A highly sensitive microtitre plate enzyme immunoassay for oestradiol-17 beta

A specific and sensitive solid-phase microtitre plate enzyme-linked immunosorbent assay for oestradiol-17 beta (E2) is described. After coating with an IgG anti-E2 fraction, we used E2-6-carboxymethyl-oxime-beta-galactosidase in a competitive binding assay and revealed the bound activity with a fluorogenic substrate. Two methods for the competitive binding assay were tested: (1) a classical one (method A) defined as a 'two-step competition' because the E2 sample was first incubated alone, and then E2-beta-galactosidase conjugate was added; (2) and a new one (method B) also performed in two steps but in which the E2 sample was evaporated to dryness. The detection limit of method A was 100 pg/ml (9 pg/well). Method B was more sensitive since 1 pg/ml (0.3 pg/well) was statistically different from 0 pg/ml. Specificity was equivalent with both methods while precision was better in B. Thus, this new method may be able to measure very low levels of oestradiol-17 beta in, for example, the blood of domestic mammals.     

Calcitonin determination by a fast and highly sensitive enzyme amplified immunoassay

A colorimetric enzyme amplification system was used to develop an immunoassay for human calcitonin (hCT) with a sensitivity of 6 pmol/l, and intra- and inter-assay CVs of 12% and 11.8% respectively for the low pool, and 10% and 11.2% for the high pool. The mean recovery of added synthetic hCT (58.5 pmol) from the plasma of 10 patients was 110% (64.4 pmol). The correlation coefficient between radioimmunoassay (RIA) and amplified enzymo-immunoassay was found to be 0.96 (p 0.001). The assay was successfully applied to the measurement of elevated calcitonin levels in plasma from patients with medullary carcinoma of the thyroid (MCT). AEIA offered a reliable and sensitive alternative to RIA for calcitonin determination with the added advantage of convenience as the label employed was much more stable.     

A time-resolved fluorescence immunoassay (DELFIA) increases the sensitivity of antigen-driven cytokine detection

In an effort to improve the quantification of the low levels of cytokines released in response to antigenic stimulation of T cells, a sandwich dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) was developed and compared to a standard sandwich ELISA. The DELFIA enhanced the sensitivity of a mouse IL-2 assay 8- to 27-fold, and a human GM-CSF assay 10-fold, as compared to colorimetric ELISA. The increase in sensitivity allows for the use of lower sample volumes per well, and the ability to run more assays per supernatant sample. This sensitive, nonisotopic alternative to other cytokine detection methods will be useful for those researchers wanting to quantitate low levels of antigen-driven cytokine production.     

A new method for fluorescence immunoassay using plane surface solid phases (FIAPS).

A method distinguished by high sensitivity, low non-specific binding, easy handling, and broad applicability with respect to various antigens is described. Films of polymethyl-methacrylate with plane surfaces were selected as solid phase for adhesive or covalent binding of different antigens (DNA, histone, human, rabbit or goat immunoglobulins). Proteins were covalently bound to the films by the azide method (Orth and Brummer, 1972). Polymethylmethacrylate films thus coated had a negligible autofluorescence and gave minimal non-specific binding of protein. Coated films were used for specificity control of FITC-labeled antibody preparations and in the double antibody and sandwich techniques for detection of antibodies or antigens in sera from man, rabbit and goat. FITC-conjugated hyperimmune antibody, in some cases purified by immunoadsorption was used as second antibody in indirect techniques. The amount of fluorescent-labeled antibody bound per unit of surface area of film was measured by incident light with a Zeiss-Axiomat fluorescence microscope equipped for fluorescence photometry and an uranyl acetate glass plate was used as a standard. The technique appears superior to present methods of quantitative immunofluorescence analysis.     

A sensitive immunoassay for von Willebrand factor

We have developed an ELISA specific for canine von Willebrand factor antigen (vWF:Ag) that also strongly reacts with the VWF:Ag of humans and many other vertebrates. This assay was designed to avoid the use of immunoreagents of human origin, however, commercially available antibodies to human vWF:Ag may also be used. von Willebrand factor (vWF) was quantitated using a modified double-sandwich ELISA with polyclonal antibodies specific for canine vWF:Ag. The assay was as sensitive for measuring canine vWF:Ag as previously published immuno-radiometric assays and the most sensitive ELISA for human vWF:Ag. Employing commercially available antibodies to human vWF:Ag in the same double-sandwich configuration, the lower limit of detection for human vWF:Ag was 4.8 x 10(-6) units/ml, lower by a factor of ten than previously reported ELISAs. In addition, a wide range of vWF:Ag levels can be determined with just a single plasma dilution. The assay readily distinguishes type III von Willebrand disease from other types of von Willebrand disease having very low levels of vWF. This vWF ELISA can be used to evaluate large numbers of plasma samples simultaneously and is therefore well-suited for large-scale screening programs.     

A sensitive sandwich-enzyme immunoassay for human endothelin

A sensitive enzyme immunoassay (EIA) for human endothelin(1-21) has been established. The assay is based on a sandwich method that uses two differing capture and detection anti-endothelin antibodies. A monoclonal anti-endothelin antibody AwETN40, which did not react with an endothelin C-terminal heptapeptide, was used as an immobilized antibody. The Fab' fragment of rabbit antibodies against the endothelin C-terminal heptapeptide was used as an enzyme-labeled detector antibody after being coupled with horseradish peroxidase (HRP). The assay is sensitive enough to detect as little as 0.2 pg/well (80 amol/well) of endothelin. Preliminary investigations indicated that the basal level of immunoreactive endothelin in male plasma (n = 24) extracted with Seppak C-18 cartridges was 1.59 +/- 0.32 pg/ml.     

A sensitive enzyme immunoassay for atrial natriuretic polypeptide

A sensitive sandwich enzyme immunoassay for mammalian alpha-atrial natriuretic polypeptide (alpha-ANP) has been developed using Fab' fragments of affinity purified rabbit anti-alpha-hANP[6-28] polyclonal antibodies (R3) conjugated to horseradish peroxidase. Immunoplates were coated with (F(ab')2) fragments of anti-alpha-hANP monoclonal antibodies (HA53). Without using a pre-concentration step the detection limit of alpha-hANP in plasma was 5 pg/ml. The assay was sufficient accuracy and reproducibility for general use of, i.e., the coefficients of within-assay and between-assay variation were 6.4-10.7% and 9.7-15.3%, respectively. Using the assay, the basal plasma alpha-hANP level of healthy persons was 15.3 +/- 8.3 pg/ml (mean +/- SD, n = 40).     

A sensitive time-resolved fluorescent immunoassay for metallothionein protein

Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins induced by a broad range of stress conditions, including exposure to transition metal ions. Biochemical and immunological methods to measure MT protein levels in tissues and cultured cells have been reported, but accuracy and sensitivity is impeded by high background levels, low specificity of currently available reagents, and relatively laborious and time-consuming multistep procedures. To address these difficulties, a protocol has been developed to measure MT protein levels using a competitive solid phase assay based on dissociation enhanced lanthanide fluoroimmuno (DELFIA) detection of anti-MT monoclonal antibody bound to solid phase MT. This assay allows time-resolved detection of antibody binding, based on binding and exchange of different lanthanide chelates followed by fluorescent detection, designed to reduce background fluorescence and increase sensitivity. The method allows measurement of low MT levels that are undetectable using current radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) protocols, and yields reproducible results with low background over a wide range of MT concentrations. Improved sensitivity of MT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function.     

A rapid and highly sensitive solid-phase enzyme immunoassay specific for human fibronectin using a characterized monoclonal antibody

A solid-phase enzyme immunoassay (EIA) was developed for the quantitation of human fibronectin in body fluids and cell culture media. In the assay a human fibronectin-specific murine monoclonal IgG1 (f-33) was used as capture antibody and polyclonal rabbit anti-fibronectin as detector antibody. The antibody showed no reactivity to purified monkey, dog, rabbit, horse, sheep, mouse, bovine or chicken fibronectins. The determinant of the monoclonal antibody was mapped to the cell-binding region of the fibronectin molecule. This localization was based on the use of purified fragments of fibronectin, immunoblotting, EIA and inhibition of fibroblast adhesion and spreading by the antibody. The detection limit of the fibronectin assay was 2 ng/ml. The assay was used for the quantitation of fibronectin in human plasma, urine and cerebrospinal fluid specimens and culture media of human cells.     

Detection of rubella virus antigen by one-step time-resolved fluoroimmunoassay and by enzyme immunoassay

A one-step time-resolved fluoroimmunoassay (TR-FIA) and a conventional two-step enzyme immunoassay (EIA) for the detection of rubella virus antigen were developed. Two noncompetitive mouse monoclonal antibodies reactive with epitopes on the E1 polypeptide of rubella virus served as immunoreagents. One of the monoclones (7A6) was used for coating the solid phase, and the other (2C3) was labeled with either Europium chelate or with horseradish peroxidase. For TR-FIA, the specimen was incubated simultaneously with the label at 4 degrees C overnight. EIA required an overnight incubation with the specimen and after washing another 1 hr of incubation at 37 degrees C with the conjugate. The sensitivity of TR-FIA was 10 pg in an assay volume of 100 microliters, and the sensitivity of EIA was between 50 and 100 pg. Antigens could be detected by TR-FIA in supernatant of cultures of Vero cells 48 hr after inoculation with approximately 1 TCID50, while cytopathogenic effect (CPE) at that time was detected only in cultures inoculated with 10(5) TCID50 or more. Virus mixed with human amniotic fluid containing antirubella-specific IgG was detectable after an incubation at 37 degrees C for 5 days. The assays may find applications in prenatal diagnosis of intrauterine rubella infection, in early identification of viral antigens in cell culture and in monitoring production, concentration, and purification of rubella antigen for antibody assays.     

A sensitive chemiluminescence based immunoassay for antibody to staphylococcal peptidoglycan

A sensitive chemiluminescence based immunoassay is described for measuring antibody to staphylococcal peptidoglycan in blood and dialysates from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Peptidoglycan was isolated from a strain of S. epidermidis obtained from the dialysate of a CAPD patient with peritonitis and after sonication used to coat polystyrene beads. The coated beads were incubated with standard or sample and bound IgG was detected by the addition of affinity-purified goat anti-human IgG labelled with acridinium ester. After a wash stage 0.1 M nitric acid containing 0.1% hydrogen peroxide was added to the beads. Subsequently the chemiluminescence produced following the addition of 0.3 M sodium hydroxide was measured over a 2 s time interval with an automatic luminescence analyser. Using this technique the optimum dilution of serum for detecting antibodies to peptidoglycan was found to be 1/800 and for overnight effluent from CAPD patients the dilution was 1/8. Initial values of serum and dialysate antibody levels from 34 subjects are presented. This method has the advantage that it will detect concentrations of anti-peptidoglycan which are less than 1% of those in sera, the reagents remain stable for long periods and large numbers of samples can be processed on the same day.     

A sensitive reverse passive haemagglutination immunoassay for hepatic caeruloplasmin.

A reverse passive haemagglutination assay for the measurement of caeruloplasmin in homogenates and organelle fractions prepared from needle biopsies of human liver tissue is described. Use of serial dilutions over a narrow range of concentrations permits accurate quantitation of caeruloplasmin down to 10 ng/ml, sufficient to detect the protein in needle biopsies with as little as 5 mg wet weight of tissue. Applied to the measurement of caeruloplasmin in human sera, the assay gives values which correlate well with those obtained by the standard radial immunodiffusion technique.     

A highly sensitive immuno-PCR assay for detecting Group A Streptococcus

A highly sensitive hybrid assay, based on immuno polymerase chain reaction (immuno-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques, was developed for the detection of pathogenic Group A Streptococcus (Strep A). Cells were disrupted by sonication and then coated onto the walls of Maxisorp microtiter plates. Next, biotinylated anti-Group A monoclonal antibody (mAb) was bound to the antigen and then linked, via a streptavidin (STV) bridge, to biotinylated reporter DNA. After extensive washing, the denatured reporter DNA was transferred to PCR tubes, amplified, electrophoresed, and used as the signal for detection of bacteria. The minimum detection limit of this assay is the equivalent of approximately one one-thousandth of a Streptococcus pyogenes cell, even in the presence of 100,000 Escherichia coli cells. The combination of multiple antigens per cell and PCR amplification provides the extreme sensitivity in this immuno-PCR assay. No cross-reaction was found with other Streptococcus species. We also directly linked the anti-Group A monoclonal antibody to DNA using succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The sensitivity using directly linked antibody-reporter DNA was approximately 10 cells. Because this assay could be adapted for detection of many different bacteria in a variety of sample types, we tested the potential for interference from substances that could be present in clinical, food, and environmental samples. Sonicated meat or human plasma did not inhibit detection; however, extracts of concentrated soil samples were somewhat inhibitory. This highly specific, sensitive, and robust assay could be applied to clinical detection of Group A Streptococcus and serves as a model for other immuno-PCR assays.     

A highly sensitive enzyme-linked immunosorbent assay for idiotype-bearing antibodies

A sensitive and specific immunoenzyme assay (ELISA) for quantitation of total and cross-reactive idiotype-bearing (CRI) anti-ABA antibodies is described. Total anti-ABA antibodies are directly assessed in ABA-BGG coated polyvinyl wells with enzyme-labelled rabbit anti-mouse immunoglobulins. By interpolation on a standard curve absorbance values give the concentration of anti-ABA antibodies with a sensitivity of 30 ng/ml. CRI+ antibodies are quantitated by inhibition of enzyme-labelled monoclonal CRI+ antibody binding to solid-phase coated rabbit anti-CRI immunoglobulins. The concentration of CRI+ antibodies, evaluated by interpolation on a standard inhibition curve, can be measured at the level of 10 ng/ml. This highly sensitive, rapid, specific and reproducible assay is easily used, with minor modifications, to detect specific antibodies in any idiotype system.     

A highly sensitive quantitative immunochromatography assay for antigen-specific IgE

We have developed a highly sensitive quantitative enzyme immunochromatography system for antigen-specific IgE, which is clinically important for the diagnosis of allergic diseases. The system uses alkaline phosphatase-labeled anti-IgE antibody (ALP-anti-IgE) and immobilized target antigen, for example cedar pollen antigen, on a membrane. Antigen-specific IgE present in the serum binds the immobilized antigen after complexing with the ALP-anti-IgE. Subsequently, the enzyme substrate migrates to the complex on the antigen line, which is stained blue. The intensity of the staining was analyzed by a quantitative detector, but can also be assessed directly by the naked eye. This system was able to detect 0.2 U/ml of IgE specific for Japanese cedar pollen. The results correlated well (Spearman's rank correlation coefficient, 0.95) to those in AlaSTAT system, which is a reliable enzyme-linked immunosorbent assay (ELISA) method. This antigen-specific IgE assay system is suitable for point-of-care testing.     

一种高度敏感的改良的IL—1检测方法

采用CTLL-2检测IL-1诱导小鼠胸腺瘤细胞系EL_4产生IL-2活性,建立了一种间接检测IL-1的方法。通过对多种影响因素进行探讨,选择了较适的诱导血清浓度,细胞浓度,诱导时间和转移诱导上清稀释度,从而使检测IL-1的敏感性达10~(-3)～10~(-4)U/ml(2.5～25×10~(-4)Pg/ml,约为小鼠胸腺细胞检测方法的10~2～10~3倍),整个检测流程可在40小时内完成。若用CTLL-2和EL_4细胞同时培养的一步法检测,敏感性约为10~(-1)U/ml,但30小时内可完成检测。此法不受高浓度rHuTNF-α、rHuTNF-β、rHuIL-6干扰,IL-2、ConA、PHA、LPS和SEB对检测系统只有较弱的影响,但A23187可明显地干扰检测系统。因此,本方法可用于基础和临床研究过程中不同来源的IL-1生物学活性检测。     

Sandwich immunoassay of small molecules. II. Effects of heterology and development of a highly sensitive and specific enzyme immunoassay for testosterone.

The effect of dimer heterology in the sandwich immunoassay of testosterone was studied using symmetrical and asymmetrical dimers prepared from testosterone-3-(O-carboxymethyl)oxime and 4-(carboxymethyl-mercapto)testosterone. The effect of antibody heterology was studied using antibodies against 3-carboxymethyl oxime and 17-hemisuccinate derivatives of the steroid and an asymmetrical dimer prepared from the same two derivatives. Using antibody against the 3-carboxymethyl oxime and the dimer of 4-(carboxymethylmercapto)testosterone, the sensitivity of direct enzyme immunoassay of testosterone in serum by this method was 0.5 pg/well and the cross-reactivity of 5 alpha-dihydrotestosterone was 5%.