Detection of biotinylated DNA probes by using Eu-labeled streptavidin and time-resolved fluorometry

Europium has been used as a label in immunoassays as it can be measured with high sensitivity by means of time-resolved fluorometry. Here we have used streptavidin labeled with europium chelates in the detection of adenovirus type 2 DNA bound to microtiter wells after hybridization with a biotinylated probe. The method gave quantitative results and a sensitivity of about 10 pg of the specific DNA.     

Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased. We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed. A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix. The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehyde-fixed, wax-embedded specimens.     

High specific activity chemiluminescent and fluorescent markers: Their potential application to high sensitivity and ‘multi-analyte’ immunoassays

The sensitivities of immunoassays relying on conventional radioisotopic labels (i.e. radioimmunoassay (RIA) and immunoradiometric assay (IRMA)) permit the measurement of analyte concentrations above ca 10⁷ molecules/ml. This limitation primarily derives, in the case of ‘competitive’ or ‘limited reagent’ assays, from the manipulation errors arising in the system combined with the physicochemical characteristics of the particular antibody used; however, in the case of ‘non-competitive’ systems, the specific activity of the label may play a more important constraining role. It is theoretically demonstrable that the development of assay techniques yielding detection limits significantly lower than 10⁷ molecules/ml depends on:

• 1 the adoption of ‘non-competitive’ assays designs;
• 2 the use of labels of higher specific activity than radioisotopes;
• 3 highly efficient discrimination between the products of the immunological reactions involved.

Chemiluminescent and fluorescent substances are capable of yielding higher specific activities than commonly used radioisotopes when used as direct reagent labels in this context, and both thus provide a basis for the development of ‘ultra-sensitive’, non-competitive, immunoassay methodologies. Enzymes catalysing chemiluminescent reactions or yielding fluorescent reaction products can likewise be used as labels yielding high effective specific activities and hence enhanced assay sensitivities. A particular advantage of fluorescent labels (albeit one not necessarily confined to them) lies in the possibility they offer of revealing immunological reactions localized in ‘microspots’ distributed on an inert solid support. This opens the way to the development of an entirely new generatio of ‘ambient analyte’ microspot immunoassays perrnitting the simultaneous measurement of tens or even hundreds of different analytes in the same small sample, using (for example) laser scanning techniques. Early experience suggests that microspot assays with sensitivities surpassing that of isotopically based methodologies can readily be developed.     

Performance of fluorescent europium(III) nanoparticles and colloidal gold reporters in lateral flow bioaffinity assay

Lateral flow (LF) immunoassays (i.e., immunochromatographic assays) have traditionally been applied to analytes that do not require very high analytical sensitivity or quantitative results. The selection of potential analytes is often limited by the performance characteristics of the assay technology. Analytes with more demanding sensitivity requirements call for reporter systems enabling high analytical sensitivity. In this study, we systematically compared the performance of fluorescent europium(III) [Eu(III)] chelate dyed polystyrene nanoparticles and colloidal gold particles in lateral flow assays. The effect of time-resolved measurement mode was also studied. Because binder molecules used in immunoassays might not behave similarly when conjugated to different reporter particles, two model assays were constructed to provide reliable technical comparison of the two reporter systems. The comparative experiment demonstrated that the fluorescent nanoparticles yielded 7- and 300-fold better sensitivity compared with colloidal gold in the two test systems, respectively. Although the two reporter particles may induce variable effects using individual binders, overall the high specific activity of Eu(III) nanoparticles has superior potential over colloidal gold particles for the development of robust high-sensitivity bioaffinity assays.     

High sensitivity immunoassays using particulate fluorescent labels.

The use of polystyrene fluorescent microspheres as sensitive labels in direct-detection (not enzymatically amplified) heterogeneous equilibrium "sandwich" immunoassays in 96-well plates is described. With mouse IgG as a model antigen, a fluorescent particulate label is more sensitive than a corresponding soluble reporter. The limit of detection of mouse IgG in the multiparametrically optimized assay was 0.2 ng/ml (7.6 x 10(8) antigens/ml) for the particulate reporter and 50 ng/ml (1.9 x 10(11) antigens/ml) for the soluble reporter. The sensitivities of assays using the particulate label were dependent on the surface densities of the capture and reporter antibodies and the concentration of reporter beads. Sensitivity was improved by adding the preformed reporter antibody/fluorescent microsphere complex to trapped antigen on the well surfaces instead of sequentially adding the reporter antibody and then the fluorescent microspheres. Maximal (equilibrium) binding of the particulate reporter to captured antigen occurred after 20 h with a concentration of 1.4 x 10(9) reporter beads/ml. Thus, particulate fluorescent labels provide high sensitivity in direct-detection immunoassays.     

Time-resolved Fluorescence in Immunocytochemical Detection of Prostate-specific Antigen in Prostatic Tissue Sections

Chelates with fluorescent lanthanides such as europium and terbium are widely used in immunofluorometric assays, e.g. for the measurement of different molecular forms of prostate-specific antigen (PSA) in serum for detection and monitoring of prostate cancer. These chelates have also been introduced as non-radioactive labels in immunocytochemistry and in situ hybridization. In the present study, sections of non-malignant prostate were investigated using monoclonal IgGs against PSA. Detection of specific immunostaining employing time-resolved fluorescence with europium-labeled streptavidin was compared with conventional detection by streptavidin conjugated to horse-radish peroxidase. The high PSA concentration in the tissue produced high intensity, specific time-resolved fluorescence signals in the epithelial cells of the prostate gland without disturbance from non-specific tissue autofluorescense. This allowed short exposure times to be used which resulted in insignificant photobleaching. Two of the three europium-chelates evaluated yielded high signal intensities. Counterstaining was found to be optimal with Gill No. 1-Haematoxylin solution and Merckoglas was the best mounting medium for the europium chelates tested. In conclusion, time-resolved fluorescence imaging is an attractive alternative to conventional detection of streptavidin conjugated to horse-radish peroxidase, as it provides linear, high intensity, specific signals subsequent to the decay of non-specific tissue autofluorescence.     

Deaminating enzyme labels for potentiometric enzyme immunoassay

Adenosine deaminase, asparaginase, and urease are examined as possible enzyme labels for immunoassays using potentiometric detection with the ammonia gas-sensing membrane electrode. Considerations of binding ability, retained activity, and stability reveal asparaginase to be the most effective enzyme label for immunoassay purposes. The utility of the potentiometric approach with this enzyme label is demonstrated via model hapten assays for dinitrophenyl groups and for cortisol.     

Enzymes as versatile labels and signal amplifiers for monitoring immunochemical reactions

Summary Enzymes have proven to be sensitive and versatile labels for immunochemical assays. The sensitivity of an enzyme label stems from its extraordinary catalytic power which in turn provides a great amplification of signals. Its versatility, however, stems from the fact that enzyme activity can be modulated by a number of substances.Enzyme labeled immunoassays can be divided into two categories: (a) heterogeneous and (b) homogeneous (non-separation). In the heterogeneous systems, the quantitation of the antibody bound and unbound fractions requires a physical separation of these two fractions, whereas the homogeneous or non-separation systems do not require such a separation. In the homogeneous systems, the unbound and antibody bound fractions can be distinguished functionally.A total of 11 unique principles used in the development of enzyme labeled immunoassays are described. The advantages, disadvantages and limitations of them are considered, as well as the future paths for research and developments.     

Europium(III) cryptate: a fluorescent label for the detection of DNA hybrids on solid support.

We report here a new detection method for DNA hybrids on dot blots. The process utilizes DNA or oligonucleotide probes labeled with biotin, followed by recognition with a conjugate of streptavidin and europium cryptate, a time-resolved fluorescent label. Unlike the other lanthanide chelates, this label is an organic molecule embedding a europium ion into an intramolecular cavity. This structure has a better stability in diluted assay media, a good sensitivity even on solid support, and an elevated fluorescence lifetime which allows elimination of most of the background generated by other species present in the assay medium. This procedure is quantitative and detects down to 2 amol of a model DNA, which is similar to other nonisotopic (especially colorimetric) methods. The main advantages of this method are easy automation, quantitation, and rapidity of measurements.     

Application of a long-term enhanced xanthine oxidase-induced luminescence in solid-phase immunoassays

The use of xanthine oxidase (XO) as a label in immunoanalysis has not been previously reported. This can be explained by the difficulties encountered in XO assays (poor sensitivity and versatility) and the competitive inhibition of the enzyme by allopurinol, a widely used hypouricemic agent. We demonstrate here that both difficulties can be circumvented. (i) The XO-dependent luminescent signal related to the oxidation of luminol is dramatically enhanced in the presence of iron-EDTA complex and sodium perborate in alkaline buffer. The mechanism of this enhancement is consistent with an O2-driven Fenton reaction, leading to the production of highly reactive OH radical. (ii) Residual inhibition of solid-phase bound XO by serum allopurinol and its metabolites is spontaneously reversible and can be prevented by the presence of folic acid or azahypoxanthine in the incubation buffer. With these two problems solved, XO can be classified as a choice label in immunoanalysis with the following properties: (i) high detection sensitivity (3 amol label), (ii) long-term luminescent signal (several days), (iii) versatile preparation and stability of conjugates, and (iv) long-term stability of the luminescence reagent. As an example of application, some data concerning total IgE and direct 17 beta-estradiol assays are described. Several other luminescent immunoassays of large and small molecules have been developed using XO conjugates as tracer (free and total T4, ultrasensitive thyroid stimulating hormone, CA 19.9, prolactin, hCG, specific IgE, anti-toxoplasma, and anti-chlamydia IgG), thus proving that XO can be classified as a universal label.     

Labeling of steroids on solid phase

Up to four tetra-tert-butyl-1-[4-aminoacetamido)benzyl]diethylenetriaminetetrakis(acetato) derivatives of Fmoc glutamic acid (1) were attached to two steroids (17alpha-hydroxyprogesterone-3-O-carboxymethyloxime 2 and 1,3,5(10)-estratriene-3,16alpha,17beta-triol-6-one-6-O-carboxymethyloxime, 3)) on solid phase using an oligopeptide synthesizer. Upon deprotection and conversion to the corresponding europium(III) chelates, these steroid conjugates were used in DELFIA-based competitive fluoroimmunoassays. The more chelates conjugated to 17-alpha-hydroxyprogesterone, the more diluted antiserum could be used in an immunoassay for 17-alpha-hydroxyprogesterone, without any alteration of the measurement range. Hence, 17-alpha-hydroxyprogesterone tracers with several chelates are useful when a high serum dilution factor is desired i.e., when only a limited quantity of antiserum is available. The result demonstrates the suitability and usefulness of lanthanide(III) chelates as multilabels in bioaffinity assays.     

Variable denaturation of ovalbumin by incorporation of amino acid analogs

A novel firefly luciferin- enhanced luminescent procedure for the quantitation of horseradish peroxidase labels has been directly incorporated into established enzyme immunoassays. The procedure is rapid and sensitive and uses readily available reagents. Light emission from the enhanced reaction is high and relatively constant and thus easily measured. The luminescence procedure has been successfully incorporated into immunometric assays for rubella antibody and human IgE and into a competitive immunoassay for digoxin.     

An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label

A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml(-1) to 27.25 microg ml(-1) in a logarithmic plot. A detection limit as low as 1 ng ml(-1) (i.e., 6 x 10(-12) M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of 5 x 10(-9) M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody.     

Multiple labeling of antibodies with dye/DNA conjugate for sensitivity improvement in fluorescence immunoassay

Fluorescence immunoassays are widely used in life science research, medical diagnostics, and environmental monitoring due to the intrinsically high specificity, simplicity, and versatility of immunoassays, as well as the availability of a large variety of fluorescent labeling molecules. However, the sensitivity needs to be improved to meet the ever-increasing demand in the new proteomics era. Here, we report a simple method of attaching multiple fluorescent labels on an antibody with a dye/DNA conjugate to increase the immunoassay sensitivity. In the work, mouse IgG adsorbed on the surface of a 96-well plate was detected by its immunoreaction with biotinylated goat anti-mouse antibody. A 30 base pair double-stranded oligonucleotide terminated with biotin was attached to the antibody through the biotin/streptavidin/biotin interaction. Multiple labeling of the antibody was achieved after a fluorescent DNA probe was added into the solution and bound to the oligonucleotide at high ratios. By comparison with fluorescein-labeled streptavidin, the assay with the dye/DNA label produced up to 10-fold increase in fluorescence intensity, and consequently about 10-fold lower detection limit. The multiple labeling method uses readily available reagents, and is simple to implement. Further sensitivity improvement can be obtained by using longer DNAs for antibody labeling, which can incorporate more fluorescent dyes on each DNA.     

Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.     

Application of europium(III) chelate-dyed nanoparticle labels in a competitive atrazine fluoroimmunoassay on an ITO waveguide

We have demonstrated the use of an optical indium tin oxide (ITO) (quartz) waveguide as a new platform for immunosensors with fluorescent europium(III) chelate nanoparticle labels (Seradyn) in a competitive atrazine immunoassay. ITO as a solid surface facilitated the successful use of particulate labels in a competitive assay format. The limit of detection in the new nanoparticle assay was similar to a conventional ELISA. The effect of particle size on bioconjugate binding kinetics was studied using three sizes of bioconjugated particle labels (107, 304, and 396nm) and a rabbit IgG/anti-IgG system in a 96-well plate. A decrease in particle size resulted in faster binding but did not increase the assay sensitivity. Flux calculations based on the particle diffusivity prove that faster binding of the small particles in this study was primarily due to diffusion kinetics and not necessarily to a higher density of antibodies on the particle surface. The results suggest that ITO could make a good platform for an optical immunosensor using fluorescent nanoparticle labels in a competitive assay format for small molecule detection. However, when used in combination with fluorescent particulate labels, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size. Different regeneration methods tested in this study showed that repeated washings with 0.1 M glycine-HCl facilitated the reuse of the ITO waveguide.     

Electrochemical immunosensors for cancer biomarker with signal amplification based on ferrocene functionalized iron oxide nanoparticles

Ultrasensitive sandwich type electrochemical immunosensors for the detection of cancer biomarker prostate specific antigen (PSA) is described which uses graphene sheet (GS) sensor platform and ferrocene functionalized iron oxide (Fe(3)O(4)) as label. To fabricate the labels, dopamine (DA) was first anchored onto Fe(3)O(4) surface followed by conjugating ferrocene monocarboxylic acid (FC) and secondary-antibody (Ab(2)) onto Fe(3)O(4) through the amino groups of DA (DA-Fe(3)O(4)-FC-Ab(2)). The great amount of DA molecules anchored onto Fe(3)O(4) surface increased the immobilization of FC and Ab(2) onto the Fe(3)O(4) nanoparticle, which in turn increased the sensitivity of the immunosensor. GS used as biosensor platform increased the surface area to capture a great amount of primary antibodies (Ab(1)) and the good conductivity of GS enhanced the detection sensitivity to FC. Using the redox current of FC as signal, the immunosensor displays high sensitivity, wide linear range (0.01-40 ng/mL), low detection limit (2 pg/mL), good reproducibility and stability. In addition, this method could be extended to the immobilization of other interesting materials (fluorescence dyes) onto Fe(3)O(4) for preparing various kinds of labels to meet the different requirements in immunoassays.     

Enhanced immunoassay sensitivity using chemiluminescent acridinium esters with increased light output

Chemiluminescent acridinium ester labels are widely used in clinical diagnostics especially in automated immunochemistry analyzers such as Siemens Healthcare Diagnostics’ ADVIA Centaur® systems. Although, chemiluminescence from acridinium compounds was discovered more than 50 years ago, details regarding the excitation process are still not well understood particularly in relation to acridinium structure and overall light output. Herein, we report an empirical study that correlates the presence of electron-donating methoxy groups at C-2 and/or C-7 in the acridinium ring with increased light output. We further demonstrate that these high light output labels can be combined with hydrophilic functional groups such as hexa(ethylene)glycol to generate unique acridinium esters that are stable and are useful in improving immunoassay sensitivity for both competitive and sandwich automated immunoassays.     

MIP-ligand binding assays (pseudo-immunoassays)

Molecular imprint sorbent assays (MIAs) have been applied to an increasing number of analytes of medical and environmental interest: the sensitivities and selectivities of these assays are comparable to immunoassays employing biological antibodies. In a number of cases complete analytical procedures starting from raw samples (blood, plasma and urine) have been demonstrated. There have been significant advances in applying MIPs in new formats and in the use of non-radioisotope labels. Progress in the field is reviewed, with particular emphasis on the technical aspects and new innovations. It is demonstrated that many of the perceived drawbacks of molecular imprinted polymers (MIPs) do not hinder their application in competitive binding assays: Many MIAs have been applied in aqueous systems and a heterogenous distribution of binding sites is not problematic, provided the recognition sites which bind the probe most strongly are selective.