Journal of immunoassay最新文献

10-Hydroxy-12(Z)-octadecenoic acid (10-OHODA) has an inhibitory effect on the tension of papillary muscles in isolated guinea-pig hearts. To establish an immunoassay for 10-OHODA a mouse monoclonal antibody (MoAb), YM-1, was produced. In order to evaluate the ability of this MoAb to recognize various 10-OHODA analogs including leukotoxin (9, 10-epoxy-12-octadecenoic acid, LTx), a sensitive enzyme-linked immunosorbent assay (ELISA) was developed using the avidin-biotin complex (ABC). The detection limit for 10-OHODA was as low as 0.5 ng in this system. In order to demonstrate the presence of 10-OHODA in living cells, macrophages derived from the human leukemia cell line THP-1 by adding 160nM phorbol 12-myristate 13-acetate (PMA) were exposed to 95% O2, and 5% CO2 for 24 h. 10-OHODA and other fatty acids were extracted from the exposed macrophages with diethylether after phospholipase A2 treatment. The 10-OHODA content was determined using the new ELISA, and 18.5 ng 10-OHODA was detected in the macrophages exposed to the high oxygen concentration (1 x 10(6) cells).

Latex agglutination assay based on monoclonal antibodies (MCAs) described in this communication may be useful for detection of Pebrine infection in silkworm. Four murine MCAs were produced against Nosema bombycis spore. In ELISA all 4 MCAs (IgM isotype) reacted with alkali treated Nosema spores and to variable extent with acetone precipitated surface protein. However, MA-310 and MA-542 showed a low degree of cross reactivity with BmNPV. In contrast, MA-503 and MA-515 were devoid of reactivity with BmNPV, B. thuringiensis, S. marcescens, Azotobactor, Rhizobium and normal hemolymph protein in ELISA. Latex beads sensitized with a combination of MA-503 and MA-515 (50 micrograms each per ml of 0.4% latex beads) could detect 1 x 10(5) Nosema spores per test. Sensitization of the latex beads with the cocktail of these two MCAs through protein-A bridge further led to a 10-fold increase in the sensitivity (1 x 10(4) spores/test) of the assay. No agglutination was observed in presence of BmNPV, Rhizobium, Azotobactor, E. coli, B. thuringiensis, S. marcescens and normal hemolymph protein indicating the specificity of the test. The results obtained by latex agglutination assay on hemolymph samples of infected as well as normal larvae collected from field, II instar larvae infected in the laboratory and from infected mother moth revealed 100% correlation with results by microscopic examination.

A polyclonal bispecific (bifunctional) antibody was prepared to develop a hemagglutination immunoassay for beta-microseminoprotein (beta-MSP), a predominant seminal protein. Three types of F(ab')2 fragments of rabbit IgG, affinity-purified anti-human red blood cell (RBC) F(ab')2 nonaffinity-purified anti-beta-MSP F(ab')2 and nonspecific (nonimmunized) F(ab')2, were mixed to obtain a F(ab')2 mixture containing 10% anti-RBC molecules and 10% anti-beta-MSP molecules. Fab' was obtained from the F(ab')2 mixture, and then reacted with maleimide-activated bovine serum albumin (BSA) at a molar ratio of 10:1. As estimated by the decrease in the maleimide content, approximately 7 Fab' molecules were introduced per one BSA molecule. The bispecific (anti-beta-MSP and anti-RBC) Fab'-BSA conjugate thus prepared was incubated successively with a human RBC suspension and with samples. In the presence of beta-MSP, RBCs become agglutinated, providing a test simple for forensic semen identification.

A fluoroimmunoassay has been developed to measure serum levels of albumin in sheep. It employs ovine albumin labelled with fluorescein as the tracer and a rabbit antiserum raised against ovine albumin. Separation of the antibody bound and free fractions is achieved using a second antiserum directed against the Fc of rabbit immunoglobulin G and, to simplify the assay, the two antisera are premixed prior to use. Assay validation parameters are satisfactory and the reagents are predicted to be stable for at least one year at 4 degrees C. In contrast to the bromocresol green method, the assay is unaffected by immunoglobulins. A reference range for serum albumin levels has been established in lambs, normal ewes and ewes undergoing immunisation. Mean serum levels were 38.8, 51.3 and 37.8 g/l respectively. The sensitivity of the assay also enabled its use to monitor albumin levels at various stages during the production of specific antibody fragments from ovine antisera for therapeutic purposes.

In order to achieve batch-to-batch consistency of whole-cell pertussis vaccines, properties relevant for protection and safety should be characterised. Therefore, ELISAs to quantify pertussis toxin (PT), filamentous haemagglutinin (FHA), 92 kD outer membrane protein (92 kD-OMP) and pertactin (PRN) in Bordetella pertussis (B. pertussis) suspensions were developed. In this paper the influence of the bacterial growth stage on antigen production and antigen release into the supernatant was studied for pertussis strains 134, 509 and CS. The levels of cell-associated and free antigens during growth were strongly strain and antigen dependent. Because of this, the proportion of cell-associated antigens changed during cultivation for all three strains. Substantial amounts of PT and PRN were released into the supernatant, while little free FHA and 92 kD-OMP were found. The amount of cell-associated FHA declined rapidly during growth, whereas cell-associated 92 kD-OMP contents increased. These findings demonstrate that, although antigen exposure and release differ from strain to strain, the main factor that determines the antigen production and release is the growth phase.

A competitive monoclonal antibody-based immunoassay which quantifies a hydrophobic hapten (Rx) in water immiscible solvents, obviating the need of a pre-extraction step, has been developed. Approximately linear dose response profiles of analyte, over the range 1-20 ugml-1 in the hydrophobic solvents, hexane, toluene and xylene were obtained. UV spectrophotometric analyses of Rx dosed hexane confirm the phenomenon of antibody-mediated transfer of analyte from the organic to the aqueous milieu. Preliminary data on the effect of water immiscible solvents on the immunoreactivity of a monoclonal antibody in free solution are presented. The potential industrial applications of water immiscible solvent based immunoassays are discussed.

An immunoradiometric assay (IRMA) of human thyrotropin (hTSH), based on magnetic solid phase separation, was studied especially in terms of its nonspecific bindings (B0) which were identified as a product of the interaction between an altered form of radioiodinated anti-hTSH monoclonal antibody (125I-mAB) and the uncoupled magnetizable cellulose particle (matrix). Preincubation with the same matrix, solid phase saturation with milk proteins, tracer storage at 4 degrees C and serum addition during incubation were found to be particularly effective in preventing their formation. These findings were used to reproducibly decrease nonspecific bindings to values < 0.1% (or < 70 cpm), thus increasing the signal-to-noise ratio (B60/B0) up to values of 300-500. This way hTSH radioassays were obtained with functional sensitivities of about 0.05 mIU/L and analytical sensitivities of the order of 0.02 mIU/L. Such sensitivities, and, more importantly, a general improvement in assay performance, were obtained in a highly reproducible manner and all over the useful tracer life.

We have developed a simple, direct radioimmunoassay for progesterone in saliva. The correlation coefficient (r) between the direct assay and an extraction procedure was 0.92 (n = 65, P < 0.001), and the correlation between concurrent serum and salivary progesterone concentrations in the luteal phases of menstrual cycles of 48 women was 0.75 (P < 0.001). Whereas certain polystyrene and polyethylene vials and tubes were found to bind and remove up to 87% of the progesterone from saliva, other plastic and glass surfaces were satisfactory for the procedure. Intraassay and interassay CVs from values greater than 300 pmol/L were 12.0 and 12.4%, respectively. The assay sensitivity was 48 pmol/L. Collection of saliva is a more convenient and less invasive technique for frequent sample collection than phlebotomy, and is useful for monitoring ovulation and assessment of luteal function in women clinically.

We describe the application of an enzyme immunoassay (EIA) for detecting bacteria bound to a solid surface. Different Yersinia enterocolitica and Escherichia coli strains, expressing the YadA protein, type 1 or type P fimbriae were used as models for this study. The assay was used to detect bacteria bound to fixed tissues or to glass slides coated with extracellular matrix molecules (collagen, laminin or fibronectin). E. coli specific antiserum (B357, Dakopatts, Glostrup, Denmark) and peroxidase conjugated antiserum (P217) were used to detect all E. coli strains used in the study. The bacterial binding could be monitored with a linear detection range between 10(5) and 10(8) bacteria. Most importantly, dose dependent inhibition of bacterial binding by soluble extracellular matrix molecules could be measured.

Recombinant bovine leukemia virus receptor, BLVRcp1, possessed the unusual property of binding plastic plates after blocking nonspecific binding sites. Adhesiveness of BLVRcp1 to blocked plates hindered development of an antigen capture and receptor binding assay with this protein. Unexpectedly, non-specific adsorption of BLVRcp1 was dramatically influenced by temperature. Optimizing incubation temperature and antigen capture at 4 degrees C instead of 37 degrees C and the use of milk as blocking solution removed nonspecific binding of BLVRcp1 allowing development of a functional immunoassay. Thus, the temperature used for antigen capture can be a critical factor that influences performance of the immunoassay.