransferase (EC 2.6.1.44 L-Alanine: glyoxylate aminotransferase) of adult human liver was examined by sucrose density gradient centrifugation. The enzyme sedimented at the same density as catalase, indicating that it was localized in the peroxisomes. Alanine-glyoxylate aminotransferase activity in the liver of patients with cirrhosis was about 65% of that of normal liver or 71% of that from patients with chronic hepatitis, but its activity in the serum of patients with cirrhosis was higher than that from patients with chronic hepatitis. Patterns of activity of alanine-glyoxylate aminotransferase in liver and serum differed from those of aspartate-2-oxoglutarate aminotransferase and ornithine carbamoyltransferase that have a different intracellular location. Serum immunoreactive alanine-glyoxylate aminotransferase (Im-AGT) was measured by enzyme-linked immunoadsorbent assay (ELISA). The Im-AGT levels (mean +/- SEM) in acute (80 +/- 13 micrograms/L) and chronic (72 +/- 4 micrograms/L) hepatitis were higher than those of normal controls (44 +/- 1 micrograms/L). However, the difference between acute and chronic hepatitis was not statistically significant. The level in liver cirrhosis (54 +/- 3 micrograms/L) was lower than those of the hepatitides but higher than that of normal controls. The apparent half-life of serum Im-AGT of patients who underwent liver lobectomy by a microwave tissue coagulation method was approximately 3-4 days.     

One step sandwich enzyme immunoassay for human type IV collagen using monoclonal antibodies

Monoclonal antibodies were used in one step sandwich enzyme immunoassay (one step sandwich EIA) for human serum immunoreactive type IV collagen. The one step sandwich EIA using either polystyrene ball or microplate was characterized by carrying out two immunoreactions simultaneously, type IV collagen reacting with both a monoclonal antibody as a solid phase and a horseradish peroxidase-labeled monoclonal antibody (Fab') against human type IV collagen as a conjugate. Sensitivity of one step sandwich EIA system by using either polystyrene ball or microplate was 0.22 ng per tube or 0.04 ng per well for type IV collagen, and linearity was obtained between 0.22-40 ng/tube or 0.04-20 ng per well, respectively. Both methods gave reproducible quantitative analysis of immunoreactive type IV collagen levels in the sera of patients with hepatocellular carcinoma and patients with liver cirrhosis, which were apparently higher than the levels in the sera of healthy subjects. Protein immunoblotting shows that the immunoreactive type IV collagen trapped in our present one step sandwich EIA system was not the 7-S and NC1 domains of type IV collagen.     

Determination of triiodothyronine concentration in human serum

A simplified method has been described for the measurement of triiodothyronine (T3) in human serum. The sensitivity was sufficient for determinations on hypothyroid as well as normal and thyrotoxic sera. The values obtained have been in reasonable agreement with a double isotope derivative assay.The normal T3 concentration in human serum approximates 0.2 mug/100 ml; the mean +/-SD of 31 normal sera was 220 +/-27 ng/100 ml. Elevations were observed in sera from 40 patients with thyrotoxicosis (752 +/-282 ng/100 ml), and diminutions were found in sera from 10 hypothyroid patients (98+/-48 ng/100 ml).In rare instances thyrotoxicosis may be due to elevated serum T3 with normal thyroxine (T4) concentration. The incidence of this condition remains to be determined.In approximately half the cases with low serum T4 after (131)I therapy, the eumetabolic state may be maintained by normal or elevated T3 concentration.From these data and kinetic studies indicating a rapid turnover it may be inferred that T3 rather than T4 may be the more important hormone in health and in disease.     

Multiple-dose pharmacokinetics of oral zidovudine in hemophilia patients with human immunodeficiency virus infection.

The disposition of zidovudine (ZDV) was examined during chronic oral dosing (300 mg every 4 h while awake) for 12 weeks in eight asymptomatic patients with hemophilia who were infected with the human immunodeficiency virus. Pharmacokinetic studies were conducted at the initiation of drug administration and after 6 and 12 weeks. Baseline liver function tests indicated normal values for bilirubin, albumin, and prothrombin time, while hepatic enzyme levels ranged from one to three times the normal levels. Initially, the mean peak ZDV concentration in plasma was 2,052 ng/ml with a range of 1,033 to 3,907 ng/ml, while during chronic dosing the peaks were 1,619 +/- 1,062 ng/ml and 1,711 +/- 786 ng/ml at weeks 6 and 12, respectively. ZDV concentrations at 4 h declined to 77 +/- 53 ng/ml, 110 +/- 43 ng/ml, and 101 +/- 49 ng/ml at weeks 1, 6, and 12, respectively. Initially, the plasma concentration-versus-time decay in three patients was linear, with a mean half-life (t1/2) of 1.3 +/- 0.5 h, while five patients had detectable concentrations in plasma after 4 h with an apparent delayed terminal-phase t1/2 of 4.8 +/- 2.8 h. At week 6 the prolonged elimination pattern was noted in all patients (terminal t1/2 = 4.1 +/- 2.0 h). No correlation between hepatic enzyme levels and t1/2 was noted. These findings suggest that ZDV may display a prolonged elimination phase during multiple dosing. Further studies utilizing a more sensitive assay may help to further define this later phase of ZDV elimination.     

Transforming growth factor-beta1 as a surrogate marker of hepatic dysfunction in chronic liver diseases.

The aim of the study was to evaluate the possible association between plasma concentrations of transforming growth factor-beta1 (TGF-beta1) and the degree of hepatic dysfunction in patients with chronic liver diseases. TGF-beta1 was measured with an enzyme immunoassay in plasma from 21 patients with chronic active hepatitis and 40 patients with liver cirrhosis. Normal values were obtained from a group of 13 healthy volunteers. Results were analysed with respect to aetiology and the degree of liver insufficiency as evaluated by the Child-Pugh classification. The mean plasma concentration of TGF-beta1 in patients (36.9+/-2.8 ng/ml) was twice that found in normal volunteers (18.3+/-1.6 ng/ml). The highest values were observed in patients with alcoholic liver cirrhosis (44.4+/-4.7 ng/ml). Plasma TGF-beta1 showed a statistically significant positive correlation with the degree of liver insufficiency. These results indicate the possible use of plasma TGF-beta1 measurement as a good marker of liver function impairment. Further observation of patients involved in this study may help to evaluate its possible prognostic value in chronic liver diseases.     

Clinical usefulness of serum tissue inhibitor of metalloproteinases (TIMP)-2 assay in patients with chronic liver disease in comparison with serum TIMP-1

Tissue inhibitors of metalloproteinases (TIMPs) are involved in liver fibrosis through impaired matrix degradation. Previous studies showed that the serum level of TIMP-1 was increased in patients with chronic liver disease, reflecting the liver TIMP-1 level, and that it is useful for assessing liver fibrosis. An enzyme immunoassay for TIMP-2 is now available. In this study, we examined the clinical usefulness of this serum TIMP-2 test for liver fibrosis in patients with chronic liver disease, in comparison with the serum TIMP-1 test. The serum TIMP-2 concentration was 61 +/- 13 ng/ml in healthy controls (n = 32), and 18% higher in the group of chronic active hepatitis (CAH) patients (n = 34), 64% higher in the liver cirrhosis (LC) group (n = 33) and 44% higher in the hepatocellular carcinoma (HCC) group (n = 61), and similar to the control level in the chronic persistent hepatitis (CPH) group (n = 23). In contrast, the serum TIMP-1 concentration was 155 +/- 17 ng/ml in the healthy controls, 18% higher in CPH, 35% in CAH, 63% higher in LC and 92% higher in HCC. The serum TIMP-2 level was related to the histological degrees of both periportal necrosis and liver fibrosis, as well as to the serum TIMP-1 level. However, the relationships for TIMP-2 were weaker compared to those of serum TIMP-1. These results suggest that compared to the serum TIMP-1 level, changes in the serum TIMP-2 level in chronic liver disease are less liver-specific, and the serum TIMP-2 level is less useful in the assessment of liver fibrosis in chronic liver disease.     

An enzyme immunoassay for the measurement of thyroglobulin in human serum.

An enzyme-linked sandwich immunoassay using silicone rods coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin F (Fab') conjugated with beta-D-galactosidase was developed for the measurement of thyroglobulin in human serum. The volume of serum needed for the assay was as little as 2 microliters. The sensitivity of the assay was 3.5 ng/ml, which is equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by the following observations: (1) The absence of crossreaction of thyroxine and triiodothyronine, (2) non-detectability of thyroglobulin in the sera of patients who underwent total thyroidectomy, (3) parallelism of the standard curve with dilutions of reference serum. The precision of the assay was proven by the demonstration of the sufficient recovery of human thyroglobulin added to sera (92--99%) and coefficients of variance in within and between assays were 6.2--9.3 and 2.5--5.3%, respectively. Furthermore, a highly significant correlation was observed between thyroglobulin concentrations measured by our enzyme immunoassay and those by radioimmunoassay (r = 0.99, p less than 0.001, n = 63). Human thyroglobulin in serum was detectable in 90% of 146 normal subjects, the concentration (mean +/- S.D.) being 13.3 +/- 10.3 ng/ml.     

Radioimmunoassay of human plasma retinol-binding protein

A radioimmunoassay for human plasma retinol-binding protein (RBP) has been developed utilizing a double antibody precipitation technique. RBP was purified 1500- to 2000-fold by procedures described previously. A specific anti-human RBP antiserum was prepared in rabbits by three once-weekly injections of purified RBP emulsified with Freund's adjuvant. RBP was iodinated with (131)I and the RBP-(131)I was purified by gel filtration on Sephadex G-100 after complex formation with human plasma prealbumin. The RBP-(131)I was completely (> 95%) immunoprecipitable in the presence of an excess of specific antiserum, it was not (< 5%) immunoprecipitable in the absence of specific antiserum, and it could be completely displaced from antibody by excess unlabeled RBP. The standard curve obtained in the immunoassay with normal plasma was identical to that with pure RBP. Duplicate samples differed from their mean by 5 +/-5% (+/-SD). There was a quantitative recovery of pure RBP added in varying amounts to normal plasma. The immunoassay accurately measured RBP in amounts of 10-100 ng per assay tube. There was no significant difference in the immunoreactivity of apo-RBP as compared to holo-RBP. The mean plasma values (+/-SEM) for a group of 76 normal subjects were 47.2 +/-1.6 mug/ml for males and 41.6 +/-1.6 mug/ml for females. Plasma RBP levels were markedly depressed (15 +/-2.3 mug/ml) in 14 patients with acute viral hepatitis. There was a highly significant correlation between the plasma levels of RBP and of vitamin A in both normal subjects and patients with hepatitis. In all subjects plasma RBP was generally saturated with retinol. The data suggest that under normal circumstances RBP circulates almost exclusively as the holoprotein.