Noncompetitive Enzyme-linked Immunosorbent Assay Research Articles

Transferrin and transferrin receptor expression in intraocular proliferative disease. APAAP-immunolabeling of retinal membranes and ELISA for vitreal transferrin

Transferrin (TF) is the major transport protein involved in human iron metabolism. The expression of the cell-surface TF receptor is associated with cellular proliferation, the dominant feature of proliferative vitreoretinal disorders with traction retinal detachment. A total of 14 retinal membranes from patients with different clinical diagnoses contained immunoreactive TF. Expression of the cell-surface TF receptor was confirmed by a monoclonal anti-human TF-receptor antibody label in 11 of the 14 specimens. We developed a noncompetitive enzyme-linked immunosorbent assay (ELISA) for TF and found it to be a significant component of vitreal protein, with a level of 65.7 +/- 33.9 mg/l. Vitreal TF as a major iron acceptor probably has a protective function, but its interaction with macrophages and its growth-promoting activity may subsequently stimulate the proliferation of fibroblasts and retinal pigment epithelial cells.

Subcellular distribution of cardiac fatty acid-binding protein in bovine heart muscle and quantitation with an enzyme-linked immunosorbent assay

Several types of the 14–15 kDa fatty acid-binding proteins (FABPs) are known to occur in the cytosol of mammalian cells. With antibodies raised against the cardiac-type protein from bovine heart, immunoblots indicated a more widespread distribution of the cardiac FABP in subcellular fractions, such as mitochondria and nuclei. A detailed view was obtained when the post-embedding protein A-gold labeling method was applied to cross-sections of heart cells and isolated subcellular fractions. Cardiac FABP in myocytes was associated with myofibrils and localized within mitochondria and nuclei. After subfractionation of mitochondria, the binding protein was recovered with matrix proteins only. A non-competitive enzyme-linked immunosorbent assay (ELISA) of the direct type was developed specifically for bovine cardiac FABP. This assay was sensitive in the range of 0.05 to 1 ng, and concentrations of cardiac FABP per mg protein were found for cytosol, matrix and nuclei to be around 3.18, 0.18 and 0.03 μg, respectively. The newly found compartmentation of cardiac FABP in the heart cell must be considered when the true functions of the protein, yet to be defined, are studied.

Characterization of two monoclonal antibodies to human apolipoprotein A-I that also bind to rabbit apolipoprotein A-I. Application to ELISA evaluation of rabbit serum apo A-I

Two monoclonal antibodies against human apolipoprotein A-I were characterized to recognize rabbit apo A-I. By immunoblotting we determined that while neither antibody reacted with the other rabbit apolipoproteins they both recognized all rabbit apo A-I isoforms. Cotitration revealed that each antibody bound to different apo A-I epitopes. Then we developed a noncompetitive enzyme-linked immunosorbent assay (ELISA) to measure the concentration of total apolipoprotein A-I in rabbit serum. The ELISA curves of the different lipoprotein class and serum showed the similarity of the expression of the apo A-I epitopes recognized in each of them and in serum. This assay, as opposed to other techniques, offers several advantages such as sensitivity, specificity, and simplicity and avoids the use of radioisotope.

Enzyme-linked immunosorbent assay of lipoprotein(a) in serum and cord blood

We have developed a new sensitive method for quantifying lipoprotein(a) (Lp(a)) in human serum, using a ‘sandwich’ type noncompetitive enzyme-linked immunosorbent assay (ELISA). The solid-phase used was a polystyrene plate. The anti-Lp(a) antibody-enzyme conjugate was labelled by linking Fab' fragments to peroxidase (EC 1.11.1.7) by the maleimide method. The minimum detectable concentration was 0.5 ng/well. Routinely, the assay was carried out with 1000-fold diluted serum, and Lp(a) was quantified between 4.0 and 500 mg/1. Within-run coefficients of variation (CVs) ranged from 3.5% to 10.4% and between-run CVs from 5.0% to 11.1%. Results by the ELISA were in good agreement with those by radial immunodiffusion ( r = 0.955). The distribution of Lp(a) in serum from 820 healthy donors was highly skewed: mean 141.1 mg/1, medium 97.9 mg/1. In cord blood, the mean and median were 15.6 and 9.8 mg/1, respectively. This ELISA for Lp(a) has the advantages of being highly sensitive and specific, simple to perform, and does not use radioisotopes.

Enzyme-linked immunosorbent assay for serum amyloid A apolipoprotein with use of specific antibodies against synthetic peptides.

We describe a noncompetitive enzyme-linked immunosorbent assay for amyloid A apolipoprotein in human serum (apo SAA) in which specific antibodies against synthetic peptides are used. Microtiter plates were used as solid phase and coated with affinity-purified antibodies raised against SAA1-(95-104) peptide. After incubation with delipidated plasmas, the bound apo SAA was revealed by labeled antibodies raised against SAA1-(58-69) peptide. The assay offers several advantages over existing techniques: sensitivity, specificity, simplicity, and non-use of radioisotopes. Results correlate well with those by a nephelometric method in which polyclonal antibodies are used.

Fibronectin quantification in plasma and vitreous by a noncompetitive ELISA technique.

Fibronectin, a high-molecular glycoprotein of the extracellular matrix and plasma, has provoked increasing interest in ophthalmology because of its possibly important role in the pathogenesis of proliferative vitreoretinopathy (PVR). A quantitative determination of fibronectin in vitreous aspirates is necessary for the evaluation of this glycoprotein in PVR. A noncompetitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of immunoreactive fibronectin in human plasma samples and vitreous aspirates. Here, procedures to establish an ELISA for the clinical laboratory routine are discussed and possible pitfalls during the assay standardization are described.

Non-competitive enzyme-linked immunosorbent assay for human apolipoprotein SAA or S

Purified antibodies against human apolipoprotein S were used to measure apo S levels in human plasma by a sandwich ELISA technique. The method developed was highly sensitive and specific. A reliable calibration of a plasma standard was obtained by reassociating the purified protein with plasma HDL. A significant correlation ( r = 0.69) was found between plasma apo S and CRP values. This correlation coefficient reached 0.97 when the plasma samples were selected according to additional clinical and laboratory criteria.

Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.

Apolipoprotein A-I (apoA-I), the major protein component of human high density lipoprotein, appears intracellularly as an intermediate precursor (proapoA-I) with a hexapeptide extension (Arg-His-Phe-Trp-Gln-Gln) at its amino terminus. To investigate the regulation of processes that regulate plasma apoA-I levels, a sensitive and simple assay for proapoA-I is required. We describe a specific enzyme-linked immunosorbent assay (ELISA) for quantification of proapoA-I using monospecific rabbit antibodies raised against the peptide: Arg-His-Phe-Trp-Gln-Gln-Asp-Glu-Pro. The monospecificity of antibodies to propeptide has been checked and no cross-reaction with mature apoA-I has been found although three first mature apoA-I amino acids (Asp-Glu-Pro) were included in the immunizing peptide. The assay is a non-competitive sandwich ELISA in which polystyrene microtiter plates were used with antibodies to propeptide adsorbed on the wells. After incubation with plasma samples, the bound proapoA-I was revealed by labeled rabbit polyclonal antibodies directed against mature apoA-I. The working range was 10 to 100 ng/ml, recovery of proapoA-I added to plasma was 94.6 to 106.5%, and the intra- and interassay coefficients of variation were 3.8% and 7.9%, respectively. A delipidation step using diisopropylether-n-butanol was necessary to expose antigen sites of proapoA-I in native lipoproteins. Mean level of proapoA-I in normal subjects was 87 +/- 15 micrograms/ml. It represented 7.1% of total apoA-I while in Tangier serum it represented 29%.

Enzyme-linked immunosorbent assay for human plasma apolipoprotein B

A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for measuring total plasma apolipoprotein (apo) B using affinity purified polyclonal and monoclonal antibodies. Microtiter plates from different manufacturers were tested with regard to their IgG binding characteristics; only one plate yielded consistent coefficients of variation of less than 5%. The optimal plasma dilution in this assay was 1:3000. IgG anti-apoB antisera conjugated to alkaline phosphatase was used as a second antibody. p-Nitrophenyl phosphate was utilized as substrate for color development, and the absorbance (410 nm) was read utilizing an ELISA reader interfaced with a microcomputer for data processing. Plasma apoB levels in plasma have been determined in 1115 male and female participants in the Framingham Offspring Study. Mean (+/- SD) plasma concentrations were 89 +/- 28 mg/dl. Significant age and sex related differences in apoB levels were noted.

Detection and quantitation of toxic shock syndrome toxin 1 in vitro and in vivo by noncompetitive enzyme-linked immunosorbent assay.

Toxic shock syndrome toxin 1 (TSST-1), an exotoxin produced by many Staphylococcus aureus strains, is implicated as the prime causal agent of toxic shock syndrome (TSS). A sensitive and specific noncompetitive enzyme-linked immunosorbent assay (ELISA) capable of detecting TSST-1 at concentrations from 0.5 to 16 ng/ml was developed. This assay did not detect other staphylococcal enterotoxins including A, B, C1, C2, C3, D, and E. Possible interactions with protein A were readily eliminated by pretreatment of test samples with 10% normal rabbit serum. The assay was adapted for rapid screening of TSST-1 production by S. aureus isolates in culture supernatants in vitro and for detection of TSST-1 in vaginal washings of TSS patients and healthy controls in vivo. All 35 S. aureus isolates confirmed to be TSST positive by Ouchterlony immunodiffusion and 59 of 60 isolates confirmed to be TSST-1 negative gave concordant results by ELISA. Interestingly, toxigenic S. aureus strains isolated from TSS patients quantitatively produced significantly more TSST-1 in vitro compared with toxigenic control strains (P less than 0.05, Mann-Whitney rank sum test). TSST-1 could be detected by ELISA in three of four vaginal washings collected within 3 days of hospitalization from three women with acute menstrual TSS, compared with 0 of 17 washings from nine TSS patients hospitalized longer than 3 days (P = 0.003, Fisher's exact test) and 1 of 15 washings from 14 healthy control women (P = 0.016). This noncompetitive ELISA should be particularly useful for rapid screening of TSST-1 production by S. aureus isolates, for the purification and biochemical characterization of TSST-1, and for human and animal studies of the pathogenesis of TSS.

Immunological quantitation of O4-ethylthymidine in alkylated DNA: repair of minor miscoding base in human cells.

Rabbit antisera specific for O4-ethyldeoxythymidine (O4-EtdThd) were developed and used in competitive and non-competitive immunoassays for the quantitation of the miscoding promutagenic adduct O4-EtdThd in DNA. High titer antiserum TB3 (affinity constant, 8.1 X 10(8)) gave 50% inhibition of the tracer antigen-antibody binding with 0.26 pmol of O4-EtdThd in radioimmunoassay and negligible inhibition with unmodified DNA components. In competitive enzyme-linked immunosorbent assay (ELISA), using ENU-poly(dT) instead of hapten-protein conjugate as antigen, inhibition of antigen-antibody binding was proportional to both, the concentration of ethylnitrosourea (ENU) and alkylated DNA. The sensitivity of detection of O4-EtdThd in DNA was enhanced to fmol of O4-EtdThd and ng amounts of alkylated DNA by using avidin-biotin linked immunoreagents in non-competitive ELISA and immunoslot blot assays. An O4-EtdThd concentration-dependent binding of specific antibody, detecting 0.12 fmol of the modified base, was observed with DNA alkylated in vitro with ENU at an O4-EtdThd/Thd molar ratio of 2.6 X 10(-6). The immunological quantitation of O4-EtdThd in ENU-treated human skin fibroblast and kidney epithelial cells indicates a gradual removal of the modified base as a function of post-treatment time in culture. In both cell types about 50% of the initial damage was repaired during a 72-h period. Earlier studies using human organ extracts ruled out the participation of an alkyltransferase or glycosylase enzyme for the repair of O4-alkylthymine. None the less, the kinetics data presented indicate that normal human cells in culture are proficient for the repair of critical pyrimidine alkylation adduct that may be associated with cellular oncogenic transformation of susceptible mammalian cells.

Rapid diagnosis of herpes encephalitis by enzyme immuno-assay

Five cases of encephalitis caused by herpes simplex virus (HSV) are described. HSV-specific IgA and IgM antibodies were detected in cerebrospinal fluid and serum by use of antibody-capture noncompetitive enzyme-linked immunosorbent assay. The tests seem suitable for rapid diagnosis in the second week after onset of neurological symptoms. The clinical importance of early diagnosis and therapy in patients of HSV encephalitis has been discussed.

A non-competitive enzyme-linked immunosorbent assay for measuring human plasma apolipoprotein B levels

A non-competitive enzyme-linked immunosorbent assay for measuring human plasma apolipoprotein B levels

Quantitative determination of human plasma apolipoprotein A-I by a noncompetitive enzyme-linked immunosorbent assay

A noncompetitive enzyme-linked immunosorbent assay (ELISA) for human plasma apolipoprotein A-I (ApoA-I) was developed. Microtiter plates were coated with purified antibodies to ApoA-I and blocked. Plasma samples from normolipidemic and hypertriglyceridemic subjects were added and ApoA-I was allowed to bind to coating antibodies. After washing, the amount of ApoA-I bound to microtiter plates was estimated with horseradish peroxidase-labeled antibodies to ApoA-I. A single step delipidization procedure was included to expose masked antigenic sites of ApoA-I in plasma. The average concentration of ApoA-I in plasma of normolipidemic subjects was 1.37 g/l. Recovery of ApoA-I added to plasma varied from 93–107%. Intra- and inter-assay coefficients of variations were 4 and 8%, respectively. The assay was also used for quantifying ApoA-I in lipoprotein density classes. There was a good correlation between this assay and electroimmunoassay ( r = 0.84–0.92). The described sandwich ELISA is a specific, precise, sensitive and relatively simple method for measuring ApoA-I levels in human plasma.

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase.

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-linked immunosorbent assay to quantify fibronectin

Fibronectin, a multifunctional soluble glycoprotein, is found in soluble form in blood and other body fluids. Fibronectin binds to biological substrata-collagen, gelatin, etc.-and to nonbiological substrata as well. An interesting finding was that fibronectin bound to polystyrene plastic quickly and tightly and further treatment of plastic with denaturing agents did not remove or denature it once it was adsorbed. Through the use of this property, a three-step noncompetitive enzyme-linked immunosorbent assay has been developed to quantify soluble fibronectin in biological samples. The following parameters were studied: (i) selective attachment of fibronectin to polystyrene; (ii) interference caused by increasing amounts of external protein; and (iii) linear response and limit of sensitivity. The procedure has been applied to measure fibronectin from different animal sources.

Monoclonal antibodies to DNA modified by a benzo[a]pyrene diol epoxide.

Monoclonal antibodies were obtained after fusion of mouse P3 X 63 Ag8.653 myeloma cells with spleen cells isolated from BALB/cCr mice immunized with either DNA modified by 7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE-I-DNA) complexed electrostatically to methylated bovine serum albumin or with BPDE-I modified guanosine conjugated with bovine serum albumin, BPDE-I-G-BSA. One monoclonal hybridoma line from each type of immunization was grown as ascites tumors or in defined media and characterized in an enzyme linked immunosorbent assay (ELISA). The antibody produced from the spleen cells of a BPDE-I-DNA immunized mouse, designated 5D11, recognizes BPDE-I-DNA and DNA modified by 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE II) but not unmodified DNA, N-2-acetylaminofluorene (AAF) or 1-nitropyrene (NP) modified DNA, BPDE-II-dG or BPDE-I tetraol. It does recognize BPDE-I-dG but with a much lower affinity than when the adduct is present in DNA. In contrast, antibody 8E11 produced from the spleen cells of a BPDE-I-G-BSA immunized mouse recognizes the monoadduct BPDE-I-dG better than BPDE-I-DNA. It also recognizes BPDE-I tetraol but not BPDE-II-DNA, unmodified DNA, AAF- or NP-DNA or BPDE-II-dG. In a noncompetitive ELISA as little as 3 fmol of BPDE-I-DNA adduct can be detected with either antibody 5D11 or 8E11. The combination of the highly sensitive ELISA and highly specific monoclonal antibodies should be valuable in the detection and quantitation of human exposure to benzo[a]pyrene.

An enzyme-linked immunosorbent assay for quassin and closely related metabolites

A microtitration plate enzyme-linked immunosorbent assay has been developed for quassin and closely related seco-triterpenes. The method is much more sensitive than conventional chromatographic techniques and enables the routine analysis of large numbers of samples with a detection limit of 5 ng quassin. Antibody production was elicited by injecting a conjugate of iso-quassinic acid linked to bovine serum albumin into rabbits. Important features of the technique are (a) the use of a double-antibody, noncompetitive enzyme-linked immunosorbent assay for a low-molecular-weight, nonantigenic compound using microtitration plates; and (b) that the initial incubation of samples with primary antiserum can be carried out in buffer containing up to 20% ( v v ) methanol with minimal loss of sensitivity. The structural requirements for recognition of the hapten by the antiserum are considered based on the levels of cross-reaction found with a wide range of other quassinoids.

Enzyme-Linked Immunosorbent Assay for Quantitative Detection of Bacillus thuringiensis Crystal Protein

Accurate measurement of the toxic protein crystal produced during deep-tank fermentation of Bacillus thuringiensis is critical for optimum process yield. The currently accepted method is a bioassay that requires more time to generate data than to complete the fermentation itself. A noncompetitive enzyme-linked immunosorbent assay has been developed with purified B. thuringiensis crystals to generate rabbit antiserum. This technique gives a quantitative crystal protein value with a colorimetric endpoint for either liquids or powders within 4 h of sampling. Reproducibility of this enzyme-linked immunosorbent assay satisfies criteria for use in a commercial process.

The enzyme-linked immunosorbent assay of meningococcal and some related Escherichia coli polysaccharides

A non-competitive enzyme-linked immunosorbent assay (ELISA) system has been developed for the quantitative, sensitive and specific determination of meningococcal polysaccharides. A prerequisite, however, is the correspondence of the O-acetyl content and the molecular size of the polysaccharides in the samples and the reference preparation. If these characteristics differ, special precautions in the use of antibodies to both the O-acetylated and the non-O-acetylated variants have to be taken, including the testing of the sample in at no less than three dilutions.