Protein Protocols Protein Protocols

Enhanced Chemiluminescence 1095
light output owing to substrate exhaustion. This cannot be avoided by alteration of the
reaction conditions. Slight reduction in enhancer concentration may give more stable light
output for assays with atypically low peroxidase activity.
10. For best results, plates should be coated with the IgG fraction of an antiserum, this can be
conveniently prepared using caprylic acid precipitation (see Chapter 138 and ref. 15). Where
this is not possible, indirect capture may be used, such as antispecies antibody on the plate,
or the streptavidin–biotin system. Any indirect capture system must be compatible with the
final label, e.g., labeled antigen or different species antisera with no cross-reaction with the
indirect coating antibody.
11. Safety data (from ref. 16):
a. Luminol (commercial-grade): Irritating to eyes, respiratory system, and skin. No
specific information is available for pure luminol or for the sodium salt.
b. Hydrogen peroxide: Contact with combustible materials may cause fire. Causes burns.
Keep in a cool place. After contact with skin, wash immediately with plenty of water.
c. DMSO: Irritant to eyes, skin, and respiratory system. Harmful by inhalation, skin
contact, and if swallowed. May cause sensitization by inhalation or skin contact.
d. p-Iodophenol: Irritant to eyes, skin, and respiratory system.
e. p-Hydroxycinnamic acid: Irritant to eyes, skin, and respiratory system.
References
1. Zomer, G., Stavenuiter, J. F. C., Van Den Berg, R. H., and Jansen, E. H. J. M. (1991) Synthesis,
chemiluminescence and stability of acridinium ester labeled compounds, in Luminescence
Techniques in Chemical and Biochemical Analysis (Bayens, W. R. G., De Kekeleire, D.,
and Korkidis, K., eds.), Dekker, New York, pp. 505–521.
2. Jansen, E. H. J. M., Zomer, G., and Van Peteghem, C. H. (1991) Chemiluminescence
immunoassays in vetinary and food analysis, in Luminescence Techniques in Chemical
and Biochemical Analysis (Bayens, W. R. G., De Kekeleire, D., and Korkidis, K., eds.),
Dekker, New York, pp. 477–504.
3. Bronstein, I. and McGrath, P. (1989) Chemiluminescence lights up. Nature 333, 599,600.
4. Bronstein, I., Voyta, J. C., Thorpe, G. H. G., Kricka, L. J., and Armstrong, G. (1989)
Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme
immunoassay of thyrotropin. Clin. Chem. 35, 1441–1446.
5. Bronstein, I. and Kricka, L. J. (1989) Clinical applications of luminescent assays for
enzymes and enzyme labels. J. Clin. Lab. Analyt. 3, 316–322.
6. Thorpe, G. H. G. and Kricka, L. J. (1986) Enhanced chemiluminescent reactions catalyzed
by horseradish peroxidase. Meth. Enzymol. 133, 331–354.
7. Kricka, L. J., Stott, R. A. W., and Thorpe, G. H. G. (1991) Enhanced chemiluminescent
detection of Horseradish peroxidase labels in ligand binder assays, in Luminescence Techniques
in Chemical and Biochemical Analysis (Bayens, W. R. G., De Kekeleire, D., and
Korkidis, K., eds.), Dekker, New York, pp. 599–635.
8. Thorpe, G. H. G., Stott, R. A. W., Sankolli, G. M., Catty, D., Raykundalia, C., Roda, A.
and Kricka, L. J. (1987) Solid supports and photodetectors in enhanced chemiluminescent
immunoassays, in Bioluminescence and Chemiluminescence. New Perspectives
(Scholmerich, J., Andreesen, R., Kapp, M., and Woods, W. G., eds.), Wiley, Chichester,
UK, pp. 209–213.
9. Matthews, J. A., Batki, A., Hynds, C., and Kricka, L. J. (1985) Enhanced chemiluminescent
method for the detection of DNA dot-hybridization assays. Analyt. Biochem. 151,
205–209.