JPH0437382B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention is directed to total bilirubin, conjugated or unconjugated, in clinical chemistry.
Concerning the colorimetric analysis of bilirubin. The present invention further relates to dry analytical elements useful for this analysis. [Prior Art] Bilirubin is a degradation product of hemoglobin. In healthy individuals, bilirubin released from aging or damaged red blood cells in the body is excreted or broken down into other derivatives. However, in some cases, excessive hemolysis or overproduction of bilirubin results in excessive amounts of bilirubin in the body, such as in the case of liver damage. The presence of excessive amounts of bilirubin in the human body is always jaundice, which is characterized by a significant increase in the concentration of bilirubin in the serum. Increasing evidence indicates that the presence of excessive amounts of bilirubin in the blood undesirably increases bilirubin concentrations within body cells and interferes with various cellular phenomena. The clinical diagnostic significance of bilirubin measurements in tests regarding the function of the liver and other related organs is clear. Bilirubin exists in several different forms in human body fluids, such as bile and serum, and these forms are generally conventionally known as conjugated bilirubin (B _c , mono- and di-conjugated forms), unconjugated bilirubin (B _u , bilirubin (also known as indirect bilirubin) and delta bilirubin (also known as biliprotein). The total bilirubin content B _T represents the sum of all forms of bilirubin. Various colorimetric assays for bilirubin are known. At one time, the most widely used analytical operation was
The diazo process used a coupling reaction between bilirubin and a diazonium salt to form a detectable pigment. This method has its drawbacks and has undergone many variations and improvements over the years. Other analytical methods include direct measurement of bilirubin, a yellow pigment. However, this technique is susceptible to spectral interference from hemoglobin and other components of the liquid sample. This method is also hampered by proteins, such as albumin, to which bilirubin can bind and shift the absorption intensity and peak wavelength. Significant advancements in the prior art include U.S. Patent No.
It is described in the specification of No. 4069017. The method for the analysis of bilirubin described herein is carried out using a dry multilayer analytical element containing an interactive mordant in the reagent layer that binds to bilirubin to produce a detectable product. Ru. No other reactive compositions are used in the analysis. The element further includes a porous diffusion layer and a radiation blocking layer. Reactive mordants include binder substances such as gelatin or its derivatives, poly(vinyl alcohol),
Poly(vinylpyrrolidone) and acrylamide polymers (i.e. homopolymers of acrylamide)
(See column 15, lines 7-39). The examples (column 23) of said patent teach the use of gelatin as the matrix material. Indeed, such analytical elements for the determination of bilirubin containing gelatin as binder material have been commercially available for many years. Problems to be Solved by the Invention Although conventional analytical elements represent an advance in the prior art, improvements are desired for bilirubin analysis. These elements typically contain 50 independent analysis elements.
It is sold as a cartridge containing up to 100 pieces.
It has been observed that due to the element's sensitivity to environmental conditions when stored for long periods of time, uniform results are often not obtained within the element, but rather deviations occur due to changes in the background color. [Means for solving the problem] The analytical element for measuring complexed, uncomplexed or total bilirubin consists of a positively charged reactive mordant having at least one binding site for bilirubin (this mordant is a material other than gelatin or its derivatives). , 2-20
a substantially gelatin-free mordant layer containing (dispersed in a hydrophilic binder material present in an amount of g/m ² do not have. The method for analyzing complexed, uncomplexed or total bilirubin consists of: A. A liquid sample suspected of containing complexed or uncomplexed bilirubin is treated with a positively charged reactive mordant having at least one binding site for bilirubin (the mordant is gelatin or its like). other than derivatives,
a colorimetric and fluorescent reactive composition for bilirubin other than a mordant, comprising a substantially gelatin-free mordant layer (dispersed in a hydrophilic binder material present in an amount of ² to 20 g/m2); and determining the amount of complexed, uncomplexed or total bilirubin bound to the reactive mordant. Embodiments The present invention relates to the measurement of bilirubin (complexed or uncomplexed form or the sum of both) in aqueous liquids. In particular, the invention can be used to analyze biological fluids of animals or humans, preferably humans. Such fluids include, but are not limited to, whole blood, plasma, serum, lymph, bile, urine, spinal fluid, sputum, sweat, etc., as well as excreta. The present invention can also analyze fluid preparations of human or animal tissue, such as skeletal muscle, heart, kidney, lung, brain, bone marrow or skin. The method of the invention is carried out using a dry multilayer analytical element comprising a support having a number of separate layers thereon. Such a support is any suitable dimensionally stable, preferably non-porous, transparent (ie, radiation transparent) material that is transparent to electromagnetic radiation at wavelengths from 200 to 900 nm. The support selected for a particular element should be compatible with the intended detection method (eg, reflectance spectroscopy). Useful supports can be made from paper, metal foil, polystyrene, polyester, polycarbonate, cellulose ester, and other materials known in the art. The outermost layer of the analytical element of the invention is a porous diffusion layer made of any suitable fibrous or non-fibrous material or a mixture of one or both. The porosity and average pore size of this zone can vary depending on the fluid to be tested. Useful diffusion layers include fibrous materials mixed with suitable binder materials or woven into fabrics as described in U.S. Pat. No. 4,292,272, U.S. Pat. and the same No.
4430436 and JP 1982-101760, using polymeric compositions or particulate materials, such as beads bonded with or without bonding adhesives. It can be manufactured using The diffusion zone is isotropically porous,
That is, it is desirable that the porosity be the same in all directions of the zone, such that it is caused by continuous spaces or pores between particles, fibers, polymer strands. The analytical element according to the invention may have more than one layer, each layer being made of the same or different materials and having the same or different porosity. The analytical element of the invention further comprises a radiation blocking layer comprising a radiation blocking pigment, such as titanium dioxide or barium sulfate, distributed in a suitable hydrophilic binder material. Such hydrophilic binder materials include gelatin and its derivatives, polysaccharides, cellulose derivatives, poly(vinyl alcohol), poly(vinylpyrrolidone), acrylamide polymers and others,
Includes conventionally known ones. This binder material is preferably gelatin. The reactive mordant needed to combine with bilirubin to produce a detectable product is present in the mordant layer located below the radiation blocking layer. The reactive mordant and the hydrophilic binder material that distributes it are detailed below. Other layers, such as subbing layers or filter layers, can optionally be included in the analytical elements of the invention. All layers in the element are generally in fluid contact with each other (meaning that fluids and non-mordanting reagents and reaction products can pass or move between overlapping regions of adjacent layers). Elements of the invention do not contain reactive compositions that produce a colorimetric or fluorometric response in the presence of bilirubin, other than the reactive mordants described below. In particular, the analytical element of the invention is compatible with the literature relating to the determination of diazonium salts and bilirubin, such as US Pat. No. 4,069,016.
No. 4,548,905, which forms the detectable species known in US Pat. No. 4,548,905. Reactive mordants useful in the practice of the present invention include the mordants described in U.S. Pat. ). Generally, these mordants have one or more binding sites for bilirubin and include a hydrophobic organic matrix and at least one group having a charge-bearing cationic group. Such mordants may be monomeric or polymeric, but preferred mordants are homopolymers and copolymers having the properties described above. These bind bilirubin in complex and uncomplexed form. Particularly useful reactive mordants have the formula: [In the formula, A represents an organic group, Q represents a chemical bond or bonding group, M ⁺ represents a quaternary ammonium or phosphorinium group, and X ⁻ represents an acid anion]. Specifically, A in the above structural formula represents a part of the polymer skeleton. A will obviously vary depending on the particular polymer backbone used. However, particularly good results have been obtained when A represents an alkylene group, such alkylene groups generally containing 2 to 10 carbon atoms. Copolymers useful as reactive mordants include 10 to 90% by weight of repeating units as described above and non-interfering (i.e., do not chemically or physically interfere with bilirubin mordant).
Includes those having 10 to 90% by weight of additional repeating units. Monomers providing non-interfering units and imparting hydrophobicity to the resulting copolymer include aliphatic and aromatic hydrocarbons such as olefins and substituted olefins, styrene and substituted styrenes, alkyl acrylates and alkyl methacrylates and derivatives thereof, and others known to those skilled in the art)
including but not limited to. Difunctional crosslinkable units can optionally be incorporated into the copolymer. In the above formula, Q represents a chemical bond or a bonding group, which generally represents a divalent hydrocarbon group, such as an arylene group, an arylene alkylene group, an alkylene arylene group, an arylene bisalkylene group,
Alkylene bisarylene groups and others are known to those skilled in the art. Preferably, Q is a group having 5 to 10 carbon atoms. M ⁺ represents a quaternary ammonium or phosphonium group having the same or different aryl, aralkyl or alkaryl groups (each having up to 20 carbon atoms). Representative groups are well known to those skilled in the art.
Preferably, M ⁺ is a quaternary ammonium group. X ^- is an acid anion, such as a halide ion (e.g. chloride or bromide ion), a nitrate ion,
Represents methosulfate ion, p-toluenesulfonate ion, and others known to those skilled in the art. Typical reactive mordants that are useful are poly(N,N,
N-trimethyl-N-vinyl-benzylammonium chloride), poly[styrene-co-benzyl(dimethyl)-p-vinyl-benzylammonium chloride], poly(styrene-co-N-vinylbenzyl)-N,N-dimethyl ammonium chloride-co-divinylbenzene), poly(N,N,N
-trimethyl-N-vinylbenzylammonium chloride-co-styrene), poly(N,N,N-
trioctyl-N-vinylbenzylphosphonium chloride) and poly(styrene-co-(vinylbenzyl)-(trihexyl)ammonium chloride). A description of such reactive mordants, including methods of making such materials, is provided in , U.S. Pat. No. 4,069,017. The mordant layer further includes a hydrophilic binder material permeable to bilirubin. As noted above, in the practice of the present invention, this binder material is permeable to bilirubin. It is essential that the substance is not gelatin or a derivative of gelatin. This binder substance must be non-interfering, i.e. it must not interfere with the mordanting of bilirubin into the mordant. In other words: The binder material must not be capable of binding or mordant to bilirubin. Useful binder materials can be easily identified by a simple test: a film of binder and mordant is
15 watt cooled white fluorescent tubes at a distance of 0.3m
Expose for 30 minutes and then measure with a spectrophotometer at 400 nm and 460 nm. These measurements are compared to similar measurements made prior to tube exposure. A concentration difference of 0.01 or more is a failure. All measurements are made at ambient temperature and humidity. Substances that pass this test as binders include poly(vinyl alcohol), poly(N-vinyl 2-pyrrolidone), poly(N-vinyl 4-pyrrolidone), acrylamide homopolymer,
Includes copolymers of acrylamide or methacrylamide and N-vinylpyrrolidone, carboxymethylcellulose and terpolymers of N-vinylpyrrolidone, carboxy-containing vinyl monomers, and acrylamide. Particularly useful binder materials are acrylamide or methacrylamide (e.g. acrylamide, methacrylamide, N-isopropylacrylamide and others known in the art) and N-vinylpyrrolidone (e.g. N-vinyl 2-pyrrolidone and N-vinylpyrrolidone). 4-pyrrolidone), as well as N-vinylpyrrolidone (as above),
carboxy-containing vinyl monomers (such as acrylic acid, methacrylic acid, itaconic acid and others known in the art, as well as functional equivalents such as anhydrides and esters thereof) and acrylamide or methacrylamide (as described above). It is a terpolymer. The appropriate molar ratios of monomers used to prepare these polymers are within the knowledge of those skilled in the art. Poly(acrylamide co-N)
-vinyl-2-pyrrolidone) is a suitable hydrophilic binder material for use in the mordant layer in the practice of this invention. The amount of reactive mordant used in the analytical elements of the invention can vary depending on the particular range of bilirubin content for which a particular bilirubin assay is designed to be useful. Details regarding useful amounts are provided in US Pat. No. 4,069,017. The amount of hydrophilic binder material in the mordant layer must be sufficient to properly disperse the mordant and form a suitable film. This amount also depends on whether a monomeric or polymeric mordant is used. If the mordant is a film-forming polymer, less binder material may be required. Generally the amount of binder material is between 2 and 20 g/m ² , preferably between 5 and 20 g/m ² . Other optional additives (buffers or surfactants) can be added to one or more layers of the analytical element of the invention, if desired. As is known in the art, one or more bilirubin effectors or promoters are also useful in the element. Such substances include sodium benzoate, caffein, gum arabic, salicylates, bile salts and mixtures thereof. Such substances are preferably included in the porous diffusion layer of the analytical element of the invention. According to the present invention, various elements can be manufactured that differ depending on the analysis method. The elements can be constructed in a variety of shapes including elongated tapes, sheets, slides or chips of any desired width. Generally, the elements are individual slides that are packaged together in a cartridge for use in an automated analyzer. The analysis of the present invention can be done manually or automatically. Generally, when using dry elements,
The element is taken from a supply roll, chip package or other source and a sample of the liquid to be tested (e.g.
Total bilirubin (i.e., in all forms) is measured by physical contact such that the sample and reagent (i.e., reactive mordant) are mixed within the element. Such contact may be achieved in any suitable manner, for example by dipping the element into the sample or by manually or mechanically dropping a drop of sample onto the element, preferably using suitable dispensing means. be done. After applying the sample, condition the element,
Subject to any treatment desired to promote or facilitate obtaining any test results, such as incubation, heating, etc. When the mordant binds to bilirubin, a detectable change occurs, which is easily measured using suitable equipment for reflection spectrophotometry. Such devices are well known in the art. The signal from the detectable substance thus measured is indicative of the amount of total bilirubin in the fluid being tested. The methods and elements of the invention are also described in U.S. Pat.
The teachings of 4338095 can be used to measure bilirubin in complexed or uncomplexed form.
In general, selective determination of one or both forms of bilirubin is made by contacting the sample and the element as described above and by measuring the absorption or emission spectra at two or more wavelengths and performing appropriate calculations. This is achieved by [Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but it goes without saying that the scope of the present invention is not limited to these Examples. Example Comparison of Elements Containing Different Binder Materials in the Mordant Layer This example compares elements of the present invention and U.S. Pat.
Comparison is made with a previously commercially available control element similar to that described in this patent. Elements of the invention have the following structure and components:

【table】

TABLE Elements of the invention include poly(acrylamide-co-N-vinyl 2-pyrrolidone) as a binder material in the mordant layer. The control element was constructed similarly except that it included gelatin as the binder material in the mordant layer. Both elements were tested in the following manner. Cartridges of each of the control and inventive elements (50 elements in each cartridge) were exposed to 50% relative humidity at 21° C. for various periods up to 10 days after manufacture. One cartridge of each type of element was tested immediately after manufacture.
All 50 elements of each cartridge were then tested for bilirubin measurements using test fluids containing moderate amounts of bilirubin (complexed and uncomplexed forms) and an EKTACHEM 700 Clinical Chemistry Analyzer. FIG. 1 shows the results of these tests, ie the measured concentrations determined from a calibration curve as a function of storage period. The numbers plotted are the average measurements of all 50 elements in a given cartridge. Elements of the invention showed little change in measured complexed and uncomplexed bilirubin (B _c and _Bu ) concentrations when exposed to 50% relative humidity at 21° C. for up to 10 days. However, the control element showed significant variation under these conditions. Accordingly, the present invention is based on U.S. Pat.
This represents an unexpected and surprising improvement over the control element taught in '4069017. Table 1 below summarizes the data shown in FIG. 1 and also shows the numerical value of the sum of B _u and B _c .

[Table] *Testing immediately after production [Effect of the invention] The elements of the invention can be used for the analysis of complexed bilirubin, uncomplexed bilirubin or total bilirubin, and changes resulting from environmental conditions (e.g. high humidity and light) not very sensitive to Therefore, in a given cartridge containing a large number of elements, there is less variation between the elements immediately after manufacture and when stored for a certain period of time. Surprisingly, gelatin was found to be the cause of this problem. This was completely unexpected since gelatin is used as a binder in the layers of many commercially available analytical elements and was not previously known to cause such undesirable effects. With the discovery of the cause of this problem, gelatin in the mordant layer was substantially replaced by 2-20 g/m ² of another hydrophilic binder material that does not cause this problem. A number of useful alternative binder materials are described above. The resulting improved element exhibits significantly less concentration change over time.

[Brief explanation of drawings]

FIG. 1 is a plot of the measured concentrations of bilirubin (complexed and uncomplexed) measured using control elements and elements of the invention over several days under certain storage conditions.

Claims (1)

[Scope of Claims] 1. In an analytical element for measuring complexed, uncomplexed or total bilirubin, a positively charged reactive mordant having at least one binding site for bilirubin (the mordant is other than gelatin or a derivative thereof, 2)
a substantially gelatin-free mordant layer containing a hydrophilic binder material (dispersed in a hydrophilic binder material present in an amount of ~20 g/m ² Analysis elements not included. 2. For analysis of complexed, uncomplexed or total bilirubin, A. A liquid sample suspected of containing complexed or uncomplexed bilirubin is treated with a positively charged reactive mordant having at least one binding site for bilirubin (the mordant may be gelatin or Other than its derivatives,
a colorimetric and fluorescent reactive composition for bilirubin other than a mordant, comprising a substantially gelatin-free mordant layer (dispersed in a hydrophilic binder material present in an amount of ² to 20 g/m2); B. A method for analyzing bilirubin comprising the step of contacting with an analytical element that does not contain B and measuring the amount of complexed, uncomplexed or total bilirubin bound to the reactive mordant.