Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2001-02-08
2002-05-21
Whisenant, Ethan C. (Department: 1655)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S456000, C204S466000, C204S469000, C204S546000
Reexamination Certificate
active
06391176
ABSTRACT:
BACKGROUND OF THE INVENTION
Alkaline phosphatase (EC 3.1.3.1.) (abbreviation: ALP) is a metalloenzyme consisting of a group of isoenzymes present in different tissues of animal organisms and in particular in man.
Alkaline phosphatase isoenzymes are important in protocols for diagnosing different conditions in adults or in children, and a number of methods have been proposed for separating and assaying such isoenzymes. ALP isoenzymes can be divided into four classes: non-specific tissue (bone, liver and kidney), adult intestinal, foetal intestinal, and placental. A number of variations can exist within a single class, namely:
hepatic: hepatic 1 (H1), hepatic 2 (H2);
ultrafast (UF);
osseous (Os);
placental: placental 1 (P1), placental 2 (P2);
intestinal: intestinal 1 (I1), intestinal 2 (I2), intestinal 3 (I3).
Thus nine principal fractions can be distinguished which have to be separated, identified and quantified in particular for their detection into hepatic and biliary disorders and into certain bone diseases, including osseous tumours or Paget's disease.
Reference will occasionally be made below to the term “fraction” to designate a class of ALP isoenzymes or a particular variant within a class of isoenzymes. On the electrophoresis support, a “fraction” corresponds to a band revealed after migration.
The most frequent routine analysis carried out on alkaline phosphatase isoenzymes consists of measuring the total enzymatic activity using a substrate of this enzyme, generally para-nitrophenylphosphate. That method, however, cannot determine the levels of the different isoenzymes.
The principal method for separative analysis of such compounds uses electrophoretic techniques. Isoelectrofocussing is occasionally used and can separate 10 to 20 bands depending on the procedure used. Identifying all of the bands is difficult, rendering clinical interpretation extremely awkward.
Zone electrophoresis enables a good separation of the principal forms of the isophosphatases. However, certain fractions are superimposed, in particular the Os, H1 and P1 fractions, and thus complementary treatments have to be carried out to separate and identify them. Such treatments must be carried out on the biological test samples to be tested before depositing them onto the gel.
Such treatments consist, for example, of thermal denaturing, incubation with specific inhibitors such as urea, amino acids, etc., enzymatic incubation with neuraminidase, ficin, phospholipase C, incubation with specific antiplacental or anti-intestinal antisera.
Several separation procedures which are in current use have been dealt with by Van Hoof V. O., De Broe Marc, E., Clinical Laboratory Sciences, vol. 31, issue 3 1994, “Interpretation and clinical significance of alkaline phosphatase isoenzyme patterns”.
One particular procedure has been proposed in United States patent U.S. Pat. No. 5,264,098 which describes the separation of ALP isoenzymes using a gel electrophoresis reaction employing a gel buffer containing at least one non ionic detergent and an anionic detergent.
Available treatments for identifying and quantifying ALP isoenzymes have certain disadvantages as regards routine analysis. In addition to high costs, they can on the one hand be long and can considerably complicate manipulation, and on the other hand, complete determination (of all of the isoenzymes) necessitates a plurality of treatments (2 or 3) for a single sample, limiting the number of samples which can be simultaneously analysed on the one gel.
Other treatments have been proposed which, for example, recommend treating the sample prior to loading onto the electrophoresis gel. In this regards, the action of the WGA lectin (wheat germ agglutinin) is particularly interesting (see Sidney B. Rosalki, A. Ying Foo, Clinical Chemistry, 30/7, p. 1182-1186, 1984, “Two methods for separating and quantifying bone and liver alkaline phosphatase isoenzyme in plasma”, European patent EP-A-0 131 606 dated 5/11/86). EP-A-0 131 606 describes the differential detection of bone and liver ALP isoenzyme comprising treating the test sample with lectin, then incubating the mixture obtained followed by separating the ALP bound to the lectin from the fraction containing free ALP and determining the ALP activity in one of the two media or in both. In a particular implementation of that patent, the two fractions (ALP bound to lectin and free ALP) are separated by electrophoresis.
With the exception of the intestinal forms, all isophosphatases possess sialic acids and are thus affected by a treatment with WGA lectin to a greater or lesser extent. The osseous fraction is the most sialated and thus is affected the most by this treatment, which under suitable conditions retards its mobility and thus causes it to precipitate in a zone which is distinct from the zone where the hepatic fraction is located.
In order to render ALP isoenzyme precipitation more selective towards the osseous isoenzyme, certain authors have used detergents such as Triton X100 (Rosalki). However, despite the presence of such detergents, residual interactions of the WGA lectin with other isophosphatases subsist, which cause co-precipitation of such fractions with the osseous fraction.
In addition to this lack of specificity, a further disadvantage of this technique is to render the analysis considerably more complicated.
The publication by Rosalki S. B. et al, cited above, alternatively proposes incorporating lectin into the buffer used to impregnate the electrophoresis gel prior to using this gel. This dispenses with prior treatment of the sample. In that case, the majority of the osseous fraction is precipitated close to where the sample has been loaded. The mobility of all of the other isoenzymes with the exception of intestinal isoenzymes is affected by the action of the WGA lectin despite the presence of the detergents mentioned above.
In the context of that treatment, the properties of the lectin used are its ability to interact specifically with the ALP isoenzymes which contain sialic acids.
SUMMARY OF THE INVENTION
The present invention proposes means for at least partially overcoming the disadvantages stated in prior art methods. In particular, the invention defines a method enabling separation and identification of ALP isoenzymes which is improved as regards specificity and sensitivity.
The present invention also provides consumers, in particular clinicians, with a process for separating, identifying and quantifying the principal alkaline isophosphatases, which process can be carried out in a single step on a single electrophoresis support which is easy to produce.
The invention thus proposes a novel process for separating and identifying ALP isoenzymes by electrophoresis, characterized in that the lectin is deposited on the electrophoresis support in a localised manner.
The lectin deposited in solution in a localised manner can diffuse into the support while remaining localised in a determined zone of this support during electrophoretic migration.
The deposit in question, located close to the zone where the sample is deposited, is distinguished from the uniform loading over an extended zone or over the whole of the electrophoresis support as described in the prior art.
The invention thus provides a process for separating alkaline phosphatase isoenzymes from a biological sample by electrophoresis, characterized in that the electrophoresis reaction is carried out on an electrophoresis support after depositing a solution of lectin onto the electrophoresis support in a given zone, under conditions which permit interaction between said lectin and the ALP isoenzymes contained in the analysed biological sample, deposition of the lectin solution further being carried out under conditions which are suitable to allow separation of the ALP isoenzymes constituted by the osseous fraction and by the hepatic fraction.
The interaction in question leads to the formation of a complex between the lectin and the ALP isoenzyme until equilibrium is obtained.
The process of the invention enables the osseous fraction of
Lerner David Littenberg Krumholz & Mentlik LLP
Lu Frank
Sebia
Whisenant Ethan C.
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