Background of the Invention

Bilirubin is a reddish yellow substance found in biological substance such as blood serum, and has the empirical formula: C ₃ 3H3 N4O ₆

Its presence in serum at too high a level indicates jaundice and its measurement is used as a liver function test. One standard assay used for the quantitive determination of bilirubin is the Jendrassik-Grof method which uses as the assay composition an aqueous solution of caffeine, a benzoate salt, an acetate salt and diazonium salt of sulfanilic acid formed by reacting sulfanilic acid and sodium nitrite in diluted hydrochloric acid. Bilirubin present in the serum reacts with the diazotized sulfanilic acid to form a chromophore in which the caffeine and benzoate and acetate salts cooperate to serve as an activator and speed the reaction. The problem with the solution used is its stability.

It is initially supplied as a three component system. One contains caffeine, the benzoate salt and the acetate salt. The second contains sulfanilic acid and hydrochloric acid.

The third reagent contains sodium nitrite and is the least stable of the three. When the three components are combined, stability is less than 8 hours.

It would be desirable, therefore, to have a stable single reagent system which enables the measurement of bilirubin. Such a system would be lower in cost as three different components need not be packaged. It would also reduce the chance of human error which can occur in combining three components to make an assay reagent. This is because better quality control exist when all components can be added to form a single reagent in quite precise amounts. Such a reagent of adequate shelf life has not heretofore been proposed in the art ready to use in liquid form.

Summary of the Invention

The present invention provides a single reagent system of an excellent shelf stability of at least 18 months at 2 to 8°C and is highly responsive to the quantitative measurement of bilirubin concentration in biological fluids such as blood serum at 540 nanometers (nm) .

The composition comprises an aqueous solution of at least about 1 gm/1 up to solution saturation of a water soluble organic sulfate which may be a long chain aliphatic sulfate, an aromatic sulfate or mixtures thereof; from about 0.05 to about 15 millimole per liter (mM) of a diazonium salt, preferably 3,5-dichlorophenol diazonium tetrafluoro-borate at a concentration of about 0.15 mM; from 0 to 100 g/1 preferably from about 2 to about 100 g/1 caffeine, and having metal complexing agent preferably ethylene-diaminetetracetic acid (EDTA) present in an amount sufficient to complex with heavy metal ions. Normally, EDTA concentration is about 2 g/1. Solution pH is from about 1.0 to about 6, preferably about 3.5 to

about 5.5, and most preferably about 4.4.

The composition is prepared by first adding the co plexing agent (EDTA) to water followed by dissolving the organic sulfate then dissolving the caffeine and finally dissolving the diazonium salt.

The resulting mixture is stable for at least 18 months at 2 to 8°C which is a time sufficient to enable manufacture, worldwide shipment, warehousing and storage until use.

Detailed Description

There is provided in accordance with the present invention a single reagent composition of excellent shelf life which enables the quantitive assay of bilirubin concentration in blood serum. The composition is an aqueous solution of at least one diazonium salt, preferably 3,5 dichlorophenol diazonium tetrafluoroborate, present in a concentration from about 0.05 to about 15 millimoles per liter (mM) , preferably more, about 0.05 to about 1.5 mM, more preferably about 0.15 mM; 1 g/1 to solution saturation of a water soluble alkali salt of an organic sulfate where the organic moiety ^" is a long chain aliphatic group, an aromatic group and mixtures thereof. The lithium salts are presently preferred and preferred organic sulfate is the lithium salt of dodecyl sulfate present in a preferred concentration of about 10 g/1. There is also present 0 to about 100 g/1 preferably from about 2 to about 100 g/1, more preferably about 10 g/1 caffeine and EDTA ethylenediaminetratetic acid (EDTA) in sodium salt form present in a concentration sufficient to complex with any heavy metal ion which are present. The current preferred concentration of EDTA is about 2 g/1.

The solution has a pH from about 1 to about 6, preferably about 4 to about 5, and most preferably about 4.4.

The composition is prepared by first adding EDTA to distilled water to complex any heavy metals which are present, the water soluble organic sulfates are added and dissolved, followed by addition of and dissolving of caffeine. The diazonium salt is added last. The solution as prepared is stable at temperatures of about 2 to about 8°C (refrigeration conditions) for at least 18 months.

In the system, the diazonium salt diazotizeε with bilirubin forming a chromogen which is red in color. The water soluble organic sulfate acts to catalyze the diazo

reaction, make the complex absorbance more intense and stabilizes the diazonium salt against degradation. Caffeine accelerates the formation of the complex and intensifies the color to make the absorbance higher. In use, approximately one milliliter of the preferred composition and about 50 microliters of the serum are combined. Any bilirubin present is diazotized to form a red chro ogen whose intensity is measured at 540 nanometers to eliminate interference attributed to hemaglobin, lipemia and the like which obscure the presence of bilirubin if absorbance is measured at 515 nanometers where maximum absorbance occurs. Making the determination at 540 nanometers, the interference is minimized and the bilirubin present reacts to provide the intense color enabling measurement.

The assay system of this invention is suited to the measurement of total bilirubin including unconjugated and/or conjugated fractions.