No abstract

A new chemiluminescence procedure was established that eliminates the need to use a separate oxidant such as hydrogen peroxide for chemiluminescence detection reactions. In the procedure called optically pumped chemiluminescence (OPC), red light energy Is used by a photosensitive dye to oxidize luminol and generate blue light. By pulsing the excitation light and measuring chemiluminescence during the off time, light scattering background was eliminated and sensitivity enhanced 10-fold. The OPC reaction mechanism was determined to be type I (direct reaction of excited dye with substrate), and the dye catalyst Is recycled In the reaction. Of the popular photosensitive dyes tested, methylene blue showed the best detection limit In the OPC system. A methylene blue dye derivative was synthesized and covalently attached to protein. Antibody proteins were labeled with up to 16 dyes per antibody. An a-fetoproteln Immunoassay was developed having a detection limit of 17 pg. This assay had sensitivity of 1.5 ng of alphafetoproteln/mL of plasma and showed good correlation (/? = 0.98). The OPC catalysts are more stable and less temperature sensitive than enzyme catalysts, and the substrate solution needed Is very stable. Thus, OPC may be valuable In applications where convenience and reagent stability are Important.

Conditions for the enhanced horseradish peroxidase (HRP) catalysed reaction between luminol and hydrogen peroxide were optimized to determine detection limits for HRP conjugated to antibody fragment (HRP-Fab) in solution phase. Light output was linear with respect to HRP-Fab concentration but became nonlinear at low HRP-Fab concentrations when an accelerator (enhancer) of the reaction was used. para-Phenylphenol was a more effective enhancer than p-iodophenol at HRP-Fab concentrations below 20 pmol/l. The detection limit for HRP-Fab was 1.2 femtomoles in the absence of p-phenylphenol and 0.08 femtomole in the presence of p-phenylphenol. The acceleration of peroxidase activity at the lowest HRP-Fab concentrations occurred after an incubation time period of up to five minutes. This lag time limited the sensitivity and the mechanism for it was sought. Preincubation experiment results indicated that the lag time phenomenon may involve a reversible alteration in HRP catalytic activity and that enhancer, peroxide, luminol and HRP-Fab had to be incubated together some time before maximum activation could occur.

Methylene blue is a very strong photoactive dye that has an absorption peak (668 nm) that corresponds well to a popular low‐cost diode laser. However, it has not been used in photodynamic tumor therapy and immunodiagnostics because it cannot be covalently coupled to protein. Therefore, methylene blue derivatives having a succinimido or maleimido functional group were synthesized and coupled to antibody, serum albumin and transfemn proteins. Incorporation of dye into antibody protein at high ratios (more than three per molecule) caused precipitation and loss of antibody activity. Inclusion of one or more carboxylic acid residues in the methylene blue derivative before coupling to protein alleviated the precipitation problem, and up to 36 methylene blue dye molecules could be attached to an antibody fragment using bovine serum protein as a carrier. Methylene blue derivatives and protein complexes formed from them oxidized luminol when stimulated with red light. The new dye conjugates were used in an optically pumped chemiluminescence immunoassay for α‐fetoprotein. These compounds and techniques should also be useful for photodynamic tumor therapy where it is desired to attach a red‐absorbing photoactive dye to antibody protein.

At very low horseradish peroxidase (HRP) concentrations, the enhanced chemiluminescence reaction is often characterized by a lag time between initiation of the reaction and beginning of light output. In this study, four treatments of luminol solution were examined in an effort to remove the lag time and to improve chemiluminescence light output. Addition of ammonium persulphate stimulated light output more than tenfold. Ultraviolet irradiation and photoactive dye pretreatment of luminol solution both increased light output fourfold. Luminol purity was the most important factor affecting detection sensitivity. Recrystallization of luminol from base improved the detection limit 13-fold although there was an improvement in the detection limit from 13 attomoles per millilitre to 5 attomoles per millilitre with highly purified luminol when photoactive dye pretreatment was utilized. The results are consistent with a simple interference mechanism whereby enhancer radicals produced by the enzyme are preferentially quenched by contaminants present in the luminol, in the enhancer and in the solvent used to dissolve the enhancer. Consumption of these interferences prior to light emission results in a lag time and a less favourable HRP detection limit.