Journal of immunoassay最新文献

Helicobacter pylori (H. pylori) is a gram-negative spiral bacteria that are associated with gastritis, peptic ulcer and gastric cancer. We have developed enzyme-linked immunosorbent assay (ELISA) that detects serum anti-H. pylori immunoglobulin G antibodies using H. pylori strains isolated from Korean patients. To assess the sensitivity and specificity of our assay system with different commercial kits, serum samples from 249 Korean patients with a variety of gastrointestinal diseases were tested. Among 249 Korean patients, 178 (71.5%) were positive in culture and/or urease test. The sensitivity and specificity between our assay system and four other commercial kits (Bio-Rad, DAKO, ROCHE, and IPR) were as follows: 97.8% and 92%, 94.3% and 53%, 56.5% and 92%, 83.3% and 96%, 58.2% and 92%, respectively. All sera showing discordant immunoassay results between different ELISA kits were confirmed by immunoblot analysis. These results indicate that our assay system showed a highly accurate and reliable results in diagnosis of H. pylori infection in Korean patients.

An enzyme linked immunosorbent assay, specific for prothymosin alpha (ProT alpha) was developed using an antibody against the synthetic C-terminal peptide ProT alpha[101-109] and isolated bovine ProT alpha for the preparation of standard solutions and immunoplates. Due to the antibody used, the ELISA developed was capable of fully discriminating between ProT alpha, the naturally occuring and partially homologous peptide parathymosin alpha (ParaT alpha) and the peptide thymosin alpha1 (T alpha1), whose sequence is identical to the [1-28] sequence of ProT alpha, and its in vivo occurrence is under question. Moreover, due to its improved sensitivity, the ELISA was capable of directly determining ProT alpha concentration in human serum and tissue extracts, without any pretreatment of the samples. ProT alpha levels were directly measured in sera obtained from 48 apparently healthy individuals and 27 patients with diagnosed breast cancer and found to range from 0.67 to 2.34 microg/ml (mean value 1.27 +/- 0.49 microg/ml) and from 0.47 to 1.74 microg/ml (mean value 1.02 +/- 0.29 microg/ml), respectively. ProT alpha levels were also measured in four breast tumor and adjacent normal breast tissue extracts and found to be elevated in the tumor extracts.

An alternative protocol for immobilization of antibodies onto plastic solid supports is presented. According to the proposed protocol, tubes are first coated with gamma-globulins from non-immunized animal of the same species as that from which the antigen-specific antibody has been developed. Then, an excess of second antibody is added to the tubes and the anti-species specific antibodies present in the antiserum are immunoadsorbed on the immobilized gamma-globulins. Finally, the antigen specific antibody is immunoadsorbed on the immobilized second antibody. We found that the coating protocol developed allows the use of antigen-specific and second antibody antisera dilutions, thus avoiding the need for affinity purification of antibodies. Additionally, it provides solid-phase second antibody with increased binding capacity compared to the directly adsorbed onto the solid second antibody. The advantages of the proposed coating protocol were demonstrated through the development of a solid-phase radioimmunoassay for the determination of total triiodothyronine in human serum samples.

BMS-191352 is a single-chain fusion protein composed of the variable regions of chimeric BR96 monoclonal antibody and the binding defective form of Pseudomonas Exotoxin A (PE40). The immunotoxin exhibits potent cytotoxicity against tumor cells expressing the Lewis antigen. A sensitive and specific double antibody sandwich ELISA has been developed and validated for the determination of BMS-191352 in rat and dog EDTA plasma. A monoclonal anti-PE40 antibody (EXA2-1H8) was used to capture BMS-191352 in plasma samples. The captured BMS-191352 was then detected using a biotinylated monoclonal BR96 antiidiotypic antibody (757-4-1) followed by the addition of streptavidin-horseradish peroxidase conjugate and chromogen 3,3',5,5'-tetramethylbenzidine. The optical density was measured at 450 nm. The standard curve range in rat and dog plasma was 2-32 ng/mL. The RSD for the inter- and intra-assay precision was within 9.2% and the accuracy was greater than 89.0%. The ELISA method was applied to the analysis of BMS-191352 in plasma samples from toxicokinetic studies conducted in rats and dogs. These studies revealed that the systemic exposure of BMS-191352 was dose proportional and the kinetics of BMS-191352 were linear between the dose range of 1.8-7.2 mg/m2 in the dog.

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease which specifically converts the inactive precursor big-endothelin-1 (big ET-1) to the vasoactive endothelin-1 (ET-1). Six different mouse hybridoma cell lines have been generated secreting monoclonal antibodies specific to human ECE-1. These antibodies have been proven useful in a fast and efficient one-step purification of membrane-bound ECE-1 as well as of artificial soluble ECE-1 by immunoaffinity chromatography. The antibodies are suitable for a quantification of ECE-1 in solution by a sandwich-ELISA and for the immunohistochemical detection of ECE-1 in the cell membrane.

Using an affinity purified sheep anti-human luteinizing hormone IgG-horseradish peroxidase conjugate (sheep anti-hLH IgG-HRP) and rabbit anti-human luteinizing hormone antiserum (rabbit anti-hLH) directed against different antigenic determinants, a solid-phase "sandwich" enzyme immunometric assay (EIMA) for human luteinizing hormone (hLH) was developed. The assay was validated and compared with a liquid phase "two site" immunoradiometric assay (IRMA) for hLH which uses same two antibodies. The sheep anti-hLH IgG, which had been affinity purified by eluting at pH 3.5 from a hLH-sepharose 4 B column, was labelled with 125I. The IRMA is based on simultaneous addition of two antibodies to standards and samples. After overnight incubation, separation was achieved by addition of Sheep anti-Rabbit Fc (SARFc) antiserum. In EIMA; partially denaturated (at pH 2.5) Sheep anti-Rabbit FcIgG (SARFcIgG) coated polystyrene tubes or microtitre plates were employed as solid-phase second antibody. The substrate was N,N'-o-phenylene diamine (2 mg/ml) and H2O2 (O.02%). Two methods, modified NaIO4 and 4-(N-maleimido methyl)-cyclohexane-1-carboxylic acid N-hydroxy succinimide ester (SMCC), were employed in the preparation of sheep anti-hLH IgG-HRP conjugate. The immunoreactivity and peroxidase activity of conjugate prepared with NaIO4 method was impared to various extends. Both EIMA and IRMA had good specificity, were not susceptible to interference from serum components and exhibited very low non-specific binding. The values determined by EIMA were independent of the serum volume employed. Standard added to serum samples was accurately determined and the results obtained from the analysis of serum samples correlated closely with those obtained by IRMA.

Virus neutralization (VN) is an important functional test for evaluating RSV vaccines, also encompassing in mucosal secretion of the respiratory tract considering the infection route. In our previous study, an immunoglobin extraction method described by Bergquist et al. was adopted for RSV ELISA, but it was not suitable for virus neutralization test due to the cell toxicity of the 2% saponin solution used for the antibody extraction. In order to overcome this problem, several solvents including distilled water were tested in the present study for the capacity to extract immunogloblins. Antibodies in the extracts were evaluated and compared by ELISA. Distilled water was as efficient as the 2% saponin solution for extraction of total IgA, RSV specific IgA and IgG. More importantly, the organ extracts obtained subsequently could be used for virus neutralization test without causing adverse effect on the cell culture. Therefore, distilled water was finally chosen as the solvent for immunoglobulin extraction from mucosal organs when both ELISA and virus neutralization test are required.

The proliferative response of human lymphocytes to stimuli such as foreign histocompatibility antigens or mitogens is generally assessed by measuring the amount of tritiated thymidine which the cells incorporated in culture. In this paper, the possibility of assessing lymphocyte proliferation and viability by an empirical assay, using measurement of light absorbance on a ELISA reader in the yellow wave length (450 nm/air-550 nm/air), has been studied. The correlation of these measurements with a colormetric viability assay using MTS/PMS, with tritiated thymidine incorporation and with trypan blue exclusion viability counting, was determined. The results showed that the light absorbance assay correlated well with cell proliferation during 48-120 hours culture period and with cell viability after a 72 hour period. The MTS/PMS colormetric assay as well as trypan blue exclusion cell counting confirmed that the light absorbance assay was not merely caused by dead cells. This data confirm that the light absorbance assay is sufficiently sensitive to low levels of proliferation to allow detection of such responses at least as effectively as thymidine incorporation. The light absorbance assay procedure avoids the expense, time and hazards associated with scintillation counting, and is simple to perform without the necessity for reagents and preparative steps required by other assays.

Induction of interleukin-6 (IL-6) secretion by whole blood cultures (WBC) was used as an in vitro assay system for pyrogen-induced inflammatory reactions. The assay system was very sensitive to Eschericia coli (E coli) endotoxin (< 10 pg/ml). The potential pyrogenic effects of human serum albumin (HSA), Fibronectin (Fn) and stabilised human serum (SHS) solutions were analyzed using this system. None of the products assayed had an effect on the sensitivity of the WBC assay. Spike recovery studies with isolated endotoxin, gram positive and gram negative bacteria showed that none of the products had an effect on the spike recovery of these pyrogenic substances. Good correlations were found between the WBC assay and the rabbit assay for pyrogens for all the production batches tested. When these samples were analysed by the limulus amoebocyte lysate (LAL) assay, the LAL test gave anomalous results for 1 out of the 22 production batches tested. This batch gave a false negative result on the LAL assay and might be indicative of the inability of the LAL assay to detect pyrogens other than endotoxin.

Immunological assays appear to be the only alternative to the microbiological method for analysis of pantothenic acid in foods and blood. In order to evaluate the influence of the linker on the immunogenicity of the hapten, we have tried to raise antisera against pantothenic acid in rabbits using different conjugates. The hapten was coupled to a carrier protein (BSA or thyroglobulin) using adipoyl dichloride (adipoyl conjugate) or bromoacetyl bromide (acetyl conjugate). Only the acetyl conjugate has induced the production of a specific antibody. With this antibody, an assay on microplate using the ELISA inhibition technique was developed to measure pantothenic acid. The use of pantothenic acid coupled to thyroglobulin with adipoyl dichloride as the capture antigen has improved the sensitivity of the ELISA. This assay was applied to food products and blood.