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.     

One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies

Six monoclonal antibodies for human thrombomodulin (TM) were prepared. All of them recognized an elastase-digested fragment of TM which contains 6 epidermal growth factor (EGF)-like structural domains. We developed a one-step sandwich enzyme immunoassay for soluble TM by using 2 antibodies; one of them, which inhibited thrombin-binding to TM, was fixed to polystyrene balls, and the other, which did not inhibit the thrombin-binding, but inhibited the protein C-activating cofactor activity of TM, was used as peroxidase-labeled conjugate. The sensitivity of this assay was 1 microgram/l for soluble TM. The level of soluble TM was found to be significantly increased in sera of patients with systemic lupus erythematosus in comparison to the level in sera of healthy subjects.     

One-step sandwich enzyme immunoassay for insulin using monoclonal antibodies

An enzyme-linked immunosorbent assay for the measurement of insulin in human serum has been developed. The test is based on the sandwich technique with two monoclonal antibodies directed against two different epitopes of insulin using coated plastic tubes as the solid phase and horse radish peroxidase as the label. The immunoreactions are completed in one step within 2 h. The horse radish peroxidase activity bound to the tube wall is measured photometrically after an additional 1-h incubation with the substrate. The standards used cover the range from 0 to 260 mU insulin/L. Employing the Enzymun-Test System ES 22 modular batch analyzer, the detection limit was found to be 3.7 mU insulin/L. Coefficients of variation (CV's) between 1.4-7.8% for intraassay precision and 5.6-10% for interassay precision were obtained over the concentration range of 17-107 mU Insulin/L. The correlation between the procedure described here (y) and a commercially available double antibody radioimmunoassay (x) is expressed by the following equation: y = 1.07x + 1.14 mU insulin/L.     

Direct measurement of calpastatin subtypes by sandwich enzyme immunoassay using monoclonal antibodies.

Six stable hybridoma cell lines secreting monoclonal antibodies to human calpastatin were established. All monoclonal antibodies belong to the IgG1 subclass and recognized different epitopes on calpastatin. At least two groups were distinguished; the first group was specific for muscle-type (M-) calpastatin and the second group recognized not only M-calpastatin but also erythrocyte-type (E-) calpastatin. The inhibitory effect of all monoclonal antibodies on calpastatin activity was relatively low even at high concentrations of antibodies. Enzyme immunoassay systems were developed for direct determination of calpastatin subtypes in human cells requiring no other sample treatment than the disruption of the cells. The assay methods were, in principle, based on the sandwich enzyme immunoassay using epitope-specific monoclonal antibodies. The enzyme immunoassay system for M-calpastatin was specific for M-calpastatin and could not detect E-calpastatin. The enzyme immunoassay system for total calpastatin detected not only M-calpastatin but also E-calpastatin. The sensitivity of these assay systems was 10 pmol l-1 of calpastatins. Antigenicity of calpastatins was found to be unchanged in the presence of EDTA and haemoglobin. Good reproducibilities of within-and between-assay series and excellent recovery of exogenous calpastatins from cell lysates were observed. From these results, it seems that our newly developed subtype-specific enzyme immunoassay systems for calpastatins are useful in biochemical studies and clinical testing for determination of calpastatin subtypes.     

A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 3 (stromelysin-1) using monoclonal antibodies.

A one-step sandwich enzyme immunoassay (EIA) for matrix metalloproteinase 3 (MMP-3; stromelysin-1) was developed. The assay system used two simultaneous immunoreactions using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The sensitivity of the assay system was 20 micrograms/l and linearity was obtained between 31 and 500 micrograms/l. The EIA system was capable of measuring both precursor and active forms of MMP-3 as well as the forms of MMP-3 complexed with tissue inhibitors of metalloproteinases. MMP-3 levels as measured by this assay are significantly higher in the sera of patients with rheumatoid arthritis as compared to those of healthy subjects and patients with osteoarthritis. Immunoblot analyses showed that in the sera and synovial fluids of patients with rheumatoid arthritis, MMP-3 is present in the 59- and 57-kDa precursor forms.     

Significance of urinary type IV collagen in patients with diabetic nephropathy using a highly sensitive one-step sandwich enzyme immunoassay

Urinary concentrations of type IV collagen in patients with diabetic nephropathy were measured by a highly sensitive, one-step sandwich enzyme immunoassay. Samples from 298 patients with non-insulin-dependent diabetes mellitus (NIDDM) and 80 healthy controls were examined. In diabetic patients with macroalbuminuria or renal insufficiency, the concentrations of urinary type IV collagen were significantly higher than those of diabetic patients with normoalbuminuria or healthy controls (P < 0.001). Urinary type IV collagen concentration in diabetic patients with microalbuminuria was significantly higher than that in diabetic patients with normoalbuminuria or that in healthy controls (P < 0.001). In contrast, there were no significant changes in the concentration of serum type IV collagen between microalbuminuric patients and normoalbuminuric patients. The area under the receiver operating characteristic (ROD) curve for the urinary type IV collagen concentration was equivalent to that of urinary albumin. It was concluded that urinary type IV collagen concentration determined using this method might be a useful marker for the early detection of diabetic nephropathy. J. Clin. Lab. Anal. 11:110–116. © 1997 Wiley-Liss, Inc.     

Sandwich enzyme immunoassay for serum integrins using monoclonal antibodies.

We produced monoclonal antibodies (mABs) against human integrins. Competitive enzyme-linked immunosorbent assay (ELISA) revealed that each mAB bound to different antigenic determinants. We then developed sandwich-type enzyme immunoassays (EIAs) to measure the concentration of fibronectin receptor (FNR) and vitronectin receptor (VNR). Serum immunoreactive integrin levels were measured using these EIAs in various liver and malignant diseases. In almost all cases of liver cirrhosis (LC) and hepatocellular carcinoma (HCC), serum integrin levels were significantly elevated, but were in the normal range in gastric, colon, lung cancer, and acute hepatitis (AH). The correlation between serum FNR and VNR levels was statistically significant in all cases of liver disease, and no correlation was observed between these integrin levels and conventional biochemical markers such as AST, ALT, and GGT. The serum integrin levels were demonstrated to be a potential diagnostic marker for hepatic fibrogenesis and carcinogenesis, and these sandwich EIAs could be useful for determination of these integrins in clinical laboratory tests.     

Concentration of serum laminin and type IV collagen in liver diseases assayed by a sandwich enzyme-immunoassay using monoclonal antibodies.

Serum laminin (P1 fragment) and type IV collagen levels were determined in patients with hepatic disorders. The method was based on a sandwich enzyme-immunoassay using two monoclonal antibodies that recognize different epitopes of either laminin or type IV collagen molecule. Laminin and type IV collagen levels in the serum of patients with chronic hepatic disorders were higher as compared with those in healthy control subjects, with the increment of serum type IV collagen being far greater than that of laminin. Since type IV collagen and laminin are major basement membrane components, it is suggested that the higher levels of these peptides may reflect a so-called capillarization of the perisinusoidal wall encountered in hepatic fibrogenesis. The assay system used in this experiment is simple and sensitive and can be applied to clinical evaluation of hepatic fibrosis.     

Enzyme immunoassay for subgrouping human rotaviruses using monoclonal antibodies

Rotavirus group-specific (common in human rotaviruses) and subgroup 1-specific monoclonal antibodies were established and an enzyme immunoassay was developed for subgrouping human rotaviruses.     

An enzyme immunoassay for isotyping mouse monoclonal antibodies

A rapid, simply performed and relatively inexpensive enzyme immunoassay for isotyping mouse monoclonal antibodies is described, based on the urease/urea system. Because of the high sensitivity (less than 0.1 microgram/ml of immunoglobulin can be detected in cell culture medium) no treatment of the hybridoma supernatant sample is required prior to assay, and the isotype of a mouse immunoglobulin can be determined in about thirty minutes.     

Multi-centre study of a new CEA enzyme immunoassay using monoclonal antibodies

The use of a new monoclonal enzyme immunoassay (EIA) for the carcinoembryonic antigen (CEA) (Enzymun-Test CEA) was evaluated in a multi-centre study. Fifteen different laboratories [participated in the study. Data from the investigation were analysed in terms of precision, sensitivity, specificity and correlation with other test methods. The intra-assay coefficient of variation was between 1.3% at 23.0 microg/l CEA and 13.9% at 1.3 microg/l CEA. Inter-assay reproducibility ranged from 3.6% to 19.2%. The apparent sensitivity of the new EIA for CEA was approx. 0.5 microg/l CEA. The findings indicate that lipaemic and haemolytic sera and samples taken from icteric, rheumatic and dialysis patients did not have any influence on the results. There was no evidence that drugs commonly used in the treatment of carcinoma patients have any influence on the assay results. A good correlation between the new EIA for CEA and six other CEA enzyme immunoassay or radioimmunoassay methods was registered. These results seem to be of significance in particular for the monitoring of therapy for carcinoma patients. The new EIA for CEA exhibits a high degree of sensitivity, specificity and reproducibility.     

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.     

Monoclonal antibodies based sandwich erythro-immunoassay and 'dot' enzyme immunoassay for human chorionic gonadotropin in urine

Two simple solid-phase sandwich immunoassays for human chorionic gonadotropin (hCG) employing monoclonal antibodies have been described. One is a sandwich erythro-immunoassay employing V-shaped well microtitration plates coated with monoclonal anti-beta-hCG antibody and monoclonal anti-alpha-hCG antibody labelled sheep erythrocytes. The second is a 'dot' enzyme immuno-assay employing dip-stick (plastic strips pasted with nitrocellulose pads) coated with monoclonal anti-beta-hCG antibody. Anti-alpha-hCG monoclonal-alkaline phosphatase conjugate was used to reveal hCG bound to solid surface. The assays can be performed by 'one-step' or 'two-step' procedures. Erythro-immunoassay as well as 'dot' enzyme immunoassay was able to detect in urine as low as 10 mIU hCG/ml. A good correlation was observed between the values obtained by these two methods as well as 'two-step' sandwich enzyme immunoassay on 47 urine samples.     

Rapid determination of C-reactive protein by enzyme immunoassay using two monoclonal antibodies

Several monoclonal antibodies for human C-reactive protein (CRP) were characterized, and two antibodies binding to separate domains were used to construct a rapid and simple immunoenzymometric assay for CRP. The assay consists of a single 15 min immunological reaction during which CRP forms a complex with a peroxidase-labelled antibody and with another antibody attached to the test-tube wall. The immobilized complex is detected by a 3 min colour reaction using peroxidase substrate. The quantitative measuring range of the assay is 0.04-5 mg/l, and no hook occurs at five-fold higher values. The sensitivity of the method allows reliable determination of low CRP levels, eg. in paediatric samples. The values obtained with the present assay correlated well with turbidimetric results.     

Highly specific enzyme immunoassay for the beta-subunit of human thyrotropin with use of monoclonal antibodies

A highly specific enzyme-linked "sandwich" immunoassay is described for determining free human thyrotropin (hTSH) beta-subunit in serum by using a anti-hTSH beta-subunit monoclonal antibody conjugated with beta-D-galactosidase (EC 3.2.1.23) and a solid phase consisting of silicone rods coated with another monoclonal antibody. We could detect as little as 0.04 ng of beta-subunit per assay. The measurable range of hTSH beta-subunit concentrations in serum was 0.4 to 50 micrograms/L. The assay demonstrated little or no cross reactivity with intact hTSH, hTSH alpha-subunit, or human choriogonadotropin. The mean CVs were 12.2% within assay, 13.9% between assay. The hTSH beta-subunit was not detectable in sera from healthy subjects, patients with hyperthyroidism, or two patients with pituitary tumors producing TSH. It was measurable (at concentrations of 0.65 to 2.70 micrograms/L) in sera from eight of 23 hypothyroid patients. In five of the hypothyroid patients examined, the concentration of hTSH beta-subunit in serum increased after administration of thyroliberin. This method may be useful in elucidating the physiological and pathological significance of the hTSH beta-subunit and in examining the function of the hypothalamus-pituitary-thyroid axis.     

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.     

Enzyme immunoassay of beta-hexosaminidase isoenzymes using monoclonal antibodies

Purified beta-hexosaminidase A (Hex A) and beta-hexosaminidase B (Hex B) were used as immunogens in mice, with the purpose of obtaining isoenzyme-specific monoclonal antibodies (mabs). A total of 60 mabs were developed, 23 specific for Hex A and 37 recognizing both isoenzymes. At low pH, two of the latter mabs reacted only with Hex B, and it was, therefore, possible to develop enzyme immunoassays for the specific determination of Hex A and Hex B. The precision of the methods was adequate with intra- and interassay coefficients of variation below 3%.     

Demonstration of placental and placental-like alkaline phosphatase in non-malignant human tissue extracts using monoclonal antibodies in an enzyme immunoassay

The occurrence and nature of heat-stable placental-type alkaline phosphatase (Pl-ALP) in extracts from a variety of non-malignant human tissues has been investigated using monoclonal antibodies in a sensitive solid-phase enzyme immunoassay. The presence of Pl-ALP was confirmed in testicular, cervical and lung tissue extracts, and trace amounts were also detected in extracts from mammary and ovarian tissue. Evidence is presented that normal testis contains at least two forms of Pl-ALP, the major component being an L-leucine-inhibitable placental-like enzyme which is not the D-variant of Pl-ALP. These results have a bearing on the occurrence of Pl-ALP and placental-like ALP activity in malignancy.