Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Glycoproteins
Patent
1988-12-22
1992-05-05
Warden, Robert J.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Glycoproteins
436 34, 436 63, 436 71, G01N 3350
Patent
active
051107465
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for quantitatively determining albumin in blood serum even in the presence of globulin and other serum proteins.
Techniques for measuring albumin in blood and other biological fluids, e.g. urine, are important in medicinal, diagnostic and clinical chemistry. Usually, total protein in serum is ascertained by the so-called "biuret/copper reaction" which relates to the formation of colored complexes (violet) between copper .sup.+2 ions and the peptide bonds of proteins. To separately determine albumin and glubulins the globulins are precipitated by salting out (e.g. with alkali or ammonium sulfate) and separated by centrifugation and thereafter, albumin in the supernatant solution is determined independently, also by the biuret reaction. This technique being time consuming, methods have been developed for the direct determination of albumin in blood serum in the presence of other proteins. For instance, a bromocresol green (BCG) dye-binding procedure for the quantitative determination of serum-albumin was introduced in 1964 by DELANEY (Proc. Australian Assoc. Clin. Biochem 64 (1964), 1). Serum is diluted with buffered BCG at pH 7.0 and the decrease in absorbance at 615 nm is measured. This change in absorbance is linear with albumin concentration up to 50 mg/ml and other electrophoretically separated proteins like hemoglobin and bilirubin do not interfere.
It was however desirable to uncover even more specific techniques, i.e. even less susceptible to disturbance by other molecules in the serum. The method summarized in claim 1 is an important step toward this objective. It was completely unexpected to find that albumin can be quantitatively ascertained by this route, even in the presence of large quantities of globulins. Indeed, reagents of the kind applicable in the present method i.e. o,p-dinitrohalobenzenes are known to undego bimolecular nucleophilic displacement with the NH.sub.2 -terminal groups of proteins (see Sanger Biochem. J. 39 (1945), 507, 1945), the rate order being F>Cl.about.Br. Thus, it was surprising, at least in the case when the halogen is fluorine, to note that albumin was practically the only type of protein in blood serum to give appreciable reaction rates even in the presence of the other proteins, e.g. globulins.
Another unexpected finding is that, instead of having to measure the reaction rates by recording changes in some optical or other properties related to complex formation, the reaction was advantageously followed by measuring the amount of fluorine released. This was surprising because although the foregoing reaction (see scheme below) involves fluorine substitution as follows (X represents fluorine): ##STR1## it is not obvious that this should appear in a directly measurable form, e.g. which can be measured with an ion-selective fluoride electrode.
One of the preferred reagents, the dinitro-fluoro-benzene compound (DNFB) is sparingly soluble in water. Therefore, for best results, DNFB organic solutions were emulsified with suitable aqueous buffered media compatible with the serum samples and the reaction was run under such conditions. For example, a solution of DNFB in benzene was emulsified with buffer, (characteristically a 0.1N to 1N solution at pH 5.5-7.7) and the sample of serum to be analyzed was stirred with the emulsion. The evolution of fluoride was followed using a fluoride ion-selective electrode of any conventional type. Calibration curves were obtained by means of a set of standard solutions of albumins and the concentration of unknowns was determined by comparison with the calibration curves. The presence of globulins and other blood proteins as well as free amino acids and urea did not interfere in the range of concentrations normally present in blood serum.
As the organic solvents for the DNFB, ether, toluene, DMF, DMSO, THF, EtOH and MeOH are also suitable but to a lesser extent because some of them behave as nucleophiles and they cause electrode drift.
The choice of the buffer is also important; it was found for
REFERENCES:
patent: 4013416 (1977-03-01), Rittersdorf et al.
N. W. Tietz, "Fundamentals of Clinical Chemistry", 1982, pp. 335-338, Philadelphia, U.S.; W.B. Saunders Co.
Chemical Abstracts, vol. 93, No. 1, Jul. 7, 1980, p. 248, col. 1, Abstract No. 2522r, Joshi et al.
Chemical Abstracts, vol. 87, No. 19, Nov. 7, 1977, p. 192, col. 1, Abstract No. 147477r, R. R. King et al.
Brochot Jean
Grey Howard
Mangan Ciaran
Siddiqi Iqbal
Alfandary-Alexander Lyle
Pharmacia Diagnostics Inc.
Warden Robert J.
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