Journal of immunoassay最新文献

The major growth factors in bovine colostrum are transforming growth factor-beta s (TGF-beta 1 and TGF-beta 2) and insulin-like growth factors (IGF-1 and IGF-2). Recently, TGF-beta 2 content of bovine colostrum was measured using a TGF-beta 2 specific ELISA (1) and now we have validated ELISAs for for bovine TGF-beta 1 and IGF-1. The concentrations of IGF-1 and TGF-beta 1 in the first milking after calving were 248-1850 ng/ml and 12.4-42.6 ng/ml, respectively, and they declined in correlation with total protein concentration to 27.0-101 ng/ml (IGF-1) and 0.80-3.49 ng/ml(TGF-beta 1) by the fifth milkings. The amount of TGF-beta 1 was on average 5.3 +/- 1.4% of that of TGF-beta 2 and there is a high correlation (r = 0.966) between the concentrations of these growth factors in the same samples. No free TGF-beta 1 form of could be detected.

Monoclonal nonspecific suppressor factor (MNSF) is a lymphokine product of a murine T cell hybridoma that inhibits the immune response in an antigen nonspecific manner. Recently, we found that a novel ubiquitin-like protein (Ubi-L), a subunit of MNSF, is responsible for its biological activity. We developed a monoclonal antibody with specific activity against Ubi-L. Inhibition experiments showed that this mAb, termed NA4, preferentially recognizes Ubi-L but not irrelevant proteins such as ubiquitin. With the use of NA4, we established an ELISA method for the quantitation of Ubi-L. By this ELISA system, approximately 40 ng/ml of MNSF was detected in the culture supernatants of concanavalin A (Con A)- or interferon gamma (IFN gamma)-activated splenocytes, whereas MNSF in the supernatant of IFN alpha- and IFN beta-stimulated splenocytes was nil. In addition, NA4 could abrogate the action of Ubi-L. Thus NA4 was confirmed to be a pertinent tool for elucidation of the underlying mechanism of action of MNSF.

A clinical study using DR-70 immunoassay for the detection of 13 different cancers have been conducted with 277 healthy subjects and 136 cancer patients. The test results showed that the DR-70 immunoassay kit was capable of detecting cancers with high degree of specificity and sensitivity. At 95% specificity level, the sensitivity of the assay was 87.8%, 92.6%, 65.2% and 66.7%, respectively for lung, stomach, breast and rectum cancers. Furthermore the test kits were shown to be stable and performed reproducibly.

A rapid and reproducible enzyme linked immunosorbent assay (ELISA) was developed for detection of canine coronavirus (CCV) specific antibodies directed to both the nucleocapsid (NC) and the spike (S) proteins. The coating antigen, a methanol-treated, S-protein enriched preparation, was produced by subjecting infected cells to Triton X-114 detergent followed by phase separation. The sensitivity of this assay was determined by following the course of infection in dogs experimentally infected with CCV. The specificity of the antibody response was determined by Western blot analysis and supported the increased magnitude of the ELISA response and the presence of serum neutralizing (SN) antibody. Due to the sensitivity and specificity of the IgG response detected by this assay it can be used to determine both virus exposure and vaccine efficacy.

Polystyrene plates (96 well) were sensitized with Brucella abortus smooth lipopolysaccharide (SLPS) antigen and then air dried at room temperature (RT) for about 1 hour to dry. Dryness was judged complete when a buffer meniscus was absent from the bottom of the well. The plates were resealed kept on the bench at RT for the duration of the study. Testing was done over 13 months by competitive and indirect ELISAs (C- and IELISA) for bovine antibody to B. abortus using panels of sera from B. abortus S19 vaccinated, unexposed and infected cattle. Testing revealed that consistent results were obtained over the test period suggesting that air drying may be a suitable alterative for storage of plates sensitized with some antigens, in particular, smooth lipopolysaccharide.

Transforming growth factor-beta 2 (TGF-beta 2) is the major TGF-beta form in bovine colostrum. A colostrum pool of the five first milkings was made to validate an ELISA specific for human TGF-beta 2 for measure TGF-beta 2 concentration in bovine colostrum samples. According to this test > 90% of total TGF-beta 2 (74.5 +/- 4.4 ng/ml) in colostrum pool was in a latent form that could be activated by acetic acid treatment, whereas the concentration of the active form was only 4.19 +/- 0.27 ng/ml. Activated colostrum samples of the first milkings of five cows contained 150-1150 ng TGF-beta 2/ml and its concentration declined in correlation (r = 0.86) with total protein concentration to 12-71 ng/ml by the fifth milkings. Most of the TGF-beta 2 (94%) was found in the whey fraction of colostrum. The ELISA results were also compared with a TGF-beta 2 bioassay, the fibroblasts migration assay. This assay detected 9.8 +/- 1.0 ng/ml and 4.4 +/- 0.7 ng/ml in the activated and non-activated samples of colostrum pool respectively.

Although various methods for the detection of autoantibodies against glutamic acid decarboxylase (GAD65-AAb) are known, no sensitive method for the quantification of GAD65 as autoantigen is available. We describe a sandwich ELISA based on monoclonal GAD65 antibodies (Mc-GAD65-Ab) of different epitope specificities to quantify GAD65 in pancreatic islets and in different organ/cell extracts and during the preparation of GAD from brain extracts. GAD65 was captured via solid phase coated Mc-GAD65-Ab and detected via a second biotin-labelled Mc-GAD65-Ab recognizing a NH2-terminal epitope of the molecule. The detection limit was estimated to be 0.03 ng GAD65/ml using alkaline phosphatase (AP)-conjugated streptavidin. GAD65 contents in islets of neonatal BB/OK rats and Lewis rats amounted to 37.4 and 43.7 pg/islet, respectively. Furthermore, GAD65 was quantified in brain extracts of pig (55.1 ng/mg protein), mouse (39.5 ng/mg), rat (243.8 ng/mg) and pig cerebellum (514.8 ng/mg) and in different organ extracts of Lewis rat.

A fusion protein between green fluorescent protein (GFP) and neuron-specific enolase (NSE) was expressed in Escherichia coli. The GFP-NSE fusion protein migrated at 62 kDa in SDS-PAGE and retained the fluorescence under non-heating conditions. However, heat-denatured GFP-NSE was non-fluorescent and migrated at 74 kDa corresponding to the theoretical value. This suggests that the special structure of GFP, which is not denatured by SDS, influences its mobility in SDS-PAGE under non-heating conditions. The fluorescence intensity of GFP-NSE was measurable over a wide range by spectrophotometry or densitometry. The competitive immunoassay for NSE was performed using GFP-NSE as labeled antigen. Under our assay conditions, the working range of this system was about 2 -60 ng. This simple and rapid fluorescence immunoassay (FIA) using GFP-tagged antigen may be applicable to many protein markers.

A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r = 0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n = 24) and 14% (n = 9) respectively. Minimal detectable amounts of rGH were 0.6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.

Production of some cytokines, such as IL-4 and IL-10, often occurs at low levels and is difficult to detect by standard ELISA techniques. In many cases the level of detection is at or near to the limits of sensitivity of the assay due either to minimal synthesis and/or cytokine consumption. In an effort to enhance the quantitation of weakly detected cytokines we have developed a unique cell culture-capture ELISA. Lymphocytes are incubated in an anti-cytokine antibody coated ELISA plate for the last 6 hours of a 24 hour in vitro activation period. Use of this cell culture capture method consistently enhanced detection of several T cell cytokines compared to conventional ELISA techniques. Moreover, this technique was found to enhance detection without altering the rate of cytokine secretion which occurred prior to the cell culture capture period. Thus, the cell culture capture ELISA may be useful for detection of a variety of cytokines which are produced at low levels and have traditionally been difficult to quantify.