Antibodies enhanced chemiluminescence

Horseradish peroxidase (HRP)-coupled secondary anti-IgG antibodies (species depending on primary antibody) enhanced chemiluminescence substrate... 

Lueking et al. (1999) arrayed recombinant proteins on NC membranes and screened them with different antibodies. Joos and coworkers (2000) printed down autoantigens onto NC membranes and compared performance relative to silylated (aldehyde) and PLL glass slides. Protein arrays could be stored at room temperature for a month without significant loss in activity. Huang (2001) hand spotted down IgC species and antibodies directed toward various cytokines onto membranes. The properties of various commercial membranes were assessed in terms of absorption, background, and sensitivity levels based upon detection by enhanced chemiluminescence (ECL). 

The separated proteins were transferred to a polyvinylidene difluoride membrane, and nonspecific IgC binding sites were blocked by incubation with 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies. Immune complexes were detected by enhanced chemiluminescence (Amersham Biosciences). 

Constantine, N. T. Bansal, J. Zhang, X. Hyams, . C. Hayes, C. Enhanced chemiluminescence as a means of increasing the sensitivity of western blot assays for HIV antibody. J. Virol. Methods 1994, 47(1-2), 153-164. 

Christofides ND, Sheehan CP. Enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine Design, development, and technical evaluation. Clin Chem 1995 41 12-23. 

A novel homogeneous assay for urinary estrone-3-glucuronide has been reported by Kim et al (K8) using an A-aminoethyl-A-ethyl isoluminol derivative (see Table 3, n = 2). In contrast to antibody enhancement, this assay exploits the fact that 2N NaOH enhances free label chemiluminescence, and this is reduced in the presence of specific antibodies. Unlabeled estrone-3-glucuronide reverses the inhibition in a concentration-dependent manner and 8 pg per tube can be minimally detected (K8). 

Other applications of this type of detection include Western blotting procedures for the identification and visualization of protein antigens (LI, L8, L9). One such recent assay for herpes simplex virus Type 2 showed that enhanced chemiluminescence is 500 times more sensitive than a colorimetric method (Dl). Antibodies to several of the HIV viral proteins have also been detected in chemiluminescent Western blots (SI6). 

Imai, K., Chemiluminescence detection in flow systems. Biomed. Chromatog. 4, R1 (1990). Imai, K., Nishitan, A., Tuskamoto, Y., Wang, W. H., Kanda, S., Hayakawa, K., and Miyazaki, M., Studies on the effects of imidazole on the peroxyoxalate chemiluminescence detection system for high performance liquid chromatography. Biomed. Chromatog. 4,100-104 (1990). Ireland, D. C., and Samuel, D., Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen. J. Biolumin. Chemilumin. 4, 159-163 (1989). 

Stiles, H. J., Bicknell, H. J., and Mathews, J. A., A rapid enhanced chemiluminescence immunoassay for allergen-specific IgE antibodies. J. Biolumin. Chemilumin. 1, 53-58 (1986). 

FIGU R E 7 A similar set of substrates is tyrosine phosphorylated during activation of either intact taenia coli, cultured VSMC, or staphylococcal a-toxin-permeabilized deal longitudinal smooth muscle. In these experiments, tyrosine.-phosphorylated substrates were detected by immunoblotting with antiphosphotyrosine antibodies and enhanced chemiluminescence technology rather than the less sensitive I-labeled protein A technology used in Fig. 2. Stimulation of guinea pig taenia coli with either 10 jcM carbachol (Carb) or 1.5 mM vanadate (Van) resulted in pronounced tyrosine phosphorylation of at least nine substrates with apparent masses of 42-45, 50, 70, 80-85, 95,100, 110, 116, and 205 kDa. In like fashion, stimulation of canine femoral VSMC with 100 jjlM phenylephrine (PE) resulted in enhanced tyrosine phosphorylation of a similar set of substrates (however, note that qualitative differences were evident with respect to some substrates, such as the one of 205 kDa). Similarly, the same substrates appeared to be tyrosine phosphorylated when permeabilized ileal smooth muscle was contracted with Ca + (pCa 4.5). From Di Salvo et al. (1994), Fig. 5, p. 1438. 

Probe with anhactive ERK 1/2 antibody (1 5,000), and donkey anhrabbit secondary antibody with horseradish peroxidase (HRP 1 10,000) detected by ECL (enhanced chemiluminescence). 

CYP3A4 protein expression can be measured directly by immunodetection (LeCluyse et ah, 2000 Luo et ah, 2002). Microsomal protein (3 jig) is resolved by SDS-polyacrylamide gel electrophoresis (12% acrylamide). Resolved proteins are transferred to nitrocellulose membranes, whieh are ineubated in 3% bovine serum albumin in phosphate-buffered saline supplemented with Tween 20 (0.1 M, pH 7.4, 0.1% Tween 20) for 45 min to block nonspecific protein binding. Membranes are then treated with primary antiCYP3A4 antibody (Gentest, Woburn, MA), followed by horseradish peroxidase-conjugated antimouse seeondary antibody. The antibody-reactive CYP3A4 protein bands are visualized using enhanced chemiluminescence detection and quantitated by photodensitometry (Desai et al., 2002). 

Total mouse liver proteins (50 were separated on a 10% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) and Western blotting was performed by electrophoretic transfer of the separated proteins onto a nitrocellulose filter (Nitropure, Micron Separations Inc., Westborough, MA, USA) using a Mini-Protean II device (Bio-Rad). The blots were probed with an anti-MTE-I antibody and subsequently with horseradish peroxidase-conjugated secondary antibodies and visuahsed by enhanced chemiluminescence (ECL, Amersham) using X-ray film. 

Proteins of bacterial pellets were separated by SDS-PAGE (Laemmli, 1970), transferred to nitrocellulose (Kyhse-Andersen, 1984), and incubated with commercially available lamin antibodies (Cat. No. 65147 Progen, Heidelberg, Germany). Antibodies bound to the filter were detected using an enhanced chemiluminescence system (Amersham Buchler) by exposure to X-ray film (X-Omat AR Eastman Kodak C., Rochester, NY, USA). 

DNA probes can be labeled using the modified nucleotide, fluorescein-11-dUTP, and subsequently detected with enhanced chemiluminescence (1) using an antifluorescein antibody conjugated to horseradish peroxidase (2). The use of enhanced chemiluminescence avoids the fading of results associated with some colormetric detection procedures, used with nonradioactive labeling and detection systems. Hard copy results showing excellent resolution are produced. 

There are several advantages to performing the denaturation, renaturation, and kinase procedures with the proteins electroblotted onto a membrane rather than with the SDS-polyacrylamide gel. First, after phosphorylation, the same blot can be used for immunoblotting depending on the antibody and the integrity of the epitope recognized. We were able to successfully use monoclonal antibodies to CaM kinase a and / and the Amersham Enhanced Chemiluminescence (ECL) Western blotting detection system to detect the isoforms after in situ renaturation (Shackel-... 

Develop the blots with horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents (GE Healthcare, Piscataway, NJ). 

Detection of HRP-conjugated secondary antibodies by enhanced chemiluminescence (ECL)... 

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