JPS6231300B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is concerned with determining the presence and concentration of unbound serum protein test substances, such as hormones, in a liquid sample such as serum. For more details,
The present invention detects free test substances of a type capable of reversibly binding to serum proteins in a sample containing free test substances, bound serum proteins, and a concentration of test substance-serum protein complexes. It concerns a method that is well adapted to the However, "competitive assay" methods for measuring the concentration of various biologically active substances for diagnostic purposes are now well established. This method involves a reaction system in which an antibody specific for the test substance to be measured is incubated with an aliquot of a test sample and a labeled analogue of the sample. The natural test substance and its analogs compete for binding to the antibody's binding site. After separating the antibody from the rest of the reaction, the antibody or supernatant can be assayed for analogues. The amount of analog associated with the antibody is an inverse function of the concentration of test substance in the sample. Two conventional methods for conducting such competitive assays are known: "liquid phase radioimmunoassay" and "solid phase radioimmunoassay." In each system, the immunogenic substance bound to the antibody is used to measure the amount of labeled analogue on the antibody.
substance) must be removed from the unbound immunogenic substance. This measurement determines the amount of immunogenic substance in the sample. In "liquid phase" radioimmunoassay systems, the antibody to be measured, the labeled analogue, and the immunogenic substance are incubated in solution. Thus, several antibody binding sites are available and the reaction is rapid. To separate the antibody-conjugated immunogen from the uncomplexed immunogen, the antibody-immunogen complex is precipitated, centrifuged, and the supernatant is decanted. "Solid phase" radioimmunoassay systems eliminate the precipitation step. The antibody binds to the inner surface of the glass chip or tube. The antibody-conjugated immunogenic material is then separated from the uncomplexed immunogenic material by centrifuging the glass chip or decanting the coated tube. however,
Since several effective active sites on the antibody are blocked by the chip or tube,
The reaction is relatively slow. Hormones, such as those excreted by the thyroid, testes, ovaries, adrenal cortex, and other glands of mammals, are often transported through the circulatory system in combination with hormone-binding serum proteins or globulins. It is often of diagnostic interest if the presence and concentration of free hormone as opposed to this serum protein-bound hormone or total hormone can be quantified. For example, thyroxine T ₄ in the bloodstream exists in dynamic equilibrium as a test substance bound to transport serum proteins (thyroxine-binding globulin, albumin or prealbumin) and as unbound serum protein (“free”). ” or unbound T ₄ ) is present in a small portion (possible values are 0.1% or less). Free _T4 is considered to be the active test substance of the thyroid hormone system. Unfortunately, free
The _T4 test methodology was lengthy, complex, costly, and prone to error. This is because it is difficult to standardize substances, and hormones that are relatively loosely bound to serum proteins are overwhelmingly larger than the minute amounts of hormones that are not bound to serum proteins. This is because it is difficult to measure.
Therefore, the concentration of free test substance cannot be determined by the competitive assay described above. Among methods for measuring free T ₄ and other hormones that exist as serum protein complexes in vivo, the dialysis membrane method shows high accuracy. In this method, a dialysis bag containing known amounts of test serum and labeled hormone is suspended in a buffer solution. The labeled hormone itself distributes equally between the free and bound states as a serum hormone. The amount of labeled hormone added must be extremely small so as not to perturb the system too much, but still be counted so that small amounts can be detected.
The rate must be high. For radioactive labels,
This requires the use of labeled hormones with very high specific radioactivity. Appropriate time (about 24 hours)
After a period of time, the label and native hormones that are not bound to serum proteins diffuse through the semipermeable dialysis bag, while the hormones that are bound to serum proteins (molecular weight >20,000) remain within the dialysis bag. To determine the amount of free hormone present in serum, an aliquot of the buffer is assayed for labeled hormone.
As a result of the assay, the fraction of labeled hormone that has entered the buffer can be calculated, and the inferred fraction of total hormone present in the free state can be calculated. Thus, in order to convert this fraction to mass units of free hormone (eg, ng/dl), a total hormone assay must be performed on the sample. Although this system can show significant results in free _T4 and other free hormone assays, it is highly unsuitable for routine use. First, two tests must be performed. Thus, the accuracy of the final result can be compromised in two ways. Also, a fairly large amount of radioactivity must be used for one test. Interfering impurities in the labeled hormone can pose particular problems, and the incubation times are long. Only serum can be used, but it must be very fresh. Furthermore, correction and standardization of all substances required for assays is a routine matter.
Not. Thus, the application of this method is limited because it is costly and requires a great deal of effort and skill. Another method of free hormone assay involves reaction kinetics and requires two separate tests. In each test, the kinetic curve of how quickly the antibody captures a hormone, such as T ₄ , away from the opposing attraction of a primary binding agent such as thyroxine-binding globulin is determined. Inaccuracies in such assay systems arise from several pathological conditions. If there are more serum protein binding sites than normal, the effective traction of globulins on hormones increases and the rate of binding to antibodies decreases. Thus, in the case of thyroxine analysis, this gives the appearance that there is very little T ₄ in the sample and it is all bound, even though in reality there is a large amount of T ₄ . Generally speaking, the present invention relates to methods, reagents, and test sets for the direct assay of serum protein-unbound hormones and other similar test substances. This assay can be performed in the presence of serum proteins and serum protein binding hormones. According to the invention, semipermeable microcapsules containing antibodies complementary to a test hormone or other test substance are incubated with a labeled analogue of the hormone and a test sample. Prior to this incubation, the analog can be introduced into the microcapsule in an amount such that the antibody is saturated at the hormone binding sites. The microcapsule wall consists of a membrane that separates the test sample from the antibody, such that the membrane is sufficient to allow the passage of free test substance and its analogues, but no antibody, natural serum protein, or serum protein binding. It has insufficient porosity to allow passage of hormones. When a test sample is added to an aliquot of this microcapsule, the free hormone diffuses through the membrane and competes with its analog for binding sites on the antibody. The distribution of analogs between the antibody and the rest of the reaction system is thus an indication of the amount of free hormone initially present in the sample. This method combines the accuracy inherent in conventional liquid phase radioimmunoassays with the simplicity and convenience of solid phase systems. The antibody, along with its bound hormone (and bound analogue), is then separated from the free test substance, and the antibody or the remainder of the reaction system is assayed for analogue.
Separation can be performed by inducing a change in osmotic pressure so that when the capsule deflates, the antibody-unbound hormone moves out of the capsule. This can be accomplished, for example, by adding serum albumin or polyethyleneimine to the system to facilitate outflow of the capsule fluid. Alternatively,
Free test substances can be removed from the microcapsules by simple washing. Results are interpreted by comparing assays for analog content to standards such as curves of free hormone concentration versus radioactivity counts, fluorescence intensity, or other marker characteristics used to indicate the presence of an analog. . This assay is used to determine the presence and/or concentration of free thyroxine, triiodothyronine, new thyroxine, testosterone, cortisol, other steroid hormones, and other substances that bind reversibly to serum proteins. be able to. This assay can also be used to assay test substances, such as drugs such as digoxin, that do not significantly bind to serum proteins. Thus, essentially any substance can be assayed as long as a complementary binding substance or labeled analog of the analyte is available or can be produced. This assay consists of a test set consisting of a microcapsule containing an analogue, an antibody standard substance, and a blank containing a test substance at a predetermined concentration, and after incubation, the unbound test substance and analogue are removed from the capsule. This can be done routinely using the reagents to be removed. For quality control, the liquid inside the microcapsules may be colored. A colored supernatant after these capsules have sedimented or precipitated by centrifugation indicates rupture of the microcapsules or possible leakage of the antibody. The present detection, unlike conventional detection, covers a wide concentration range of serum proteins, hormones and interfering substances, eliminating loss of clinical correlation with the condition being diagnosed. It is an object of the present invention to provide a rapid, simple and reproducible assay method for determining the presence and concentration of test substances unbound to serum proteins in a liquid sample, reagents useful for such assays, and Another object of the present invention is to provide a test set that is suitable for easy execution. Another object of the invention is to provide a method for measuring free _T4 in a serum protein-bound T4 _- containing sample extracted from human blood. Another object of the invention is to provide a competitive method for measuring immunogenic substances that combines the advantages of intrinsic radioimmunoassay systems with those of liquid phase radioimmunoassay systems. Yet another object of the present invention is to provide a reliable method for measuring the amount of digoxin in serum. The above and other objects and features of the invention will become apparent from the following description. Preferred Embodiments In the method of the present invention, a sample containing a test substance is treated with an antibody complementary to the substance (or other substance capable of reversibly binding to the substance) and a label for the substance. It is necessary to incubate with the labeled analog and to separate the sample and antibody by one or more semipermeable membranes that exclude the passage of high molecular weight proteins. Antibodies against a selected test substance can be produced according to known techniques, including injecting laboratory animals with hormones, optionally with adjuvants, and then extracting and purifying the produced antibodies. Many such antibodies are commercially available.
Additionally, many of the labeled analogs of test substances that can be detected by the methods of the present invention are commercially available or can be produced using known techniques. As used herein, the term "labeled analog"
When used, it refers to a molecule that is similar to the test substance to be detected, preferably having the same antibody binding properties as that substance, and characterized by properties that facilitate the determination of the concentration of the test substance. A preferred analogue consists of a sample of the test substance labeled with a radioactive atom. For example, thyroid hormone
¹²⁵ and then quantified by measuring gamma radiation.
However, it is contemplated that analogs of other substances may be used as long as they have a molecular weight and resulting dimensions that are well below the native protein and antibody used. Thus, analogs can be detected by labeling a sample of the analyte with a relatively low molecular weight enzyme, fluorescent moiety, or other moiety that allows the concentration of the analog to be quantified by physical or chemical means. can be manufactured. To perform the assay, use sufficient to block the passage of antibodies and natural proteins (which uniformly have a molecular weight greater than 20,000 Daltons) but allow free passage of the analyte and its analogues. Antibodies and analogues must be separated from the sample by a membrane or membranes that are permeable enough to In a preferred embodiment of the invention, the membrane is in the form of semipermeable microcapsules containing the antibody and analogue. It is noteworthy that the semipermeable microcapsules used have a permeability similar to the dialysis membranes described above. However, the semipermeable microcapsule wall is several hundred times thinner than a conventional dialysis membrane, and its effective surface area per unit weight is larger than that of a dialysis membrane. Free _T4 or other free hormones freely enter the microcapsules, and in proportion to their concentration, _T4
Replaces labeled _T4 from antibodies. Thus, during incubation, the inside of the capsule is free T ₄
and approaches equilibrium with labeled _T4 . Suitable methods for encapsulating biological materials within membranes having the permeability properties described above are described in U.S. Pat.
(August 4, 1978) and 30847 (April 17, 1979)
) and F. Lim, US Patent Application No. 24600 (March 28, 1979). The currently preferred method of manufacturing such membranes is to produce semipermeable polyamide membranes by interfacial polycondensation techniques. Firstly, a mutually immiscible solvent or solvent system is selected, for example a solvent based on water and cyclohexane. One complementary pair of monomers constituting the copolymer is dissolved in water together with the substance to be encapsulated, and then the aqueous solvent containing the substance to be encapsulated is mixed into another solvent. The liquid is emulsified to form a large number of droplets that are dispersed. Another complementary monomer (comonomer) is then added to the continuous phase of this emulsion.
is added to initiate polymerization around the droplet at the phase boundary. The membrane permeability and uniformity of the polymer precipitate are determined by changing the affinity of the emulsion continuous phase for the encapsulated monomers during the polymerization process.
It is also controlled by adjusting the concentration of reacting monomers and the polymerization time. One strategy is to initially make the continuous phase of the emulsion a solvent or solvent system that has a relatively high affinity for the encapsulated monomers, thereby allowing a relatively thick polymer network to polymerize around the droplets. Generate it in the first step. Thereafter, the continuous phase is removed such that the affinity of the continuous phase for said (first) monomer is reduced, for example by diluting said phase with another solvent or replacing said phase with a fresh solvent. change. Upon addition of the second monomer, post-polymerization occurs selectively within the previously precipitated polymer network, filling the macropore voids and thereby allowing the diffusion of solutes smaller than a certain molecular weight. This results in a uniform capsule membrane that allows for. In another approach, the continuous phase is initially selected such that it has a low affinity for the encapsulated monomers so that a thin, relatively dense film forms in the first stage of polymerization. The affinity of the continuous phase for the encapsulated monomer is then increased to draw additional monomer from the membrane and deposit another outer layer of insoluble polymer. Encapsulation preserves most of the biological activity of labile substances when the discontinuous aqueous droplet phase contains buffering agents to present a suitable environment for labile biological substances such as antibodies. It can be implemented in the following manner.
The operability of the encapsulated material also increases the ability of the antibody to add increments of the second monomer in the continuous phase of the emulsion over the course of the polymerization such that its concentration is relatively low at any given time. Preservation is achieved by avoiding exposure to high concentrations of destructive substances. In a preferred reaction system, aqueous droplets containing the diamine, high molecular weight filler, and antibody are formed in a continuous cyclohexane phase. Therefore, the affinity of this continuous phase for monomers dissolved in the droplet phase is
Denatured by adding chloroform as a diluent. Addition of diacid halide to the system produces semipermeable polyamide microcapsules.
This microcapsule formation method pushes the upper limit of permeability.
It is effective in forming membranes having a molecular weight in the range of 2,000 to 30,000 Daltons. Thus, the capsule
It can be easily designed to allow diffusion of many hormones. To carry out the assay, the test sample and microcapsules of the above type are mixed and preferably about
Incubate at 37°C. Prior to incubation, the capsules can be suspended in a solution of analog of sufficient concentration to load the antibody with a detectable concentration of analog. However, the assay can be performed by adding the analog to the reaction system during or after incubation with serum. Serum proteins and serum protein-test substance complexes in the sample cannot pass through the walls of the microcapsules. The antibody is then separated from the rest of the reaction system (excluding any optional microcapsules) and the antibody or the rest of the system is assayed for analog. A preferred method of performing this separation is to add a high molecular weight hydrophilic material, such as a solution of polyethyleneimine or serum albumin, to the extracapsular volume. This is the osmolality
The microcapsules deflate, and the intracapsular antibody-unbound hormone and its antibody-unbound analogue migrate into the supernatant. The result is a packed pellet in which the antibody is encapsulated, which can be isolated after optional centrifugation by aspirating or decanting the supernatant. Alternatively, free test substance and analog can be separated from the capsule by washing. Free _T4 antibody, ¹²⁵ -labeled thyroxine as an analog and free _T4 solution of known concentration
The results of assays embodying the invention designed to test for the presence of T ₄ are disclosed in Tables 1 and 2 and corresponding FIGS. 1 and 2. Testing of unknown samples performed in parallel with the method used to collect this data can be interpreted by reference to a standard curve.

【table】

【table】

TABLE The invention will be better understood by the following examples. But it is not limited to that. Production of microcapsules Mix 17.7 ml of 1,6-hexanediamine and 32 ml of water, and add _CO2 to this solution for about 1 hour or until the pH value
Prepare a hexanediamine carbonate solution at such a PH level by bubbling to 8.5±0.1. TCl by adding 20 g of terephthaloyl chloride (TCl) in 200 ml of an organic solvent consisting of 4 parts of cyclohexane and 1 part of chloroform.
Prepare the solution. At this time, TCl is dissolved by vigorous stirring. The obtained solution
Centrifuge for 10 minutes at 2600 rpm. Discard all sediment. Mix 750 ml of cyclohexane and Span 85 (emulsifier, fatty acid ester or sorbitan) in a 2-mixer equipped with a magnetic stirring bar. The cyclohexane was combined with 1 ml of antiserum against thyroxine (4% in phosphate-buffered saline, obtained from RF Laboratories, Hyuston, Texas, or Rayday Attachment Systems Laboratories, Carson, Calif.), polyvinylpyrrolidone-4%. A mixture of 25 ml of bovine serum albumin and 30 ml of hexanediamine carbonate solution is added under stirring. When droplets of the desired size are formed, add 70 ml of TCl solution. After 30 seconds, add 37.5 ml of TCl. After 60 seconds, add 25 ml of chloroform. Three additional 25 ml aliquots of chloroform are added at 30 second intervals. The two-phase reaction system was centrifuged, the supernatant was decanted, and the microcapsules and Tween 20 (a polyoxyethylene derivative of fatty acid partial esters of sorbitol anhydride)
Capsules are recovered by mixing with NaHCO ₃ buffered emulsifier) and phosphate buffered saline. The capsule retains polyvinylpyrrolidone, bovine serum albumin filler and thyroxine antibodies. Saturation of antibodies with analogues in microcapsules prepared according to the above procedure.
^125- labeled thyroxine (obtained from Cambridge Nuclear Corporation, Billerica, Mass.) can be loaded by the following steps. 1. In each of 100 standard test tubes, add 0.5 ml of microcapsule suspension and T ₄ ( ¹²⁵ ) (5-6000μ
Add 1.0μCi (high specific activity of Ci/μg), 37
Incubate for at least 30 minutes at °C. 2. Wash the microcapsules with twice their volume of phosphate buffered saline (NaCl 0.15M, PH=7.5, phosphate buffer 0.015M). 3 Centrifuge at 2000xG for 15 minutes and decant the supernatant. 4 Repeat steps 2 and 3 twice. 5. Dilute the microcapsule suspension with 1.6 times its volume of phosphate buffered saline. Total volume is equal to 80ml. 0.8 ml of microcapsule suspension is used per test. Thus,
100 tests can be performed on every capsule. Test Method 1 Place 25 ml of the test sample and five samples of known free T ₄ concentration in separate test tubes. The standard curve in Table 1 used concentrations of 0.5, 1.3, 3.0, 5.0 and 7.7 ng% free _T4 , but any free _T4 concentration series can be adapted according to well-known experimental techniques.
In addition, as another test for verification accuracy, salt water
Control tubes containing μ may also be included. 2. Pour T ₄ ( ¹²⁵ ) presaturated microcapsules (commercially available as is) into each test tube using an 800μ pipette. 3 Swirl each tube and incubate at 37°C for 120 minutes. 4 After incubation, 1.0 ml of 2.0% polyethyleneimine (molecular weight 40,000-50,000) in phosphate buffered saline is added after each test. 5 Incubate for an additional 20 minutes. 6 Decant the supernatant. 7. Count each test tube for 1 minute in a gamma counter. Calculation of Results 1 Each time an assay is performed to determine the unknown free T ₄ concentration in a sample or samples, standards should be run to generate a standard curve. 2. When the test is completed, create a standard curve as shown in Figure 2 or 3 using the values obtained from the standard sample tested at the same time as the unknown sample. The counts/min (cpm) of each value can be plotted on the y-axis against the free _T4 concentration (ng%) on the x-axis (logarithmic scale) of a 3.2-cycle semi-logarithmic graph paper. An alternative way to plot cpm to free T ₄ concentration is to plot % antibody binding (relative) to free T ₄ concentration. This is accomplished by calculating the (relative) percent antibody binding for each standard, control, or unknown sample and plotting these values on two-cycle semi-log graph paper in the same manner as described for cpm. sell. % Antibody Binding (Relative) is calculated as follows: % Antibody Binding (Relative) = Antibody Binding
opm/antibody binding average of standards at lowest free _T4 concentration
cpm. FIG. 4 is a graph of free _T4 concentration (ng%) for approximately 200 test samples. Thus, each sample was assayed by the method according to the present invention and the dialysis method. As shown, there is a high degree of correlation between the two test methods. FIG. 5 is a frequency graph of free T ₄ concentration versus a given free T ₄ concentration based on approximately 200 test samples assayed according to the method described above. FIG. 6 is a graph of free T ₄ concentration for approximately 100 test samples. Each sample was assayed by the method of the invention and dynamic radioimmunotechniques. As shown in the figure, there is a high correlation between these two test methods. Table 3 shows the consistency of the intra-assay results:

Table 4 shows the consistency in results between tests:

[Table] Digoxin assay The method for encapsulating digoxin-specific antibodies is shown below. First, the reagents used and their suppliers are listed below: Sodium bicarbonate Cyclohexane chloroform All (manufactured by Fischer Chemical) Sodium chloride Monobasic Sodium phosphate Dibasic Sodium phosphate 1,6-hexanediamine Span 85 Tween 20 Lager・Chemical (Ruger
Chemical (New Jersey) Anti-digoxin rabbit antiserum Arnel Products (Brutklin, New York) Bovine serum albumin Comassie Sigma Chemical Brilliant Blue R Polyvinylpyrrolidone 40 Aldrich Chemical ) Terephthaloyl chloride Eastman Kodak The amounts of each ingredient are as follows: 1,6-hexanediamine carbonate 30 ml Phosphate buffered saline 40 ml Polyvinylpyrrolidone 40/Comassier Blue 25 ml Anti-digoxin rabbit antiserum 5 ml Cyclohexane, ACS 750 ml Span 85 125 ml Terephthaloyl chloride solution 107.5 ml Chloroform 100 ml Tween 20 wash solution QS 200 ml Phosphate buffered salt water QS 8 Procedure: Rinse all glassware with distilled water before use. 1 Place the glass mixer into the hood above the magnetic stirrer. 2 Place the microscope on the table next to the hood. 3. Span 85 with a 250ml glass graduated cylinder.
Carefully measure out 125ml. 4 Weighed span in the glass mixer in the hood
Pour in 85. 5. Weigh 750ml of cyclohexane in a 1000ml glass measuring cylinder. Pour this into a glass mixer in the hood. 6 Cover the mixer. 7 Activate the magnetic stirrer. 8 Hexanediamine carbonate 30ml, PBS
30ml, 15% PVP/Comassier Blue/4% BSA
Measure out 25 ml of PBS and 5 ml of antibody, and mix 40 ml of PBS and 5 ml of antibody in a 50 ml graduated cylinder. 9. 400 2 inch stirring rod with spin ring
Pour into a ml beaker. Place on magnetic stirrer. 10 Put 30ml of hexanediamine into a beaker.
Start the magnetic stirrer. 11 Add 15% PVP to hexanediamine in a beaker.
25ml of Komasier Blue/4% BSA followed by PBS/
Add 35 ml of antibody solution. Mix for 2 minutes. Mix the solution for 3 minutes. 12 While mixing the solution, add 70 ml portions of TCl and 37.5
Measure out ml portions in a separate 100ml glass graduated cylinder. Cover with a watch glass and place in the hood. Measure four 25 ml portions of chloroform into separate 25 ml glass graduated cylinders. Cover with a watch glass and place in the hood. 13 Add the contents of the 400ml beaker to the glass mixer in the hood through the side arm of the vial. 14 As quickly as possible, take a sample of the solution in the mixer with a disposable 1 ml glass pipette and place it on a microscope slide. Check to see if the droplets are of acceptable size (10-80 microns diameter). 15 When the droplet is of acceptable size (T=0), add a weighed 70 ml of TCl through the side arm of the mixer. T=30 After exactly 30 seconds, add another (second) 37.5 ml portion of TCl to the mixer through the side arm. Mix exactly for 60 seconds. 16 in 60 seconds (T=90), the first 25 of chloroform
Add ml part. Mix for 30 seconds (T=120). Add a second 25 ml measure of chloroform and mix for 30 seconds (T=150). Add a third 25 ml measure of chloroform and mix for 30 seconds (T=180). Add a fourth 25 ml measure of chloroform and mix rigorously for 30 seconds (T = 210 seconds). Stop the mixer. 17 Pour the contents of the mixer into two plastic centrifuge bottles that have been rinsed three times in distilled water. Centrifuge at 500 rpm for 3 minutes. 18 Carefully decant the super solution. 19 Add approximately 50 ml of Tween 20 solution to each bottle (ie approximately the same volume of Tween 20 as the capsules). Mix well with a stir bar retriever for about 5 minutes. 20 Add approximately 10 to 15 ml of PBS to each bottle and stir well. 21 Repeat step 20 4-5 times. 22 Add 400ml of PBS and stir thoroughly. 23 Balance the two bottles and centrifuge at 3000 rpm for 20 minutes. Aspirate the supernatant. Add approximately 800 ml of PBS to each bottle, mix well, and cap. 24 Repeat step 23 10 times. After the final aspiration, add 100 ml of PBS to each bottle, combine the contents of the two bottles, and shake well.
Pour into a 500ml glass graduated cylinder and add 500ml
Add enough PBS to reach ml. 25 Store in 1 glass reagent bottle at 4°C. Tween 20 wash solution is prepared as follows. However, it may be prepared the day before use. Procedure: 1 Weigh 6.06 g of sodium hydrogen carbonate on a three-ring balance. 2. Carefully place 6.06 g of this sodium bicarbonate into a 250 ml volumetric flask equipped with a stir bar. 3 Add about 200ml of purified water and stir on a magnetic stirrer until the sodium bicarbonate is dissolved. 4 Remove the stirring rod and add enough purified water to make 250ml. 5 Carefully pour into a 500ml Erlenmeyer flask equipped with a stir bar. 6. Tween 20 to 250 in a 250ml graduated cylinder.
Measure out ml carefully. 7 Add to Erlenmeyer flask containing sodium bicarbonate. 8 Mix on a magnetic stirrer until completely mixed (about 1 hour). 9 Place in a polyethylene bottle with a screw cap, tightly seal, and store at approximately 25°C. A terephthaloyl chloride solution is prepared as follows. However, it should be prepared on the day of use and should not be frozen. 1 Record the weight of terephthaloyl chloride (TCl) in the bottle containing TCl. Weight of TCl (g) to 10
Calculate the volume (ml) of the cyclohexane/chloroform solution to be added to TCl by multiplying by Calculation method: Total volume of cyclohexane/chloroform (ml) = Weight of TCl (g) x 10 Then, make sure that this total volume is sufficient for the following steps. 2 The cyclohexane/chloroform solution consists of 4 parts of cyclohexane and 1 part of chloroform. Calculate the volume of chloroform by dividing the total volume of the cyclohexane/chloroform solution (step 1 above) by 5. Multiply the volume of chloroform by 4 to determine the volume of cyclohexane. Calculation method: (a) Volume of chloroform (ml) = Total volume of cyclohexane/chloroform (ml) ÷ 5 (b) Volume of cyclohexane (ml) = Volume of chloroform (ml) x 4 3. Calculated volume of cyclohexane in graduated cylinder. and chloroform. Put them together in an Erlenmeyer flask. Mix by gently swirling. Cover with a watch glass and place in the humid hood. 4 Place the magnetic stirrer inside the fume hood. 5 Place the bottle containing TCl on a magnetic stirrer.
Uncap the bottle and add the magnetic stirrer bar and cyclohexane/chloroform solution as soon as possible. Cap the bottle. 6 Stir on a magnetic stirrer until all TCl is dissolved. It may be necessary to tilt the bottle to dissolve the TCl, which adheres to the upper inner wall of the bottle. 7. Fill a 200 ml glass centrifuge bottle with as much TCl solution as needed and as quickly as possible and cap. Centrifuge at 2600 rpm for 10 minutes at room temperature. 8 Pour the supernatant liquid into a 500ml amber bottle,
Seal well. 9 Store tightly sealed at approximately 25℃. The procedure for preparing 15% PVP40/Comassier Blue containing 4% BSA is as follows. However, it must be prepared on the day of use. No refrigeration required. Procedure: 1. Polyvinylpyrrolidone 40 to 7.5 using a three-ring balance.
g, 2 g of BSA and 0.1 g of Komasier Blue.
(100mg) Weigh accurately. 2. Place polyvinylpyrrolidone 40 in a 50ml glass beaker equipped with a stir bar. 3 Add approximately 20ml of PBS. Place a glass cover plate on top of the beaker. 4 Stir on a magnetic stirrer until dissolved. 5 In another 80ml glass beaker, add Komasier Blue.
Add 0.1g and 2g of BSA. 6 Add approximately 20ml of PBS. Place a glass cover plate on the beaker. 7. Stir while heating slightly using an electromagnetic hot plate/stirrer #10 with the heating device set to 2. Stir until dissolved (about 10 minutes). 8. Carefully add the entire contents of the beaker containing the polyvinylpyrrolidone 40 solution and the beaker containing the Komasier Blue solution to a 50 ml glass volumetric flask equipped with a stir bar. 9 Mix the combined solutions with a magnetic stirrer for 10 minutes. Remove stir bar. 10 Add enough PBS to reach 50ml. 11 Adjust the pH to 7.5±0.5 with 1N sodium hydroxide. Original PH - Final PH - Amount of NaOH used - 12 Filter the solution using a disposable membrane Nalgene filter unit (0.45μ). 13 Seal in a 60ml polyethylene bottle with a screw cap and store at approximately 25℃. The procedure for preparing phosphate buffered saline is as follows. In addition, it may be prepared the day before use. Procedure: 1 Carefully measure 1000 ml of phosphate buffered saline in a 1000 ml glass graduated cylinder. 2 Pour this into 20 large polyethylene bottles. 3 Measure 9000 ml of deionized water in a 1000 ml glass graduated cylinder and add to a large polyethylene bottle containing phosphate buffered saline. 4 Stir using a magnetic stir bar retriever. 5 Check the pH and adjust it to 7.5±0.05 if necessary. Final PHH = - 6 The phosphate buffered saline is stored in tightly sealed large 20 ml polyethylene bottles at approximately 25°C until use. The procedure for producing 1,6-hexanediamine carbonate is as follows. In addition, it may be manufactured the day before use. Procedure: 1. Place the bottle of 1,6-hexanediamine in 3 beakers. Add enough tap water to the beaker to bring the level of hexanediamine in the bottle. 2 Loosen the lid of the hexanediamine bottle. 3 Place the beaker on an electromagnetic stirrer/hot plate equipped with two heaters to completely melt the hexanediamine. 4 Accurately measure 17.7 ml of hexanediamine using a 25 ml glass graduated cylinder. Carefully pour into a 500ml amber bottle. 5. Accurately measure 32 ml of purified water using a 50 ml glass graduated cylinder. Add to hexanediamine in an amber bottle. 6 Leave in the solution for about 1 hour until the pH becomes 8.5±0.1.
Bubble _CO2 . Final PH-. 7 Seal the amber bottle and store at approximately 25℃. A microcapsule and its working principle are schematically shown in FIG. As shown in this figure, the digoxin-specific antibody 9 is fixed inside the wall 10 of the microcapsule 12. However, the walls 10 of the microcapsules 12 have openings small enough to allow free passage of digoxin. Thus, digoxin 16 from the sample and labeled digoxin 18 freely pass through the microcapsule wall and compete with each other for binding sites of antibody 9. Both unbound and bound digoxin 16 or 18 can be washed away after incubation. As mentioned above, some of the digoxins are labeled. Thus, it is the labeled digoxin that competes with unlabeled digoxin from the sample for the binding site of the digoxin-specific antibody. A preferred labeled digoxin is ( ¹²⁵ )digoxin, which is available from New England Nuclear Corporation, Billerica, Massachusetts. Test method 1 A serum reagent to be tested is obtained by a method well known in the art. In this example, a control is used. 2. Add 0.1 ml each of the standard, control, or unknown patient serum sample to each tube containing 0.5 ml of the digoxin antibody microcapsule suspension prepared as described above.
(100μ) into the pipette and label the test tubes accordingly. 3 Pipette 0.5 ml (500μ) of ^125- labeled digoxin (in phosphate buffered saline) into each test tube. Swirl for a minimum of 3-4 seconds. 4. Incubate all reaction tubes in a water bath (37°C) for a minimum of 15 minutes. [Alternatively, incubate all reaction tubes at room temperature (20-25°C) for a minimum of 3 minutes]. 5 Add 0.5ml (500μ) of washing solution to each tube and add at least 3~
Swirl for 4 seconds. 6. Incubate all reaction tubes at room temperature (20-25°C) for 5 minutes. 7. Place all tubes at a minimum of 1600 x G for 10 minutes at room temperature (20-25
Centrifuge at 10°C, decant the supernatant into a suitable container, and blot the last drop onto blotting paper. 8. Count all tubes for 1 minute using a gamma counter adjusted for ¹²⁵ . 9. Plot the known amount of digoxin against cpm to create a standard curve, then read the unknown amount of digoxin from this curve. Reagents used Digoxin-antibody is encapsulated in semipermeable nylon microcapsules suspended in phosphate buffered saline prepared as described above. For quality control, antibody microcapsules were coated with blue dye (Comassier Blue).
_R250 ). A blue supernatant after the microcapsules have been sedimented or precipitated by centrifugation indicates microcapsule rupture or possible antibody leakage. ¹²⁵ I-labeled digoxin 10μ of ^125I digoxin with less than 4.2 μCi in each ml of solution
Contains g. Buffer Solution Phosphate buffer 0.015M at PH7.5 containing 0.15M sodium chloride, 0.5% bovine serum albumin (BSA) and 0.1% sodium azide. Washing solution 20% polyethylene glycol 6000 solution in barbital buffer 0.5M with pH 8.9 containing 0.15M sodium chloride. Calculation of results The results of this test, which is a representative test, are shown in Table 5.
This is also schematically shown in FIGS. 7 and 8. In Figure 7, the final y for the two-cycle semilogarithmic graph
CPM is plotted on the axis, and digoxin concentration (ng/ml) is plotted on the x-axis (logarithmic scale). Figure 8 shows % antibody binding (relative) plotted instead of cpm versus digoxin concentration. It calculates the % antibody binding (relative) for each of the standards, controls or unknown samples and also converts these values into
It is made by plotting two cycles on semi-logarithmic paper in the same manner as described for cpm. The % antibody binding (relative) is calculated as follows: % antibody binding (relative) = (0 ng digoxin standard/
ml antibody binding average cpm) x 100

[Table] The assay system of the method of the present invention was found to be extremely accurate. Intra-assay variation is less than 7% and inter-assay variation is less than 9%. Intraassay Variation The CV of the two control samples tested 25 times each in one experiment was found to be: Average digoxin ng/ml CV Control 1 1.54 4.9% Control 2 2.95 6.2% Interassay variation Variability The percent variation for the two control samples, each tested three times in separate experiments 18, was found to be: Average digoxin ng/ml CV Control 1 1.50 7.3% Control 2 3.04 8.8% Table 6 shows very high specificity for this digoxin and for the exclusion of other potential interfering substances.

[Table] Quantitative Recovery The method of the present invention was found to be very quantitative as more than 96% of the added sample digoxin was recovered.

[Table] It is particularly important to note the high correlation between the results of the method of the present invention and the values measured for digoxin by other assay methods. Table 8 presents a comparison of the test results obtained by the method of the invention and other assay results. System A and System C represent liquid assay systems with precipitation reagent separation, and System B uses solid phase separation. As shown in the table, the correlation coefficient is 0.97 to 0.98.

[Table] From the above table, it can be seen that the present invention can be used to determine the presence and concentration of any free test substance, as long as a complementary substance capable of specifically binding to the test substance and its labeled analogue is available. It is clear that the disclosed assay can be used. Another requirement is that species and their analogues can be used to provide microcapsule membranes that selectively allow the diffusion of these substances and block the passage of high molecular weight substances such as natural proteins. This means that the molecular weight of is sufficiently low. Fortunately, steroid hormones, thyroid hormones, many drugs and other substances of clinical importance are characterized by much smaller molecular sizes than natural proteins. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments and examples described above are only illustrative of the invention in all respects and should not be considered as limiting. The scope of the present invention is defined by the above claims rather than the above description.
It is shown by. Therefore, the “scope of claims”
All changes that come within the meaning and range of equivalence are intended to be embraced by the present invention.

[Brief explanation of the drawing]

FIG. 1 is a standard curve prepared according to the method of the invention, with free T ₄ concentration (n
g/dl) versus count/min on the y-axis
(cpm) (×10 ³ ) (see Table 1 above). FIG. 2 illustrates another standard curve for the free T ₄ assay of the present invention. The data for this figure can be found in Table 2 above. Figure 3 shows free T ₄
from _T4 ( ¹²⁵ ) bound to _T4 antibody contained within semipermeable microcapsules soaked in a standard solution of
This is a graph of cpm versus time. As serum T ⁴ displaces T ₄ ( ¹²⁵ ) at T ₄ antibody binding sites, the residual cpm associated with that antibody decreases. Of note is that the rate of displacement is more or less proportional up to 2 hours of incubation at 37°C. FIG. 4 graphically illustrates the correlation between the results of the dialysis assay and the microencapsulation method of the present invention. FIG. 5 graphically illustrates the standard range for free T ₄ established by the microencapsulation assay system.
FIG. 6 graphically illustrates the correlation between the dynamic radioimmunoassay and the microencapsulated type assay system of the present invention.
Figure 7 shows the x-axis (logarithmic scale) of the 2-cycle semi-logarithm.
A standard curve of antibody-bound digoxin (cpm) plotted on the y-axis against digoxin (ng/ml) above (data in Table 5) is shown. FIG. 8 shows % antibody binding (relative) of digoxin versus digoxin concentration. Antibody binding (relative) % is antibody binding (relative) %
= Average antibody binding opm/0 ng/dl Calculated as average antibody binding cpm of digoxin standard x 100 (data in Table 3). FIG. 9 shows retention of antibodies and free passage of immunogen in microcapsules formed according to the present invention. In the figure, the explanations of the symbols representing the main parts are as follows: 9: Antibody, 12: Microcapsule (10: Wall), 16: Digoxin from sample, 1
8: Labeled digoxin.

Claims (1)

[Claims] 1. A method for measuring the presence of a test substance that is not bound to serum proteins in a serum protein-containing sample, the method comprising: The sample and the antibodies are incubated with a labeled analogue of the test substance such that the sample and the antibodies are incubated in a manner that allows passage of the unbound serum protein and the labeled analogue thereof. the antibody and a labeled analogue separated by at least one membrane having sufficient porosity to allow passage of the antibody or serum protein-bound test substance present in the sample; is contained in a microcapsule made of the membrane, and the test substance unbound to serum proteins in the sample is passed through the membrane and labeled with the test substance at the binding site on the antibody. removing the antibody-unbound test substance and the analog from the microcapsules by increasing the osmotic pressure outside the microcapsules and separating the microcapsules from the rest of the reaction system; and measuring the amount of the labeled analog bound to the antibody, which amount is indicative of the amount of serum protein-unbound test substance in the sample. . 2. The method according to claim 1, wherein the labeled analogue comprises a test substance labeled with a radioactive atom. 3. The method according to claim 1 or 2, wherein the test substance is L-thyroxine. 4. The method according to claim 1 or 2, wherein the test substance is L-3,5,3'-triiodothyronine. 5. The method according to claim 1 or 2, wherein the test substance is selected from the group consisting of cortisol, testosterone, and neonatal thyroxine. 6. The method according to claim 1, wherein the separation step is performed by washing the unantibody-bound test substance and the antibody-bound analogue from the microcapsules. 7. The method according to claim 1, wherein the test substance is digoxin. 8. A test set used to determine the amount of serum protein-unbound test substance in a liquid sample, comprising:
The set comprises a plurality of microcapsules containing a labeled analogue of the test substance and an antibody complementary to the substance to allow passage of the substance and its labeled analogue. microcapsules for the absorption of serum protein-free test substance from said sample, comprising a membrane having a permeability sufficient to permit the passage of said antibodies or natural proteins; a reagent capable of removing an unantibody-bound test substance and its labeled analog unbound to the antibody from the microcapsules by increasing the external osmotic pressure, and a predetermined amount of the test substance; A test set consisting of a combination of standard materials for comparison with a sample. 9. The test substance is thyroxine, the labeled analog is thyroxine labeled with a radioactive atom, and the standard consists of an aliquot of a substance containing a known amount of free thyroxine, and the substance is sampled. 9. The test set according to claim 8, which is capable of creating a standard curve when testing parallel to the . 10. The test set of claim 9, wherein the reagent comprises an element selected from the group consisting of serum albumin and polyethyleneimine. 11. The test set of claim 8, wherein the microcapsules contain antibodies against digoxin and the labeled analogue is ¹²⁵ -labeled digoxin. 12 Used in quantifying the unbound test substance that can form antibodies and reversibly bind to serum proteins in the sample as an unbound test substance in a liquid sample. A reagent comprising a plurality of microcapsules containing an antibody complementary to the substance and a labeled analogue of the test substance, the microcapsules containing the test substance and its label. The reagent comprises a membrane having sufficient permeability to allow the passage of the analog but insufficient to allow the passage of the antibody and the native protein. 13. The reagent of claim 12, wherein the antibody is complementary to thyroxine and the labeled analogue is thyroxine labeled with a radioactive atom.