WO1996010177A1

WO1996010177A1 - Method and device for determination of proteins

Info

Publication number: WO1996010177A1
Application number: PCT/IB1995/000805
Authority: WO
Inventors: Johan Jan Scharstuhl; Nisar A. Shaikh
Original assignee: Spectral Diagnostics Inc.
Priority date: 1994-09-28
Filing date: 1995-09-28
Publication date: 1996-04-04
Also published as: AU3483495A

Abstract

Methods and devices for quantitatively determining the amount of protein in a body fluid such as blood which contains proteins and other substances as contaminants.

Description

METHOD AND DEVICE FOR DETERMINATION OF PROTEINS

FIELD OF THE INVENTION

In invention relates to a method and device for the separation of selected proteins from body fluids. In a preferred embodiment, the apparatus of the present invention is used for the separation of low density lipoproteins from whole blood.

BACKGROUND OF THE INVENTION

Elevated plasma cholesterol and triglycerides levels are associated with increased risk for coronary disease. Among cholesterol containing lipoproteins in plasma, elevated levels of low density lipoproteins (LDL) and decreased levels of high density lipoproteins (HDL) are described to be independent risk factors for premature coronary heart disease (CHD) .

The National Cholesterol Education Program (NCEP) guidelines recommend the identification of individuals at risk for CHD by plasma cholesterol measurements and suggest a direct LDL cholesterol test for more accurate assessment and management of patients at risk. Primary criterion for treatment decisions require means for differentiation and accurate measurement of different lipoproteins' cholesterol levels. The three methods most frequently used to estimate LDL are the ultracentrifugation method, the Friedewald formula method and the antibodies methods. In the following discussion, the term LDL refers to a group of heterogeneous particles (buoyant density greater than 1.019 Kg/L but less than or equal to 1.063 Kg/L) comprised of LDL+IDL (intermediate density lipoprotein or VLDL rem ants) + Lp(a) fractions. The ultracentrufugation method for LDL cholesterol is based upon density differential centrifugation of serum or plasma for 18 hours at 109,000 x g and isolation of 1.006 Kg/1 fraction (chylomicron and very low density lipoprotein, VLDL) and 1.006 Kg/1 infaranate (LDL and HDL fractions) . Cholesterol is then measured in 1.006 Kg/1 infranate before and after precipitation of LDL by the addition of dextran-Mn or heparin-Mn. LDL cholesterol is calculated as follows:

LDL Choi. = 1.006 Kg/1 infranate Choi minus HD1 Choi. (LDL precipitated by dextran-Mn or heparin-Mn)

Such method is both time consuming and labor intensive and is not available in most clinical laboratories.

Friedewald Formula method which is well known and understoo is most commonly used by clinical laboratories and is based upon determination of total cholesterol in serum before and after precipitation of VLDL and LDL fractions by dextran-Mn (HDL remains in solution) and estimation of VLDL cholesterol by measuring total fasting serum triglyceride (TG) levels. LDL chol. is then estimated as follows:

LDL Choi. = Total Choi, minus (HDL CHol. + VLDL estimate) VLDL Choi, estimate = TG levels/5

There are a number of problems associated with the use of this method. Samples must be from fasting patients. The indirect estimation of VLDL Choi, is invalid if the TG levels ar high i.e above 400 mg/dL. Even though the method does not require ultracentrifugation, it involves multiple assays and steps which result in increased variability in the measurements. The direct measurements of LDL with antibodies is based upon selective retention of VLDL and HDL lipoproteins in plasma or serum and quantification of LDL in the effluent with chemical or enzymatic method. The measurement of LDL cholesterol is effected as follows:

LDL Choi = plasma or serum Choi, (after removal of VLDL & HDL fractions)

The only other technique that provides measurement of LDL directly is based upon specific removal of lipoportein other than LDL from serum/plasma by affinity purified goat polyclonal antisera coated to latex particles. In the technique, serum or plasma is obtained by routine laboratory techniques and added to a small cup containing immunoseparation reagents. After brief incubation followed by centrifugation, LDL is recovered and quantitated in the filtrate.

While such a method can be used with non-fasting patients, it still requires initial centrifugation to obtain plasma or serum followed by another centrifugation to isolate LDL fraction.

As presently contemplated, the methods and devices of this invention will be principally of interest in identifying and quantifying protein in blood and blood components such a plasma and serum. It is recognized however, that the invention is also applicable to other body fluids such as urine and saliva and to body fluid components other then proteins. For convenience , the invention will be described principally as applied to the analysis of blood.

SUMMARY OF THE INVENTION

This invention provides a process for identifying and quantitatively determining the amount of a selected protein in a sample of body fluid containing the protein together with contaminating proteins. The process is applicable, for example, and is particularly useful for determining the presence and amount of low density lipoprotein (LDL) in blood in the presence of contaminants such as very low density lipoproteins (VLDL) intermediate density lipoproteins (IDL) , high density lipoproteins (HDL) , and others.

In accordance with the process, the sample to be analyzed i filtered through a composite membrane structure in which a porous, hydrophylic, separator membrane is laminated to the top surface of a porous, hydrophylic reactor membrane to which one o more antibodies reactive with the contaminating proteins or othe contaminants are irreversibly bound or immobilized. The antibodies are selected so that they will react with and irreversibly bind the contaminants but will not bind the protein to be recognized and quantitatively determined.

The first membrane will remove particles such as red blood cells. The second membrane will remove other contaminants as a result of antigen/antibody reactions. As a result, the body fluid which exits the reaction membrane will contain the protein to be determined. They may be bound directly to the membrane or to latex or equivalent beads which are placed in the membrane.

To collect the protein to be determined, the bottom surface of the reaction membrane is brought into capillary contact with a third porous, hydrophilic, collector membrane of known volume. The result will be that the collector membrane will collect a known volume of the body fluid containing the protein in question. The collector membrane is then separated and analyzed by any of a number of known methods to determine the amount of the selected protein present in the original sample.

A number of structures can be designed to conduct the process of the invention. These structures will normally include a separator membrane and a reaction membrane suitably supported together with a collector membrane supported so as to be moved into a capillary relationship with the reaction membrane and to be removed for analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a sketch showing the arrangement of the membranes in the device of this invention.

Fig. 2 is a schematic representation of a presently referred device of the invention. The figure also illustrates one method of practicing the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

The process and devices of the present invention will be understood from the following discussion taken together with the figures.

Fig. 1 is a sketch showing the arrangement of the membranes as they are employed to carry out the process of this invention. The membranes will always be in this configuration no matter how they are supported in a specific device. In the figure, 1 is optional course filter membrane constructed of glass or other equivalent material. Its function is to assist in the removal of large particles such as red blood cells. It is not essential to the invention since in most instances the course particles can be removed by filtering the body fluid through the separator membrane 2.

The separator membrane 2 is a porous, hydrophlic asymmetric membrane. By an asymmetric membrane is understood a membrane having asymmetric pores wherein the average pore size at the top surface of the membrane is larger than the average pore size at the bottom side of the membrane. The techniques for the preparation of asymmetric membranes useful in this invention are known. The asymmetric separator membrane 2 is used with the larger pores directed to the fluid to be separated. This allows the body fluid together with the particulate materials, if any t filter through the membrane with retention of the larger particles in the asymmetric pores. If blood is the body fluid under analysis, the resulting plasma passes through the separating membrane 2 into the reaction membrane 3.

The reaction membrane is also a porous hydrophilic membrane but is not necessarily asymmetric. Its top surface is in capillary contact with the bottom surface of the separator membrane. "Capillary contact" means that the membranes are in sufficient contact, usually complete, laminar surface contact, s that the fluid in one membrane flows into the adjacent membrane because of the influence of capillary forces. In most instances, there will be complete contact throughout the total surface area of the adjacent membranes maintained by the use of spot adhesives.

The function of the reaction membrane is to remove contaminating substances which will interfere with the measurement to be performed. This is accomplished by irreversibly binding to the reaction membrane a sufficient numbe of antibodies so that substantially all of the contaminating materials react and thereby become immobilized or irreversibly bound to the separator membrane.

If for example, blood is under analysis for LDL content, th separator membrane may carry any of a number of known antibodies to HDL, VLDL, IDL, cholesterol and the like. Many of these antibodies are known and readily available, some from commercial sources. These include, for example, anti APOAI, A2, E, C, anti HDL and the like. There will be no antibody to LDL in the reaction membrane with the result that the plasma containing LDL is free to flow into the collector membrane. The collector membrane is not initially in contact with the bottom surface of the reaction membrane. This is to allow sufficient incubation time for the antibodies in the reaction membrane to react with and bind the contaminating substances. This is normally from about 5 to 15 minutes, preferably 8 to 12 minutes. A 10 minute incubation period is usually selected.

At the end of the incubation period, the collector membrane is brought into capillary contact with the reactor membrane. The collector membrane is of a preselected volume. The LDL or other protein under study will be concentrated in the collector membrane. The collector membrane is then separated from the lower surface of the separator membrane and analyzed for the amount of the protein of interest using any known procedure.

The pore size in the separator membrane varies from about 20-30 μm at the upper surface to form about 1.8 to 3.0 μm at the lower surface. The average pore size in the other membranes is from about 3 to 10 μm.

Since only a few drops of blood or other body fluid e.g. about 100 μl is necessary for the practice of this invention, the volume of the membrane employed is very low.

Hydrophilic, porous membranes including asymmetric varieties are well known in the art. They are described, for example, in European Patent 261734 and in U.S. Patent 5,240,862. One suitable membrane is prepared from a mixture of hydrophobic polymers such a polysulfones, polyether sulfauoues and polyetheramides. Polyvinyl pyrrolions are examples of suitable hydrophilic polymers. Nitrocelluloce membranes are also suitable.

A number of known procedures are available for determining the amount of LDL in the collector membrane. These include a wide variety of colori eteric assay based enzymes labels which are visualized for example with hydrogen peroxide. These assays are well know and understood by the skilled artisan. Since the method of assay is not a part of this invention, it need not be discussed in detail.

While the process has been specifically described for the determination of LDL, it will be readily apparent that it is applicable to the determination of other materials by selection of antibodies which are irreversibly bound to the reactor membrane so that the only product to be determined passes throug to the collector membrane.

A number of devices are possible for the practice of this invention. All will provide means for filtering the body fluid to be analyzed through a composite membrane structure including an asymmetric separator membrane in capillary contact with a reaction membrane to which selected antibodies are bound and means for bringing a collector membrane of defined volume into capillary contact with the bottom surface of the reaction membrane after an incubation period. Optionally, there may be a coarse membrane of glass or equivalent material in capillary contact with the top surface of the separator membrane to aid in the removal of coarse particles.

Figs. 2 illustrates the presently preferred devices for concluding the process of this invention. The figure also outlines the complete process in a schematic way.

As shown in Fig. 2, 5 is a lower number which functions as a base for the device. A holder 6 for the collector membrane 7 includes a downwardly extending support 8. The reaction membrane 9 and the separator membrane 10, although shown as exploded in the sketch will be laminated together in capillary contact in the upper housing 11 which will be formed with an aperture for admitting the blood or other body fluid to be analyzed. The figure shows a glass capillary 1. This capillary, or an equivalent device may be used for drawing blood and feeding it through aperture 12 for filtration.

When assembled for use is shown in Fig 2B, the separator membrane 10 and the reaction membrane 9 will be laminated together and permanently fixed in the upper housing 11 which will also contain the collector membrane 9 spaced apart from the laminate comprising the separator membrane 10 and the reaction membrane 9. The distance between the upper surface of the collector membrane 7 and the lower surface of the reaction membrane 9 will be equivalent to the vertical length of the downwardly extending support 8. A glass fiber membrane, not shown, may be laminated to the upper surface of the separator membrane 10 to aid in the filtration process. Typically, the average pore size in the glass membrane is from about 20 to 60 μm.

After applying the sample of the composite of the glass membrane (if used) and the separation and reaction membranes 10 and 9, the fluid under test containing proteins or other contaminants migrate quickly to the reaction membrane wither these produces bind to the irreversibly fixed antibodies and, themselves, become irreversibly fixed. The incubation period for the immobilization reaction to take place is, as indicated above, about 5 to 15 minutes.

At the end of the incubation period, the collector membrane 7 is brought into capillary contact with the reaction membrane 9 and a fixed quantity of the body fluid containing the protein in question flows into the collector membrane 7. To accomplish thi capillary contact, the upper housing 11 is removed from the lowe housing 5 and placed on a flat surface as shown in Figs. 2C and 2D. The support member 8 protrudes from the upper housing 11 as shown in Fig. 2D. The holder 6 is force fit into the upper housing 11 and, as aforesaid, spaced from the lower surface of the separator membrane by a distance which is equivalent to the length of the support member 8. A small amount of pressure on the support member 8 forces the upper surface of the collector membrane 7 into capillary contact with the lower surface of the reaction membrane 9 to permit capillary flow from the reaction membrane 9 into the collector membrane 7 as explained above. This configuration is shown in Fig. 2F.

The collector membrane 7 on holder 6 is them removed from the upper housing 11 as shown in Fig. 2F so that the body fluid may be analyzed. If blood is being analyzed, the fluid will be plasma containing LDL.

Fig. 2G shown the holder 6 with the collector membrane 7 placed in a reaction fluid in a cuvette 14 of a colorimeter 15 for analysis. The reaction fluid will contain all of the necessary reagents to conduct a colorimetric analysis. Of course, other methods of analysis can be employed.

Another presently preferred device of the invention is essentially similar to the device shown in Fig. 2 except that th support 8 of the device of Fig. 2 is replaced with vertical handle which extends outwardly from the holder 6 from the collector membrane 7. In the operation of this device, the holder 7 is again force fit in the upper housing 11 in a positio such that the collector membrane 7 is spaced away from the

-10- reaction membrane 9 until the end of the incubation period whereupon slight finger pressure is applied to the holder 6 to bring the collector membrane 7 into capillary contact with the lower surface of the reaction membrane 9. After a selected volume of filtered body fluid containing the protein of interest has been collected in the collector membrane 7, the holder 6 is removed for analysis of the filtered body fluid by any selected method.

It should be understood that the invention has been described in connection with specific embodiments thereof. Various modifications will occur to those skilled in the art without departing from the spirit and scope of the invention as set forth in the appended claims. All such embodiments are within the scope of the invention.

Claims

1. A process for identifying and quantitatively determinin the amount of a selected protein in a sample of body fluid containing said protein in admixture with other contaminating proteins; said process comprising filtering the sample through a composite membrane structure comprising a first separation membrane and a second reaction membrane; the separation membrane being a porous, hydrophilic, asymmetric membrane having top and bottom surfaces with decreasing average pore size running from the top surface to the bottom surface whereby large particles in the sample may be removed; the second reaction membrane being a porous hydrophilic membrane having top and bottom surfaces, said top surface being in capillary contact with the bottom surface o the separation membrane, said reactor membrane containing irreversibly bound antibodies reactive with said contaminating proteins; and after the contaminating proteins have reacted with the antibodies and thereby become immobilized, bringing a third porous, hydrophilic, collector membrane of predetermined volume having top and bottom surfaces into capillary contact between th bottom surface of the reaction membrane and the top surface of the collector membrane thereby to collect in the collector membrane a measured volume of filtered sample containing the selected protein to be quantitatively determined and, thereafter quantitatively determining the amount of the selected protein in the collector membrane.

2. A process as in claim 1 wherein the filtration efficiency of the composite membrane is improved by placing a glass fiber membrane on the top surface of the separation membrane.

3. A process as in claim 1 wherein the body fluid to be analyzed is blood.

4. A membrane structure useful for the quantitative determination of a selected protein in a body fluid which contains the selected protein in admixture with other contaminating proteins which structure comprises a first separating membrane which is a porous, hydrophilic, asymmetric membrane having top and bottom surfaces with decreasing average pore size running from the top to the bottom surface the pore sizes being selected to permit separation of large particles in the sample; the second reactor membrane being a porous, hydrophilic membrane having top and bottom surfaces, the top surface being in capillary contact with the bottom surface of the separator membrane, said reactor membrane containing irreversibly bound antibodies reactive with said contaminating proteins, the reaction thereby immobilizing the contaminating proteins in the reactor membrane; and a third porous, hydrophilic membrane having top and bottom surfaces of predetermined volume the top surface of which is supported so as to be brought into capillary contact with the bottom surface of the reactor membrane after the contaminating substances have been immobilized in the reactor membrane by reaction with the antibodies to permit flow into the reactor membrane of a fixed volume of the filtered body fluid containing the protein to be determined and free of contaminating proteins.

5. A membrane structure of claim 3 wherein the filtration efficiency is improved by placing a glass fiber membrane on the top surface of the separation membrane.