Nonmetallic tetrapyrrole molecules are shown to catalyze the production of light by chemiluminescence in the presence of a signal solution at a pH from about 10.0 to about 14.0, having an appropriate oxidant or combination of oxidants and a luminescent reactant. The addition of an electron transport facilitator, a surfactant, a carbohydrate, and a chelating agent to the signal solution increases the output of light. These tetrapyrrole molecules are used alone or attached to haptens or macromolecules and are utilized as labels in the preparation of chemiluminescent, homogeneous or heterogeneous assays. They are also used in conjunction with other chemiluminescent label molecules to produce multiple analyte chemiluminescent assays. A chemiluminescent signal solution which comprises at a pH ranging from about 10.0 to about 14.0 trans, trans-5-(4-Nitrophenyl)-2,4-pentadienal, sodium di-2-ethylhexyl sulfosuccinate, glucose, benzyltrimethylammonium hydroxide, cumene hydroperoxide, trisodium para periodate, potassium superoxide and EDTA with or without a luminescent reactant is also disclosed.

The present invention refers to a process for the preparation of an activated metallo-porphyrin derivative and to novel activated metallo-porphyrin derivatives obtainable by said process. Further, the present invention refers to conjugates of metallo-porphyrins with molecules having at least one primary amino group, especially biomolecules. The conjugates are used in a procedure for the detection of a biological substance, especially in an immunoassay or a nucleic acid hybridization assay.

A method for immunoassay for a ligand suspected to be present in a fluid includes use of an enzyme, a metal ion catalyst for an indicator reaction and a blocked modulator for the catalyst. Ligand present in the fluid binds to an antiligand. The resulting bound fraction activates the enzyme to unblock the modulator. The free modulator activates or inhibits the catalyst thereby modulating the rate of an indicator reaction between a substrate and a redox reagent. The presence of absence of the ligand in the fluid is indicated by a signal, such as a color change or a rate of color change, consequent to the indicator reaction. The invention includes a kit of materials useful for performing the method of the invention.

Assays, e.g. immunoassays, based on the chemiluminescent reaction between a peroxidase enzyme, e.g. horse radish peroxidase, a chemiluminescent 2,3-dihydro-1,4-phthalazinedione, e.g. luminol or isoluminol and an oxidant, e.g. hydrogen peroxide or a perborate, are improved by carrying out the reaction in the presence of a 6-hydroxybenzothiazole, especially 6-hydroxybenzothiazole itself or firefly luciferin. Light emission from the reaction is significantly enhanced by the 6-hydroxybenzothiazole.

An enzyme-free diagnostic reagent, methods of its use and diagnostic systems containing that reagent are disclosed. The enzyme-free diagnostic reagent comprises an enzyme-free catalyst coupled by a linking group to a first binding agent, binds in aqueous medium to a second binding moiety to form a binding complex, and indicates the amount of second binding moiety present in the complex by means of its catalytic reactivity with co-reactant molecules.