Europium labeled antibodies

The wells were blocked with 100 pL of 1% BSA-PBST at 37°C for 30 min. After removal of the blocking buffer, 75 pL of primary antibody in PBST-1% BSA were added and incubated for 1.5 hr at 37°C (or overnight at 4°C). The wells were then washed three times with 300 pL DELFIA wash buffer, and 100 pL of secondary europium-labeled anti-rabbit (1 4000) or anti-mouse (1 500) antibody in DELFIA assay buffer added and incubated at 37°C for 1 hr in darkness. Following three washings with 300 pL DELFIA wash buffer, 100 pL DELFIA enhancement solution were added into each well and plates were incubated on a shaker at room temperature for a minimum of 15 min. The plates were read using the Victor2. 

Pro-inflammatory cytokines are important mediators of inflammation and tissue destruction. This section describes two cell-based assays that were used to screen for inhibitors of cytokine production and some of the compounds discovered using these screens. The two screens were important elements of a collaboration between Xenova Ltd and the Suntory Institute of Biomedical Research to find microbial metabolites with potential utility for the treatment of rheumatoid arthritis. Both screens were cell stimulatory assays with similar formats, the principle of which is illustrated in Figure 3. Treatment of cells with a particular stimulus activates a signal transduction pathway, one of the end results of which is production of a cytokine, which is secreted into the assay medium. After a separation step, the cytokine of interest is measured quantitatively in the supernatant by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) using a europium-labeled tertiary antibody. At the same time, cytotoxic properties of test substances are determined by assessing their effect on proliferation of the separated cells. 

In time-resolved fluorescence (TRF) (Maundrell et al., 1985), europium chelates are excited at 340 nm to emit two types of fluorescence, a shortlived background fluorescence (< 0.1 ms) and a fluorescence due to emitted photons of Eu " lasting up to over 1 ms. This difference in fluorescence decay rate can be exploited by measuring fluorescence only after background fluorescence has completely decayed to obtain a very high signal to noise ratio (detectability down to 10 5 M) as shown in Fig. 7.5. Originally, anti-hapten antibody was labeled with Eu " but in more recent procedures Eu " is directly attached to the nucleic acid (Sections 7.3.2.1 and 7.8.1). 

Fig. 6 A typical competitive immunoassay of a small antigen (cAMP) with TR-FRET. Sample cAMP competes in the assay with europium-labeled tracer for limited amount of anti-cMAP antibody. In the absence of sample cAMP, maximum TR-FRET signal is produced...

Fig. 7 A scheme of an assay of tyrosine kinase activity measurement as done in kinase inhibitor screening with TR-FRET. Acceptor-labeled peptide substrate is phosphorylated by the kinase under investigation, and the resulting phosphorylated peptide is quantitated with europium-labeled antiphospho-specific antibody...

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Organic fluorescent dyes with the appropriate spectral properties also can be paired with lanthanide chelates in FRET systems. For instance, many rhodamine dyes and the cyanine dye Cy5 have ideal excitation wavelengths for receiving energy from a nearby europium chelate. The LeadSeeker assay system from GE Healthcare incorporates various Cy5-labeled antibodies for developing specific analyte assays. In addition, if using a terbium chelate as the donor, then a Cy3 fluorescent dye can be used in assays as the acceptor. 

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

Another antibody-based immunosorbent assay that can be used to determine HAT activity uses a secondary anti-IgG antibody, which is directed against the primary antibody and is labeled with the lanthanide Europium (Eu). After another washing step that removes all nonbound secondary antibody, one last incubation step is performed, which releases the lanthanide ion from the antibody, so that the final detection of time-resolved fluorescence (340/615 nm) caused by the released metal ion correlates with the acetylation level of the oligepeptide histone substrate, which is correlated with enzymatic activity. So far. 

Self (S4) first proposed the concept of noncompetitive assay for haptens utilizing an adequate combination of an a-type and a jS-type anti-idiotype antibody, in which he used the term, selective antibody for the a-type antibodies. Then, Barnard and Cohen (Bl) applied this assay principle for the determination of serum E2, naming the assay system an idiometric assay. Figure 12A illustrates the assay procedure of the idiometric assay of E2. The target hapten is captured by excess anti-E2 antibody immobilized on microtiter strips by incubation at room temperature for 1 h (step i). After washing the strips, the /3-type anti-idiotype antibody was added in order to saturate (or block) the unoccupied paratope of the anti-E2 antibody (incubation, room temperature for 30 min) (step ii). The a-type anti-idiotype antibody, which has been labeled with a europium chelate (H4), was then added to the plate and incubated at room temperature for a further 2 h (step iii). Finally, fluorescence intensity due to bound europium was measured with a time-resolved fluorometer. Because of large steric hindrance around the bound jS-type antibody (MW 150,000), the labeled a-type antibody would. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

These assays are based upon the use of a europium or terbium chelate (a transition metal-ligand complex displaying long-lived fluorescent properties) and labeled anti-phosphopeptide or anti-phos-photyrosine antibodies that can bind to phosphorylated peptides. The antibodies are usually labeled... 

---