Luminol Derivative Research Articles

Improved Enzyme Immunoassay for Human Basic Fibroblast Growth Factor Using a New Enhanced Chemiluminescence System

An enhanced chemiluminescence reaction has been incorporated into an enzyme immunoassay (EIA) for human basic fibroblast growth factor (hbFGF). We developed a new luminol derivative, designated L-012 and a new enhancer, 4-(4-hydroxyphenyl)thiazole. Using these compounds, the detection limit of hbFGF was improved to 0.1 pg/assay, which was 10 - 20 and 2 times better than the o-phenylenediamine colorimetric and luminol chemiluminescence assays, respectively. The average concentration of bFGF in sera from 25 normal volunteers was 5.9 pg/ml. On the other hand, serum bFGF levels were elavated in renal, lung and brain tumor patients. The data presented here indicate that the serum bFGF level could be a useful diagnostic marker for these tumors. Furthermore, these new compounds could easily be applied to any other EIA that uses horse radish peroxidase to improve sensitivity.

Chemiluminescent Assays for β-d-Galactosidase and Alkaline Phosphatase Using Novel Luminol Derivatives as Substrates

Chemiluminescent assays for β-D-galactosidase and alkaline phosphatase are described which use novel chemiluminogenic substrates, o-aminophthalylhydrazide-β-D-galactoside and o-aminophthalylhydrazide-O-phosphate, respectively. Each substrate is enzymatically hydrolyzed under the optimum conditions to release luminol, which luminesces in the presence of potassium hexacyanoferrate(III) in an alkaline medium. The detection limits (S/N=3) of the enzyme activities (μU per tube) were 50 for β-D-galactosidase and 100 for alkaline phosphatase.

CHEMILUMINESCENT DETERMINATION OF AMINO-TERMINAL RESIDUES OF PEPTIDES AFTER DERIVATIZATION OF ANILINOTHIAZOLINONE FROM EDMAN DEGRADATION WITH <i>N</i>-(4-AMINO- HEXYL)-<i>N</i>-ETHYLISOLUMINOL

We derivatized 2-anilino-5-thiazolinone amino acids obtained from Edman degradation of peptides with a chemiluminescent primary amine, N-(4-aminohexyl)-N-ethylisoluminol. Luminol derivatives of the amino acids were separated by reversed-phase high-performance liquid chromatography and detected as chemiluminescence using a hydrogen peroxide-microperoxidase system in a flow cell. The detection limit of the luminol derivative by this method was 500 attomol. This chemiluminescent method would provide a simple and sensitive determination of amino acid sequences of peptides.

Alkali-labile luminol derivatives as labels for quantitative binding studies with polyionic macromolecules.

Acylated derivatives of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) bearing a carboxyl or amino group can be linked by amide bonds to a macromolecule requiring labelling. Though themselves of low quantum yield these compounds are alkali-labile and can be detected at a similar level of sensitivity to the parent compound luminol. These cheap, readily accessible compounds are less hydrophobic than other currently employed chemiluminescent labels. They also lack a positively charged nitrogen atom which could complicate their covalent linkage to polyanionic compounds. They thus appear well suited for labelling heparin and other macromolecules which interact with the luminal surface of blood vessels.

Chemi- and bioluminescent immunoassay.

The application of a highly sensitive immunoassay, using chemi- or bioluminescent reaction is reviewed. Their advantages of these reactions are that they can be quantiated rapidly, are relatively stable, possess high specific activity, and can take part in an amplification reactions. They can be employed in the immunoassay in three different ways. 1. The label can be a chemiluminescent substances such as luminol derivatives. 2. Product from label enzyme can be monitored using chem- or bioluminescent reaction. Examples of these application of a luminescence in the immunoassay are for instance, the use of oxidase enzyme or dehydrogenase enzyme labels that produce hydrogen peroxide or NADH. Those products are monitored using chemi- or bioluminescent reaction. 3. The label can be a catalyst or cofactor for bioluminescent reaction.

Chemiluminescence-linked immunoassay

Covalent complexes of a chemiluminescent compound (a luminol derivative), protein and a steroidal hapten have been used as a labelled antigen in a way analogous to the radioactive label in radioimmunoassay. The antibody-bound complex emits light when treated with hydrogen peroxide and copper acetate at high pH. The chemiluminescent complex bound to antibody can be displaced by free steroid to produce a standard curve in a way analogous to radioimmunoassay.