WO1987006345A1

WO1987006345A1 - Colorimetric ratioing immunoassay

Info

Publication number: WO1987006345A1
Application number: PCT/US1987/000569
Authority: WO
Inventors: Miles G. Hossom
Original assignee: Murex Corporation
Priority date: 1986-04-11
Filing date: 1987-03-19
Publication date: 1987-10-22
Also published as: AU7358187A

Abstract

A method for the determination of the relative amount of a component of an analyte of interest, present in a specimen, said analyte being composed of at least two components comprising: (a) insolubilizing an antibody directed against said component on a solid support material in a delimited area defining a reaction zone; (b) contacting with said insolubilized antibody a specimen containing said analyte to such that substantially all of said component binds to said antibody while said specimen is retained on or within said reaction zone; (c) reading said reaction zone to determine the relative amount of analyte present; (d) washing said reaction zone to remove only substantially all material not bound to said antibody; (e) reading said reaction zone to determine the relative amount of component remaining that is bound to said antibody; and (f) calculating from said readings the ratio of the said component to said analyte.

Description

COLORIMETRIC RATIOING IMMUNOASSAY

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to an immunological method of determining the relative amount of a component of an analyte of interest present in a specimen.

PRIOR ART Immunodetection has been used in the field of electophoresis to measure the amount and location of separated protein bands on a nitrocellulose sheet (Blangarin, et al . , Clinical Chemistry, vol. 30, No. 12, 1984, p.2021-2025). In such a method a first antibody, directed against the different fragments or classes of proteins (e.g., human im unoglobulins), is added to the nitrocellulose. A second antibody, directed against the first antibody and labelled with a signal generating material, is allowed to react, thus forming a protein:first antibody: labelled second antibody-label complex. The label, such as an enzyme, is contacted with an amount of an appropriate substrate and a color appears at the given bands where protein is present. The quantity of color formed is measured photometrical ly. In a Western Blot technique for qualitative gel electrophoresis analysis, where a mixture of antigens is being analyzed, a labelled antibody, directed against the antigens of interest, is added to the separated bands. When the label is developed the color indicates the presence of the band, which is compared to a sample mixture run simultaneously which has been developed with a dye or stain, such as Coomasie blue, which will stain all the protein bands in the sample. By comparing the two sample results one can determine the presence or absence of a particular band, and, therefore, antigen.

While these techniques utilize the principle of colorimetric development and measurement they have significant drawbacks in terms of their effectiveness in a rapid diagnostic test. In addition to time, cost and complexity, these techniques do not have the accuracy needed for a highly sensitive immunoassay for the quantisation of individual antigens or analytes.

In the field of blood analysis and monitoring tests, glycosylated hemoglobin is an important substance of interest. Its proportion to the total hemoglobin in a blood sample is a useful indicator for maintenance of a diabetic patient where diet and insulin therapy must be carefully balanced to maintain the patient in reasonable health. In these, as well as healthy individuals, total hemoglobin content in the blood may vary due to nondiabetic related influences. A healthy individual will normally have a glycosylated hemoglobin level of about 4-5% of the total hemoglobin in the blood, whereas a poorly controlled diabetic may demonstrate glycosylated hemoglobin levels which are as much as 15% of the total hemoglobin. Current methodologies for measurement include elution from resin columns, high performance liquid chromatography, and, recently, PBA affinity chromatography. Typically, such tests involve two sample preparations and two measurements, both of which have their own inherent errors and variables. Using two tests, then, multiplies the inaccuracy of the method. There is a need for a test that can conveniently combine both testing sequences into an integrated testing procedure; that is fast, inexpensive, reliable and specific for glycosylated hemoglobin or similar ratio-type analyte measurements.

SUMMARY OF THE INVENTION

The present invention provides a method for colorimetrical ly quantifying the relative amount of a component as a part of a total sample within a single test format. Briefly stated, the present invention comprises a method for the semi-quantitation of a component of an analyte of interest, present in a specimen, where the analyte is composed of at least two components. In particular, the method consists of (a) immobilizing an antibody directed against the component on a solid support material in a delimited area defining a reaction zone; (b) contacting with the immobilized antibody a specimen containing the analyte such that substantially all of the component binds to the antibody while the specimen is retained on or within the reaction zone; (c) reading the reaction zone to determine the relative amount of analyte present; (d) washing the reaction zone to remove substantially all material not bound to the antibody; (e) reading the reaction zone to determine the relative amount of component remaining bound to the antibody; and (f) calculating from the readings the ratio of the said component to the analyte.

DESCRIPTION OF THE INVENTION An immunoassay method which can provide in a single test format an quantitative ratio measurement of a specimen and a constituent thereof can be highly useful where current testing procedures require two or more separate tests to be performed. In certain biological systems the amount of a component part as a percentage of the total amount of substance can provide an indication of the presence or progression of a disease state, such as diabetes, sickle cell anemia, heart disease, or the like.

In a preferred embodiment the assay can be performed in a three dimensional inert porous filter matrix means such as glass fiber, scintered glass, paper, or the like. Immobilized or insolubilized on or within a delimited area of the filter defining a reaction zone is an antibody directed against the component part of the total sample. When fluid specimen is added to the filter the component part will immunofogically bind to the antibody, thereby becoming bound to the filter. A first absorbence reading of the filter area containing the specimen composed of the component part of interest and the-total sample, which correlates with the amount of total sample present in the specimen, is taken. Subsequently, the reaction area is washed with an appropriate buffer or other solution to remove the unbound sample. A second absorbence reading is taken of the reaction zone, which yields a measurement of the quantity of component part that is bound to the antibody retained on or within the filter. The ratio of the two measurements gives the percentage of component part to the total sample. By conducting both the measurements in a single reaction zone in one test procedure the sampling and user error are reduced. The absorbence readings are calibrated with respect to each other requiring no external standard; therefore, any instrument drift, reagent or user variances, or the like, are cancelled out. Where the sample is inherently capable of absorbing light, such as hemoglobin, the measurements may be taken without further treatment. However, where the analyte is not naturally "colored", or capable of absorbing a significant fraction of light, the material may have bound to it an absorptive molecule, such as a stain or dye.

Sensitive dyes such as fluorescein, Sudan Black B, Oil Red 0, or the like can be employed; the particular staining or coloring material is chosen on an individual basis and optimized depending on the analyte. While the method of this invention is suitable for coioπ^'metric or spectrophotometric measurement of material absorption, other measurement techniques that produce a detectable signal are suitable. Coioπ^'metric measurement presents certain advantages over other methods in that no second antibody is required, as compared to a sandwich immunoassay which uses a capture antibody and an antibody-label conjugate which binds to the analyte, thereby "tagging" the analyte with a measurable signal. Since fewer steps are involved the assay procedures is simplified for the user; moreover, expensive antibody-label conjugate is not needed, since the staining or coloring material serves the same purpose. There are circumstances, however, where other detection techniques are preferable to staining, such as when very low concentrations of component part are being measured which would not be detectable without some kind of signal amplification technique more effective than a stain or dye. Under such conditions a signal generator such as an enzyme and corresponding substrate can be used, following one of several enzyme immunoassay procedures known in the art. Alternatively, fluorescent, luminescent, chemi-or bio-luminescent, ferromagnetic or radioisotopic materials can be used with their appropriate assay methods. The novelty of using a ratio measurement, however, remains. In one embodiment of this invention the percentage of 5 glycosylated hemoglobin ("HbAlC") in total hemoglobin can be measured. The determination of such a ratio is of great importance to patients with diabetes, as the varying percentage indicates to what extent and how controllable their diabetes is. Clinically, HbAlC values are used most frequently to assess long

10 term glucose control in diabetes, especially in insulin-dependent diabetics whose glucose levels are very labile, and in whom blood and urine glucose measurements exhibit significant daily variation. HbAlC measurements reflect the level of control present over the proceeding 100-120 days, and is especially helpful when renal

15 thresholds are high or low. A healthy individual will normally have an HbAlC level of about 4-5% of the total hemoglobin in the blood, whereas a poorly controlled diabetic may demonstrate HbAlC levels which are as much as 15% of the total hemoglobin. (Jacobs, et al., Laboratory Test Handbook, Lexi-Comp Inc., Ohio,

201984, p. 119). Moreover, because hemoglobin is a molecule inherently colored with the he e moiety, it lends itself well to colorimetπ^'c measurement.

In the performance of such an assay for HbAlC, a patient's blood sample containing red blood cells is lysed to expose and release the

25 hemoglobin. This can occur prior to, during or after addition to the filter matrix containing immobilized antibody. The sample solution is added to the matrix in a volume sufficient to saturate an area of the matrix. The total sample is then colorimetrically read by measuring the absorbence of the light passed through the filter matrix area containing the reactants. The measurement is recorded. The matrix is incubated for a sufficient length of time to permit the HbAlC to bind to the antibody on or in the matrix. Preferentially, the incubation period should be of adequate duration to permit the antibody to bind substantially all of the HbAlC on or in the matrix; however, a controlled incubation time which permits binding a representative portion of the HbAlC is also satisfactory. The filter matrix is subsequently washed with an appropriate solution to remove substantially all unbound hemoglobin and other unbound material. A second absorbence reading is then taken of the HbAlC remaining in the filter matrix. The ratio of the absorbence readings is proportional to the ratio of glycosylated hemoglobin to total hemoglobin in the sample. The precise concentration of HbAlC can, if necessary, be determined by applying the absorbence ratio to a separately determined measurement, employing a standard method.

Another preferred embodiment is the determination of the percentage of hemoglobin S ("HbS") present in a blood specimen as an indication of sickle cell anemia. This inherited disease is caused by a defect of a single amino acid in the hemoglobin molecule. Individuals who inherit this abnormal gene from only one parent are called heterozygous individuals possessing the sickle cell trait, and have erythrocytes (red blood cells) that contain only 25% - 45% HbS, exhibiting no abnormal health patterns. Individuals who inherit the abnormal gene from both parents are ho ozygous, and have the sickle cell anemia disease. Their erythrocyte population contains 75% or more of HbS. Thus, the amount of HbS which is present is a prominent factor in determining the severity of the disease. (Seiverd, He atology For Medical Technologists, 4th ed., Lea & Febiger, Philadelphia, 1972, p. 608.) A ratioing immunoassay using HbS as the component follows the same procedure as that for HbAlC.

Similarly, the amount of other abnormal hemoglobins or abnormal concentrations of normal hemoglobins, such as but not limited to fetal hemoglobins ("HbF"), can be determined as a fraction of total hemoglobin.

In another embodiment a noncolored nonabsorptive protein can be measured using a sensitive protein stain. Lipoproteins ("LPs") are complexes or compounds containing lipid and protein. Almost all lipids in plasma are present as lipoproteins, are characterized by their densities, and can broadly categorized as high density lipoproteins ("HDL") or low density liproproteins ("LDL"). Their presence in plasma is normal, but the ratio of LDL to total lipoproteins ("TLP") has been shown to be an indication of the relative risk of coronary arterial disease, particularly coronary atherosclerosis. High LDL is a direct risk factor for such disease, because LDL is the major transport protein for cholesterol in plasma. The LDL:TLP ratio provides a better indication of increased risk than either measurement alone.

The present invention can determine the ratio of LDLrTLP by combining a serum sample and a mixture of at least two of monoclonal antibodies, one directed against LDL and another directed against HDL. These two monoclonals are designed so that one of them is acid-switchable or acid-labile; i.e., the antibody dissociates from the antigen at a particular pH, while being associated to the antigen at a different pH. These monoclonal antibodies are designed for this purpose by screening hybridoma cell populations to isolate those clones which are acid sensitive at a certain pH. After incubating to facilitate immunological binding of LDL & HDL to their appropriate monoclonal antibody, the mixture is added to a filter matrix, which traps and immobilizes the immune complexes. Alternatively, the monoclonal antibodies can be pre-immobilized on or within the filter in a delimited reaction area, to which is added the serum sample. In either case the reaction area washed with a neutral solution, e.g., pH 7.4, to remove unbound material from the reaction area. The complexes are contacted with a sensitive specific stain which will color only the lipoprotein molecules. An illustration of a suitable stain or dye, but not a limitation, is Sudan Black B, which is a diazo dye specific for fats and lipids. Another example is Oil Red 0, which is a weakly acidic diazo dye used to identify neutral fats. Neither stain will color the antibody. By using a stain the need for a second antibody conjugated to a label is obviated. These are, though, certain instances, such as the analyte being present in extremely low concentrations, where a signal amplification means is a arranged, such as an enzyme and substrate, a fluorophore, or any other commonly used label.

After the stain has been applied and the excess washed away, an absorbence reading is taken of the reaction zone to determine the total amount of lipoprotein present (LDL plus HDL). The reaction zone is subsequently washed again, this time with an appropriate solution having a lower pH, such as pH 4.0, which causes the acid switchable monoclonal antibody to dissociate from its bound component, either LDL or HDL. In this manner either LDL or HDL will remain not both, depending on which is bound to the acid sensitive monoclonal antibody. The reaction zone is read again to provide an absorbence measurement of the remaining component, preferably LDL, bound to the acid stable monoclonal antibody. From the absorbence readings the desired ratio of LDL: or present in the patient sample can be calculated by subtracting the LDL measurement from the TLP measurement, which gives the HDL; in other words, HDL = TLP - LDL.

Alternatively, rather than using an acid switchable monoclonal antibody, Staphylococcus Protein A ("Protein A") can be used to bind to the complexes formed of LDL and an appropriate labelled monoclonal antibody, and, HDL and an appropriate labelled monoclonal antibody. For example, one of the antibodies can be IgG2a and the other could be IgG2b . An unique property of Protein A is that at a pH greater than 4.5 it forms a stable con.plex with IgG2a and IgG2b, but at a pH less than 3.5 IgG2a dissociates from Protein A, thereby permitting its removal from the reaction area with the correlative decrease of label present, a means for detecting the amount of LDL or HDL present as the remaining monoclonal antibody complex still bound to the, Protein A is provided.

The analyte can be any substance that is inherently colored or capable of being colored. By colored it is meant that the analyte, or molecule attached thereto, is capable of being quantified by colorimetric means. Naturally colored materials which exist in the body include hemoglobin, ferritin, cytochrome C, and the like. Hemoglobin is perhaps the most commonly analyzed fluid in the body, and the hemoglobin molecule can provide the basis for a quantitative absorbence measurement. Typically, the analyte is a protein, or of a proteinaceous structure. The amino acid structure of noncolored proteins has been well researched, and many methods have been developed to stain, dye or otherwise color proteins. Generally, these methods involve the covalent or ionic bonding of a dye or staining molecule, such as bromocresol green or Biuret's reagent, where the intensity of the color is proportional to the amount of compound it is bound to.

Other substances include drugs, hormones, vitamins, enzymes, antibodies, polysaccharides, bacteria, protozoa, parasites, fungi, viruses, cell and tissue antigens, and the like.

Samples can be obtained from any of the biological fluids commonly used for analysis such as but not limited to, whole blood, serum or plasma, saliva, feces, sputum, mucus, cerebral spinal fluid, urine, pus, wound exudate, cell or tissue extracts, and the like. It is preferable that the sample not contain extraneous undesirable colored matter which might interfere with an accurate absorbence measurement of the colored analyte.

Colorimetry is a convenient and accurate means of measuring the optical density, also called the absorbence, of a solution. If light is passed through a solution containing a colored compound, certain wavelengths are selectively absorbed. The resultant color observed is due to the transmitted light. Measuring light absorption aids scientists in both the identification and quantification of substances. The amount of absorption of a compound at a given wavelength is a function of compound concentration.

The assay of the present invention is conducted using a solid phase medium that will lend itself to colorimetry. A preferred embodiment employs a filter matrix material that is inert, porous and light transmitting. Glass fiber filter paper is convenient, inexpensive and reliable, but other materials such as nitrocellulose, paper, polymeric or other plastics, and the like, as well as other structures such as beads or particles, wells, tubes, and the like, are usable so long as they are inert, porous and light transmitting. For the purpose of this invention, a filter matrix will be described as an illustration of a solid support surface.

The matrix is three-dimensional wherein the interstitial voids therein define and can hold a volume. When the sample mixture to be analyzed is added to the matrix the mixture will generally fill the void volume of the matrix and the volume of the mixture in unit area of the matrix will be defined by the void volume. In this manner, an absorbence reading can be taken of a constant volume retained by the matrix, thereby affording reproducible results.

The analyte being measured is preferentially immobilized and retained within the matrix by trapping or binding to the matrix a capture antibody directed against the analyte of interest prior to or concurrent with the addition of sample containing the analyte. By acting as a selector, the antibody recognizes the particular spatial and polar configuration on the analyte molecule, called an idiotypic determinant or an epitope. By specifically selecting and binding only a certain component the antibody is useful in an assay for quantifying one component contained in a solution of any components.

Monoclonal antibodies provide even greater selectivity and specificity because of their unique structure and properties. Moreover, monoclonal antibodies can be designed to recognize only one specific protein whereas a polyclonal antibody will cross-react with proteins that are similar yet not identical in structure. Monoclonal antibodies are prepared according to the method described by Milstein and Kohler in Nature 256:495-497, 1975. The method is well known and need not be reviewed here. The filter matrix is washed with a relatively colorless solution such as but not limited to water, salt solution, buffer, detergent and the like, and the material not bound to the immobilized antibody is removed and replaced by the colorless wash solution. During this process the volume of liquid contained in the matrix remains relatively constant and equal to the matrix void volume.

To improve the retention of analyte or to reduce nonspecific binding a blocking agent, such as gelatin, serum, B.S.A., skim milk, egg albumin, Tween-20, or the like, can be added to the solid support surface.

It is intended that the ratioing method be applicable to a broad range Of analytes and systems where a measurement of the proportion of component to total sample is required.

The invention will be further described in connection with the following examples which are set forth for purposes of illustration only.

EXAMPLES Example 1

Assay procedure for determination of the ratio of glycosylated hemoglobin:total hemoglobin.

One hundred microliters of a monoclonal antibody directed against glycosylated hemoglobin (HbAlC) are added to glass fiber filter paper (Micro Filtration Systems D) defining a delimited reaction area and incubated so that the antibody becomes immobilized on or in the matrix pores. Five drops of a patient blood sample are spotted in the reaction area containing the immobilized monoclonal antibody. An absorbence reading of the reaction area is then taken as an indication of the amount of total hemoglobin. The reactants are incubated to permit the formation of a complex between HbAlC and the immobilized monoclonal antibody. The reaction area is then washed with Tπ^'s buffered saline to remove unbound hemoglobin and other unbound materials. A second reading of the absorption of the reaction area is taken as an indication of the amount of HbAlC present that has bound to the immobilized monoclonal antibody. The ratio of absorbencies of HbAlC to total hemoglobin is calculated and the per cent of HbAlC present in the patient is thereby obtained.

Example 2

Assay procedure for determination of the ratio of hemoglobin S (HbS):total hemoglobin.

The procedure according to Example I is followed wherein substituted for HbAlC is HbS.

Example 3

Assay procedure for determination of the ratio of hemoglobin F

(HbF):total hemoglobin using Protein A binding.

Staphylococcus Protein A is first immobilized on a filter matrix. In a separate reaction vessel a specimen containing HbF is mixed at neutral pH with fluorescin-labeled mouse IgG2b monoclonal antibody directed against HbF and fluorescein-labeled mouse IgG2a monoclonal antibody cross-reacti e with all hemoglobin antibody.

The mixture is allowed to incubate to permit binding of antibody to hemoglobin until equilibrium is reached. The reaction mixture is then added to a delimited area of the filter matrix containing the immobilized Protein A and incubated so as to permit binding of Protein A to the Fc portion of IgG2a and IgG2b. The reaction zone is then washed with a 0.05M Tris buffered saline solution ("TBS") at pH 7 to remove unbound material. The reaction zone is read for the amount of fluorescent signal generated. Subsequently, the reaction zone is washed with TBS at pH 4.0 to elute out IgG2a:total hemoglobin complex. The reaction zone is again read for the remaining amount of fluorescent signal associated with the IgG b: HbF remaining bound to immobilized Protein A. From these two measurements the ratio of the second measurement correlative with the amount of Hb ) to the first measurement (correlative with total hemoglobin) produces the relative amount of HbF present in the specimen.

Example 4

Assay procedure for determination of the relative amount of low density lipoprotein ("LDL") present in a specimen using Sudan Black B stain:

A specimen containing LDL is combined at neutral pH with a mixture of a monoclonal antibody directed against LDL and a monoclonal antibody cross-reactive with all lipoprotein. The latter monoclonal antibody is selected so as to be acid switchable; i.e., the antibody binds with the analyte in a neutral pH range, while it will dissociate from the analyte within, an acidic pH range. The mixture is incubated to permit immunological binding to occur. The resultant reaction mixture is added to glass fiber filter paper (Micro Filtration Systems, Dublin, California) in a delimited area. Subsequently, an effective amount of Sudan Black B stain is added to the reaction zone so as to stain the lipoprotein present. The area is washed with neutral pH buffer solution to remove unbound material. Colorimetric reading is taken of the reaction area to measure the absorbence of the material immobilized therein. Then, a buffer solution at about pH 4.5 is added to facilitate the dissociation and elutin of the acid switchable monoclonal antibody and the lipoprotein bound thereto. A second absorbence reading is taken of the reaction zone to measure the remaining stained LDL bound to the immobilized monoclonal antibody. From the two readings the amount of LDL to total lipoprotein (or LDL:HDL) can then be calculated.

While the invention has been described in connection with certain preferred embodiments, it is not intended to limit the scope of the invention to the particular form set forth, but, on the contrary, it is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for the determination of the relative amount of a component of an analyte of interest, present in a specimen, said analyte being composed of at least two components comprising: (a) insolubilizing an antibody directed against said component on a solid support material in a delimited area defining a reaction zone; b) contacting with said insolublized antibody a specimen containing said analyte such that substantially all of said component binds to said antibody while said specimen retained on or within said reaction zone;

(c) reading said reaction zone to determine the relative amount of analyte to present;

(d) washing said reaction zone to remove substantially all material not bound to said antibody;

(e) reading said reaction zone to determine the relative amount of component remaining that is bound to said antibody; and

(f) calculating from said readings the ratio of said component to said analyte. 2. The method of claim 1 wherein said analyte and said component are any substances capable of absorbing a detectable amount of light.

3. The method of claim 1 wherein said analyte and said component are capable of selectively binding to a stain or dye.

4. The method of claim 3 further comprising the steps of staining said component and said analyte with any suitable substance selective for said analyte and said component.

5. The method of claim 1 wherein said antibody is a polyclonal or monoclonal antibody. 6. The method of claim 1 wherein said antibody is insolublized by immobilizing it on or within a filter matrix means.

7. The method of claim 6 wherein said filter matrix means is a three dimensional inert porous structure capable of transmitting light while containing a defined volume of fluid within its interstices.

8. The method of claim 7 wherein said filter matrix means is composed of a material that is or can be made light transparent or light translucent.

9. The method of claim 8 wherein said filter matrix means is composed of a material selected from paper, glass fiber filter paper, cellulose, nitrocellulose, polymers or other plastics, nylon, or scintered glass.

10. The method of claim 9 wherein said antibody is immobilized on or within said filter matrix means prior to the addition of said specimen to said filter matrix means.

11. The method of claim 9 wherein said antibody is immobilized on or within said filter matrix means simultaneously with the addition of specimen to said filter matrix means.

12. The method of claim 1 wherein said antibody is bound to insoluble particles capable of being entrapped on or within the pores of said filter matrix means.

15. A method for the determination of the relative amount of glycosylated hemoglobin (HbAlC) present in a specimen containing total hemoglobin, comprising: (a) insolubilizing a monoclonal antibody directed against HbAlC on or within a porous inert glass fiber filter paper in a delimited area defining a reaction zone; b) contacting with said monoclonal antibody a specimen containing said HbAlC in a manner favoring the formation of a complex of monoclonal antibody and HbAlC while unbound hemoglobin is retained in said filter; (c) measuring colorimetrically said reaction zone for the absorbence of material present therein;

(d) washing said reaction zone to remove unbound material;

(e) measuring colorimetical ly said reaction zone for the absorbence of material present therein; and (f) calculating from the measurements of step (c) and step (f) the ratio of HbAlC to total hemoglobin in said sample. 16. The method of claims 1 or 15 further comprising the step of blocking said solid support surface with any suitable blocking agent to prevent the non-specific binding of specimen or analyte. 17. The method of claim 15 wherein said specimen is hemoglobin.

18. A method for the determination of the relative amount of a first component with respect to a second component of an analyte of interest, present in a solution, said analyte being composed of at least two components, comprising: (a) combining a specimen containing first and second components with a mixture of a first and second monoclonal antibodies, which are each directed against only one of said components in a manner favoring the formation of two different complexes of said first component with said first monoclonal antibody and said second component with said second monoclonal antibody; b) adding any of the complexes formed in step (a) to a delimited area of a solid support surface capable of retaining said complexes thereby defining a reaction zone; (c) adding an effective amount of Staphylococcus Protein A

("Protein A") under conditions favoring the binding of said complexed first and second monoclonal antibodies to said Protein A and the binding of said Protein A to said solid support surface; (d) washing said reaction zone to remove unbound material;

(e) reading said reaction zone for the amount of first complexed component and second complexed component;

(f) washing said reaction zone to remove substantially all of only one of either first complexed component or second complexed component;

(g) reading said reaction zone for the relative amount of the remaining complexed component; and

(h) calculating the relative proportion of either said first component to said second component or either said first component or second component to the sum of said components.

19. The method of claim 17 wherein said first and second monoclonal antibodies are labelled with any suitable labelling material.

20. The method of claim 19 wherein said label is selected from an radioactive isotope, an enzyme, fluorescent, luminescent, chemiluminescent, bioluminiescent or ferromagnetic material.

21. The method of claim 18 wherein said first monoclonal antibody is IgG2a and said second monoclonal antibody is IgG2b.

22. The method of claim 21 wherein either first or second component is low density lipoprotein and the other component is high density lipoprotein. 23. The method of claim 18 wherein the washing in step (c) is accomplished using a solution with a pH above about 4.5 and the washing of step (e) is accomplished using a solution that is less than about pH 4.0 so as to remove substantially all of said complexed IgG2b.

24. The method of any one of claims 1, 19 or 21 wherein said analyte or said component is applied to said filter matrix in a manner so as to concentrate the flow of fluid onto said reaction zone sufficient to produce a readable signal if a detectable quantity of material is present.