SE445680B - IMMUNO ANALYSIS PROCEDURE AND DEVICE FOR DETERMINATION OF BONDED SUBSTANCES - Google Patents

Description

7905101-7 10 15 20 25 30 35 2 In the "liquid" phase radioimmunoassay systems the antibody, a labeled analog, and the immunogenic substance to be determined in solution. A number of binding sites on the antibody are available and the reaction time is short. To separate with anti- body complex immunogenic substance from non complexed immunogenic substance precipitates the complex of antibody and immunogenic substance, after which the complex centrifuged and the supernatant decanted.

Radioimmunoassay systems with "fixed solid" lack the precipitation step. The antibody binds to a piece of glass or the inside of a pipe surface. Centrifugation of glass pieces or decantation of the coated tubes distinguishes the complex of antibody and immunogenic substance from the non-complexed immunogenic substance. Emel- however, the reaction time is relatively long due to a number of available active sites on the antibody blocked by the piece of glass or tube.

Hormones, such as the hormones that are secreted from thyroid gland, testicles, ovaries, adrenal cortex and other mammalian glands, are often transported by circulatory system together with hormone-binding proteins or globulins. For diagnostic purposes it is often it is important to be able to determine the presence and concentration of free hormones as opposed to protein bound hormone or the total amount of hormone. For example, thyroxine exists (T4) in the blood flow in dynamic equilibrium as a substance, which is bound to a transport protein (thyroxine binding globulin, albumin or prealbumin) and a small proportion (potentially 0.1% or less) as the unbound substance ("Free" or unbound T4) Free T4 is considered the active one the substance in the thyroid hormone system. Unfortunately is the way to analyze free TA tiring, complicated, expensive and errors easily occur due to the difficulty to analyze the materials and measure very small amounts of unbound hormone in the presence of the predominant amount hormone, which is relatively loosely bound to protein. The 10 15 20 25 30 35 7905101-7 3 specified competitive analysis does not allow determination of the concentration of free substance.

Of the methods for measuring free T4 and other monons that exist as protein complexes in vivo dialysis membrane procedure high degree of precision. A dialysis sack containing known amounts of the serum to be tested, and labeled hormone, suspended in a buffer. It marked the hormone is distributed between free and bound state in the same proportions as the serum hormone. The appointed the amount of labeled hormone must be extremely small so that one avoids seriously disturbing the system and yet the pulse rate must be so high that it becomes possible to detect small amounts. In case of radioactive labeling it is then required that labeled hormone with very high specific activity white was used. After a suitable period (about 24 hours) dif- consider the labeled and natural hormone, which is not bound to protein, through the semipermeable dialysis bag, while hormone, which is bound to protein (molecular weight over 20,000 »remain in the dialysis bag. To decide the amount of free hormone in the serum is analyzed an aliquot amount of buffer with respect to labeled hormone. Som re- results of the analysis, the part of the labeled hormone, which entered the buffer is calculated and one can calculate it amount of total hormone, which is present in the free state.1ür to convert this quantity into mass units (eg ng / dl) of free hormone, need an analysis with regard to total hormone is also run on the test.

This system can provide meaningful results in sees free T4 and other free hormones but the method is bad suitable for routine use. Two tests must be performed and the precision of the end result can be compromised at both the tests. Quite a large amount of radioactivity must used per test. Disturbing pollutants in the label The hormone can cause special and long problems incubation times are required. Only serum can be used and this must be extremely fresh. Furthermore, the country and the standardization of all the materials, which 10 15 20 25 30 35 7905101-7 4 is necessary fi for the analysis, not a routing purpose. Thus the application of this method has been limited to due to the high cost and due to the fact that it is labor intensive and that trained staff is required.

Another type of analysis with respect to free hormones based on reaction kinetic conditions -Dchrequires two- ratu tester. At each test, a kinetic curve is made, which shows how quickly an antibody captures a hormone, such as T4 from the opposite pull of the primary binding the substance, such as thyroxine-binding globulin.

In such an analysis, errors arise due to many pathological conditions. About several protein binding sites than usual is present will globulin effective attraction with regard to the hormone to be greater and the rate of binding to the antibody will decrease.

In the case of thyroxine analysis, this would give the impression that the sample have low T4 then in fact a high content of T4 could exist, but all bound.

Summary of the invention The present invention relates to an immunoassay method and a test equipment for direct analysis of unbound hormones and other such substances. The analysis can be performed in the presence of serum proteins and bound hormone.

According to the invention, semipermeable microcircuits are incubated which contains a complementary antibody to or the other substance to be detected, a distinguishable analogue of the hormone and the sample. capsules, the hormone with boats The analog is introduced into the microcapsule before incubation in such amounts that the antibody is saturated with respect in hormone-binding sites. The microcapsule walls contain take membranes which separate the sample from the antibody, and has sufficient permeability to pass through it free substance and its analogue shall be permitted but sufficient permeability for the antibody, natural proteins, or protein-bound hormone must pass.

When a sample is added to an aliquot of micro- the capsules diffuse free hormone through the membranes 10 15 20 25 30 35 7905101-7 5 and competes with its analogue in terms of adhesion sites on the antibody. Thus the distribution of the analogue between the antibody and the rest of the reaction system a measure of the amount of free hormone, which originally was present in the sample. This method combines the inherent accuracy of conventional liquid phase radioimmunoassay and the simple and convenient one Ffastfas "system.

The antibody is then separated together with its bound hormone (and its bound analogue) from the free sub- punch and either the antibody or the remainder of the reaction. the analysis system is analyzed with respect to analogue. Separate can be carried out by providing one osmotic change so that the capsules collapse and unbound hormones migrate out of the capsules. This can, for example is achieved by adding serum albumin or polyethyleneimint fl dj.sysumet, which favors utfiöae av liquid in the capsule. Alternatively, the free substance can simply washed out of the microcapsules. The results are interpreted by comparing analysis of the analog content with a standard dard, against radioactive pulses, such as a curve of the concentration of free hormone, fluorescent intensity or another marker, which was used to detect the presence of the analog.

The assay can be used to detect the presence of and / or determine the concentration of free form horizontal thyroxine, triiodothyronine, neonatal thyroxine, testosterone, cortisol, other steroid hormones, and others substances that bind reversibly with protein. Analytical then can also be used to detect substances, such as not bound to protein to a greater extent, e.g. drugs such as digoxin. Virtually anything such material may be determined provided that a complement-binding substance and a distinguishable analogue until the material is available or can be produced.

The analysis can be performed routinely using of a test kit comprising analog, microcapsules, containing antibody standards and control samples, such as 7905101-7 IC 15 20 25 30 35 6 contains predetermined concentrations of major and a reagent which removes unbound sub- the apple after completed incubation the substance, punch and analog from k tion. In order to be able to control the quality can the solution in the microcapsule be colored. A colored supernatant would then the capsules have sunk or settled through centrifugation can be a measure of the degree of micro- capsule rupture and possible leakage of anti-body. In return conventional analyzes are avoided according to underlying analysis lack of clinical correlation with diagnostic conditions over a wide range in terms of concentrations of protein, hormone and disruptive substances stops.

An object of the invention is to provide one simple and reproducible method of detecting presence which are not bound fast, and determining the concentration of substances, to protein, in a proteinaceous sample, and a test equipment, which is suitable for performing such analyzes in a fast and convenient way. Another purpose is to provide a way to determine free T4 in samples, taken from human blood and containing protein bound T4.

Another purpose is to designate a competitive method for determining an immunogenic substance, which method combines the benefits of solid phase radioimmunoassay and the benefits of liquid phase radioimmunoassays. Another The purpose is to provide a highly reliable method for determining the amount of digoxin in serum. These and other objects of the invention will become more apparent of the description, which follows.

Brief description of the drawings Fig. 1 shows a standard curve, made according to present procedure. The pulses have been plotted in the curve Per minute (X103) on the y-axis on a linear scale against the concentration free T4 in ng / dl on the X-axis on a logarithmic scale (see table l for data). ._ ..-....-. ~. 15 20 25 30 35 7905101-7 7 Fig. 2 shows a second standard curve for analysis of according to the invention, depositing cpm (linearly) free T4 Data for this fi- against ng / dl free T4 (logarithmic scale). gur are given in Table 2.

Fig. 3 is a graph of cpm from T4 to T4 ~ antibody, which enclosed the semipermeable the microcapsules, which are immersed in standard solutions of free T4, vs time. As serum T4 replaces T4 (1251) at the T4 binding sites on the antibody, decreases cpm, which is left in combination with the anti ~ body. (1251) bound Note that the displacement speed is large seen linear at 2 h incubation at 37 ° c.

Fig. 4 graphically shows the correlation of the results between the dialysis method and the microcapsule system according to the invention.

Fig. 5 graphically shows the normal range for free T4 according to the assay system with microencapsulation.

Fig. 6 graphically shows the relationship between kinetic radioimmunoassay and the microencapsulation assay system according to the invention.

Fig. 7 shows a standard curve with pulses per minute (cpm) for digoxin bound to antibody, deposited on linear scale against digoxin in nanograms / ml on the logarithmic scale on a semi-logarithmic paper (data from Table 5).

Fig. 8 shows digoxin as percent bound (relative) vs digoxin concentration. Percent bound (relatively) is calculated as a percentage bound (relative) = average value cpm bound / mean cpm bound for 0 ng / dl digoxin- standard x 10 0 (data from Table 8).

Fig. 9 is a diagram showing the retention of anti ~ body and the free passage of immunogenic substance in the micro- capsules formed according to the invention.

Description of a Preferred Embodiment The method of the invention requires that the samples which contains the substance to be detected is incubated with antibody, which is complementary to the substance (or other substance, which has the ability to be reversibly bound ._ a- * v-a. y _ _ ... 7905101-7 10 15 20 25 30 8 to the substance) and a distinguishable analog of the substance, and that the sample and the antibody are separated by a semi- permeable membrane or membranes, which exclude passage of high molecular weight proteins.

Antibodies to the selected substances can be produced set according to well-known methods, which are based on that injects the hormone laboratory animals, possibly together adjuvant, and then extract and purify the the antibodies. Many such antibodies exist commercially available. Many discernible analogs gives to substances which can be detected by the present method, are also commercially available or can produced in a known manner. The expression "discernible analog "in this context means a molecule which has antibody binding properties similar and preferred thirdly identical with the properties of that substance one seeks to demonstrate and which is characterized by a property, which allows one to easily make a measurement of its con- centration.A preferred analog comprises a sample of the substance to be detected and labeled a radioactive atom, such as thyroid hormones, which are labeled appropriately with l25I and then quantitatively can be determined by measuring the gamma radiation. Emeller- time, other types of analogues can be used as long as the analog has a molecular weight and resulting dimensions, which are clearly under the corresponding characteristics of the proteins and the antibody used. Analogs can thus be prepared by labeling a sample of the substance to be detected, with a relative low molecular weight enzyme, with a fluorescent component or other component, which allows quantitative measurement of the concentration of the analog with physical or chemical agents.

To perform the assay, the antibody and the analog is separated from the sample by one or more membranes, which has sufficient permeability to exclude sage of antibodies and natural proteins (as 10 15 25 30 7905101-7 9 species have molecular weights over 20 000 daltons) sufficient permeability to allow free passage of the substance to be detected and its analogue. According to but who has a preferred embodiment of the invention has membranes in the form of semipermeable microcapsules, which contain smiles the antibody and the analog.

It should be noted that the semipermeable microcapsules, used has a permeability similar to that of those the dialysis membranes described above. However, the semi- permeable microcapsule wall 100 times thinner than conventional national dialysis membranes and its available specific surface area is considerably larger per unit weight. Free T4 or others free hormones enter freely into the microcapsule and replace proportional to its concentration, labeled T4 from T4- the antibody. Thus one approaches a balance between free T4 and labeled T4 within the capsule during incubation period.

Suitable methods for encapsulating biological material membranes with the specified permeability properties 931 177 and 30 847 and rial i shown in detail in US PA 606 l66, in US PA 24,600. According to the presently preferred method to produce such microcapsules are obtained semipermeable polyamide membranes by means of an interfacial polycondensation method. mutually immiscible solvents or solvents means system is selected, such as water and a cyclohexane based solvent, and a monomer in a complementary pairs, which form a copolymer, are dissolved in the water together with the material to be encapsulated. The aqueous the solvent containing the material to be encapsulated, then emulsified in the second solvent so that a plurality of individual droplets are formed. The other one the complementary monomer is then added to the the continuous phase of the emulsion to initiate poly- merisation around the droplets at the phase boundary. Membrane permea- The ability and uniformity of the polymer precipitate to be controlled by varying the affinity of the emulsion continuous phase with respect to encapsulated monomer 7905101-7 IG 15 20 25 30 35 10 during the polymerization process and by regulating the concentration of the reacting monomers and the the length of the merisation.

According to the method, the continuous phase is formed from jan fl no solvent or a solvent system need relatively high affinity with respect to the encapsulated the monomer so that in a first step of the polymerization a relatively thick polymer network is formed around the droplets.

Thereafter, the continuous phase is changed so that its affinity for the first monomer is reduced, for example by diluting the continuous phase a second solvent or replaces the continuous one phase with a new solvent. When adding it the second monomer, further polymerization takes place before within the initial precipitated polymer network, macroporous defects are patched together and you get an even capsule membrane, which allows diffusion of solutes below a certain molecular weight.

According to another method, the continuous one is selected phase from the beginning with low affinity with respect to encapsulated monomer so as to thin relatively dense membranes formed in the first step of the polymerization. Then increase the affinity of the continuous phase with respect to encapsulated monomer so that additional amounts of monomer pulled through the membrane and so that a second outer layer of insoluble polymer precipitates.

Then the discontinuous aqueous droplet phase buffered in order to create an environment that is bar with labile biological materials, such as antibodies, the encapsulation can be carried out in such a way that one preserves a large part of the biological material of the labile material activity. The properties of the encapsulated material are preserved also if one adds the second monomer to the con- tinuary phase portionwise during the polymerization so that the concentration at any given time is relative low and so that the antibody is not exposed to high concentrations of potentially destructive substances. 10 15 20 25 30 35 7905101-7 ll In a preferred reaction system, aqueous halide is formed. drops containing a diamine, a high molecular weight filler material and the antibody in a continuous phase of cyclohexane, whose affinity with respect to it monomer, which is dissolved in the droplet phase, modified by addition of chlorophone seed diluent. The additive of a diacid halide to the system results in permeable polyamide microcapsules are formed. This microin- encapsulation method works when it comes to manufacturing membranes with an upper permeability limit of 2000-3000O dalton. Thus, the capsules can be manufactured so that they are allows diffusion of many hormones.

When performing the assay, the sample is mixed with micro- capsules of a specified type and are preferably incubated at about 37 ° C. Prior to incubation, the capsules can be suspended in a solution of the substance analogue with sufficient concentration tration to fill the antibody with a detectable amount of analog concentration. However, the analysis can is carried out by adding the analogue to the reaction medium. during or after the incubation with serum.

Protein in the sample and protein substance complex can not penetrate through the microcapsule wall.

The antibody is then separated from the rest reaction system (possibly with the exception of micro- the capsule membranes) and either the antibody or the remainder of the system is analyzed with respect to analog. According to a preferred method of carrying out the separation is added a high molecular weight hydrophilic material, such as a solution of polyethyleneimine or serum albumin, to the extra- capsular volume. This gives an increase in osmolality, leading to the collapse of a microcapsule and to intracapsular unbound hormone and its analog migrate in in the supernatant. This leads to a packed mass of in- encapsulated antibody, which after any centrifugation treatment can be isolated by aspirating or edges the supernatant. Alternatively, the separation can take place by digesting the free substance and its analogue from the capsule. 7905101-7 10 15 20 25 30 35 12 Tables 1 and 2 and corresponding figures 1 and 2 in the drawing shows the results of analyzes according to the invention intended to demonstrate the presence of free T 4, wherein 51 labeled thyroxine as an analog Tests off used free Til antibody, and solutions with known concentration of free T4. unknown material running in parallel with the process and which used to collect this data can be interpreted as display to the standard curves.

TABLE l Standard analysis curve for free TOE Free T 4 CPM CPM (average) 0.5 ng / dl 57097 57 285 57472 1.3 ng / a1 54256 54 839 54717 3.0 ng / al 50960 50 953 50946 5.0 ng / a1 48467 48 302 46117 7.7 ng / a1 45982 46 025 46067 Total pulses T4 (1251) added 86 403 incubation 2 h, 37 ° c 10 15 20 25 30 35 TABLE 2 13 7905101-7 Standard analysis curve for free T, 0.1 ng / dl 0.5 ng / dl 1.0 ng / dl 2.0 ng / dl 4.0 ng / dl 6.0 ng / dl Total pulses T4 (125 incubation 2 h, I) added 37 ° c 61219 60398 61183 58020 55618 56389 52096 52671 53705 48483 50126 49971 44853 45108 44235 42008 41344 41015 86 903 CPM (average) 60933 56675 52824 49536 44732 41456 7905101-7 10 15 20 '25 30 35 14 The invention will be explained by the following non-limiting examples.

Manufacture of microcapsules Hexanediamine carbonate solution (pH = 8.5 in 0.1) prepared was prepared by mixing with 17.7 ml of 1,6-hexanedianyl 32 ml of water and CO 2 were bubbled through the solution for about 1 hour or until the pH level is reached.Terephthaloyl chloride solution (TCl) is prepared was prepared by adding 20 g of TCl in 200 ml of organic technical solvent, which consisted of 4 parts of cyclohexane and part chloroform. TCl was dissolved by stirring vigorously and the solution was then centrifuged for 10 minutes at 2600 rpm. Any precipitate was discarded. 750 ml of cyclohexane were mixed with 125 ml of SPAN-85 (emulsion preservative, fatty acid ester or sorbitan) in a 2-liter mixer, which was equipped with a magnetic stirrer.

While stirring, a solution prepared was added of 1 ml of antiserum to thyroxine (4% in phosphate buffered saline) saline, R.F. Laboratories, Houston, Texas or Radioassay Systems Laboratories, Carson, CA), 25 ml polyvinylpyrrolidone - 4% serum albumin from livestock, and 30 ml of hexanediamine carbonate solution, to hexane. Then drops of the desired size formation were added 70 ml TCl solution. Thirty seconds later, 37.5 ml of TCl was added. 60 s later, 25 ml of chloroform was added. Three additives of aliquots of 25 ml of chloroform was added with, 30 s intervals.

The microcapsules were recovered by centrifugation of two phase reaction system, decantation of the supernatant and mixing the capsules with TWEEN-20 (polyethylene oxide derivatives of fatty acid partial ester of sorbitol anhydride - emulsifier buffered with NaHCO 3) and phosphate buffered saline solution. The capsules retained the polyvinylpyrrolidone and the bovine serum albumin filler as well as thyroxine antibody.

Saturation of antibody with analog Microcapsules, made as specified method, can be filled with 125 I-labeled thyroxine (Cambridge Nuclear Corporation, Billerica, Massachusetts) by to perform the following steps: 10 15 20 25 30 35 7905101-7 15 l. Add to each one of 100 standard tubes 1251) 0.5 ml microcapsule suspension and 1.0 microCurie T4 ( (high specific activity of 5-6000 pCi / pg). Man incube- at 37 ° C for at least 30 minutes). 2. The microcapsules were washed with 2 times the volume of phosphate buffered saline (0.5 M NaCl, pH = 7.5, 0.015 M phosphate buffer). 3. Man decanted 4. Man 5. Man their volume with the indicated phosphate buffered saline.

The total volume was 80 ml. 0.8 ml microcapsule suspension was used per test. Thus, 100 tests can centrifuged at 2000 xg for 15 min and the supernatant. 2 times diluted the microcapsule suspension by 1.6 times repeated steps 2 and 3 carried out with the capsules.

Test procedure 1) Test samples of 25 μm and 5 samples with known con- centrationfri'T4 placadesi.separatarör. In standard the curve from Table 1 used concentrations of 0.5, 1.3, 3.0, 5.0 and 7.7 ng / dl free T4, but whichever any series of free T4 concentrations can be used according to well-known experimental methods. A control tubes containing 25 μl of saline may also be included such as further control of the analysis precision. 2) Pipette 800 μl of T4 (125 I) in pre-saturated microcapsules (supplied as such) in each tube. 3) Shake each tube and incubate for 120 min at 37 ° C. 4) After the incubation, 1.0 ml of 2.0% was added polyethyleneimine (molecular weight 40,000-50,000) in phosphate buffered 5) Incubate for another 20 min. saline to each tube. 6) The supernatant was decanted. 7) Each tube was counted for 1 min in a Y-counter.

.Calculation of results 1) Each time an analysis is run to determine unknown concentration of free T4 in sample (s) should 79o51o1 + 7 10 15 20 25 30 35 16 standard tests are carried out to establish a standard dardkurva. 2) When the analysis is completed, a standard curve is made of the type shown in Figures 2 or 3, wherein using the values obtained from the standard samples, as analyzed simultaneously with unknown samples. 3) The number of pulses per minute (cpm) for each value can be deposited in the linear scale in one (2 cycle) semi-logarithmic diagram the concentration of free T4 in nanogram percentage on the log scale.

An alternative to allocating cpm v. Concentrates tion free T4 is to set aside the percentage bound (relatively) vs free T4 concentration.

This can be done by calculating the percentage bound (relatively) for each standard, control or unknown amount and sets these values on two-cycle semi-logarithmic paper in a way similar to that of previously described for cpm. Percent bound (relative) calculated as follows: percent bound (relative) = cpm bound / cpm mean bound for standard with lowest concentration free T4.

Figure 4 shows a curve of the concentration free T4 in ng% for about 200 samples, each of which was analyzed according to the invention and the dialysis method. As shown there is a high degree of correlation between the two samples methods.

Figure 5 shows the frequency of a given concentration tion free T4 vs. concentration free T4 based on approx. 200 samples, which have been analyzed according to the .the.

Figure 6 shows the concentration in ng% of free T4 in about 100 samples, each of which was analyzed according to the invention and according to the kinetic free method. As there is a high degree of correlation between the two the lighting methods.

Table 3 shows the agreement between the results within the analysis. 10 15 20 25 30 35 17 "TABLE 3 7905101-7 Variation within the analysis (values in ng / dl) Content A (1,2) 1.5 1.3 1.1_ 1.1 1.2 1.4 1.4 1.4 1.4 1.4 Content B (2.0) 2.3 2.1 2.2 2.1 2.1 Content C (5.3) 3.6 _ 4.2 4.2 -3.3 3.3 .3.9 4.1 3.7 3.5 3.5 4.2 3.4 3.8 4.2 4.6 uàoh oh then ~ ~ UWKU h) o 7905101-7 18 Variation- Number x + s.a. Variation coefficient coefficient A 28 1.34 and 0.13 9.6% B 29 2.2 and 0.17 7.7 a; 5 c 29 4.1 i 0.38 '9.3 æ Table 4 shows agreement between results from various analyzes.

TABLE 4 10 Variation between analyzes (value ng / dl) Test No. Level A (1.2) Level B (2.0) Level C (5.3) fl 1.3 2.4 4.6 1.2 1.8 4.4 15 2 1.4 1.5 4.6 1.5 2.6 4.7 1.2 2, 4.0 20 1.3 2.0 _ 3.7 1.4 2.5 3.8 1.2 1.9 4.0 3 1.3 2.2 3.9 25 4 1.4 2.0 3.2 1.2 2.1 4, 5 1.5 l, 8 4.2 30 6 1.3 2.0 4.2 1.2 2, 4.5 7 1.3 2, 3.5 1.35 2.5 3.7 1.1 1.1 1.7 4.0 “I 1.2 2.0 3.5 1.4 4.2 1.4 4.0 U? 10 15 20 F) V1 30 7905101-7 19 Variation- Number x + s.a. Variation coefficient coefficient A 20 1.32 1 0.11 8.41% B 18 2.1 1 0.27 13% C 20 4.0 1 0.4 10% Digoxinanalgs A method for encapsulating antibody specific dig against digoxin is shown below.

The reagents used and their manufacturers are counted up in the following: Sodium bicarbonate Cyclohexane Chloroform All from Fisher Chenical Sodium chloride Sodium phosphate, monobasic Sodium phosphate, dibasic 1,6-hexanediamine Span-85 Tween-ZO Fnger Chemicals, New Jersey Antidioxin antiserum from rabbit ArnelProducts, ßrooklyrnNewYork Serum albumin from cattle _ Sigma Chemical Aldrich Chemical Easümn1Kakm Comassie Brilliant Blue R Polyvinylpyrrolidone-40 Terephthaloyl chloride The amount of each ingredient was as follows: 1,6-hexanediamine carbonate 30 ml Phosphate buffered saline 40 ml Polyvinylpyrrolidone 40 / Comassie Blue 25 ml Antidioxin antiserum from rabbit 5 ml Cyclohexane, ACS 750 ml SPAN ~ 85 125 ml Terephthaloyl chloride solution 107.5 ml Chloroform 100 ml Tween-20 washing solution Q.S. 200 ml Phosphate buffered saline Q.S. 8 l lO 15 20 25 30 35 7905101-7 20 Method: Rinse all glass in distilled water before the use. 1. A two-liter glass mixer was placed in a cover on a magnetic stirrer. 2. A microscope was set up on the bench next to the cover. 3. In a graduated glass cylinder (250 ml) was measured accurately 125 ml SPAN-85. 4. The measured amount of SPAN-85 was poured in the glass mixer in the cover. 5. 750 ml of cyclohexane were measured in a graduated form glass cylinder (1000 ml). The cyclohexane was poured in the mixer in the cover. ' 6. Put the lid on the mixer. 7. The magnetic stirrer was turned on. 8. 30 ml of hexanediamine carbonate were measured; 30 ml PBS; 25 ml 15% PVP / Comassie Blue / 4% BSA; 5 ml antibody; and they mixed with 40 ml of PBS 5 ml of antibody in a graduated cylinder (50 ml). 9. A 5 cm long stir bar was placed spinning ring in a beaker (400 ml). They placed it all on a magnetic stirrer. l0. 30 ml of hexanediamine was added to the beaker.

The magnetic stirring was started. ll. To the hexanediamine in beakers was added the following Ingredients in the order indicated: 25 ml l5% PVP / Comassie Blue / 4% BSA; 35 ml PBS / antibody solution. They mixed for 2 min. The solution was allowed to mix for 3 minutes. 12. While the solution was mixing, a portion was measured of 70 ml and a dose of 37.5 ml of TCL in separate graduated glass cylinders (100 ml). It was covered with ur- glass and placed in a cover. 4 portions were measured about 25 ml of chloroform in separate graduated glass cylinders.

They covered watch glasses and placed inside the cover. 13. Through the side armrest bottle was added 400 ml of the contents of the beaker to the glass mixer in the cover. 14. Take as soon as possible with a 1 ml pipette a sample of the solution in the mixer and put the sample on a disk to a microscope. It was checked that the drop- 10 15 20 25 30 35 7905101-7 21 pairs size wolf acceptable. (10-80 Pm in diameter). l5. When the droplets had an acceptable size, T = 0, 70 ml of measured TCL was added through the side of the mixer. arm. At T = 30, exactly 30 s later, it was added second portion (37.5 ml) TCL to the mixer through side arms. The mixture was allowed to proceed for exactly 60 s. l6. After 60 s (T = 90) the first was added the 25 ml portion of chloroform. They mixed for 30 s.

(T = 120). The second measured portion was added (25 ml) chloroform, mixed for 30 s (T = 150). MAN added the third measured portion (25 ml) of chloroform. Mix for 30 s (T = 180). They added the fourth measured portion (25 ml) of chloroform.

The mixture was mixed for exactly 30 s (T = 210 "). ningen. 17. The contents of the mixer were poured into two centrifuges. bottles (1 liter) of plastic, which are rinsed in distilled water 3 times. It was centrifuged at 500 rpm for 3 minutes. 18. The supernatant was carefully poured off. 19. Approximately 50 ml was added to each bottle Tween-20 solution (dxrs the same volume of Tween-20 as the capsule na). Mix thoroughly with the stirrer rod for approx 5 minutes. 7. Approximately 10 ~ 15 ml of PBS was added to each bottle. Stirring thoroughly. 21. The step was repeated 4-5 times. 22. 400 ml of PBS was added. They mixed care fully. 23. The bottles were balanced and centrifuged for 20 min at 3000 rpm. The supernatant was aspirated. MAN added approximately 800 PBS to each bottle. They stirred carefully and put a lid on the opening. 24. The step was repeated 23 10 times. After the last the aspirator, 100 ml of PBS was added to each bottle and the contents of the two bottles were combined. They shook carefully. The contents were poured into a graduated cylinder. (500 ml) and make up to 500 ml with PBS. 10 15 20 25 30 35 79osío1-7 22 25. Store in a glass reagent bottle (1 liter) at 4 ° C.

The wash solution of Tween-20 was prepared according to following.

Procedure: Can be done the day before use. 1. Weigh in 6.06 g of sodium bicarbonate balance scale. 2. 6.06 g of sodium bicarbonate were carefully charged in a bottle (250 ml) 3. Approximately 200 ml of purified water and with magnetic rod. stirred with the magnetic stirrer to the sodium bicarbonate solved. 4. The stir bar was removed and filled up 250 ml with purified water. 5. Carefully pour the whole into an Erlenmeyer ~ flask (500 ml) containing stirring rod. 6. 250 ml of Tween-20 was carefully measured in one graduated cylinder (250 ml). 7. Add this Erlenmeyer flask, containing kept the sodium bicarbonate solution. 8. Mix with a magnetic stirrer until the whole was completely mixed (about 1 h). 9. The whole thing was stored well sealed in a one-liter polyethylene bottle with screw cap at approximately ZOOC.

The terephthaloyl chloride solution was prepared as follows method: Should be done the day before use. Do not cool. The weight of terephthaloyl chloride (TCl) was recorded on the bottle, which contained TCl. The TCl weight was multiplied in grams by 10 to calculate the volume in millimeters of it solution of cyclohexane and chloroform, which would be added to TCl.

Calculations: _____g. TCl x l0 = mold solution. Check that the total volume is ml total volume of cyclohexane / chloro- sufficient for the method to be implemented. The cyclohexane / chloroform solution is 4 parts of cyclohexane hexane and part chloroform. Divide the entire volume of cyclohexane / - chloroform solution (step 1 above) with five to calculate 10 15 20 25 30 35 7905101-7 23 the volume of chloroform. Multiply the chloroform volume by four to determine the cyclohexane volume.

Calculations: (a) _____ ml total volume - 5 = ml chloroform. cyclohexane / chloroform ml chloroform x 4 = 3. The calculated volumes were carefully measured (b) _____ _____ml cyclohexane. cyclohexane and chloroform in graded cyiindrarcx fi idethela Satsadeß in an Erlenmeyer flask. They shook gently for mixing and covered with watch glass. You put it whole in fume cupboards. 4. A magnetic stirrer was placed in the fume cupboard. 5. The vial containing TCl was placed on the magnetic the stirrer. The bottle was opened and added as soon as possible one. magnetic stir bar and solvent the cyclohexane / chloroform. They put the lid back on the bottle. Stir until all TCl is dissolved. It can be necessary to tilt the bottle to loosen any TCl around the top of the bottle. 7. So many glass centrifuge bottles were filled (ZOO ml) which was necessary with TCl solution so fast as possible and as equal as possible and one connected with lid. It was centrifuged for 10 minutes at 2600 rpm at room temperature. 8. The supernatant was poured into yellow bottles (500 ml) and sealed carefully. 9. It was stored while the bottles were well closed at about 25 ° C.

The process for producing 15% PVP 40 / Comassie Blue with 4% BSA is available as follows: Procedure: Done on the day of use. Fridge on balance scale 7.5 g of polyvinylpyrrolytic and 0.1 g (10 mg) of Comassie Blue. not. l. We weighed lidon 40; 2 g BSA; 2. Polyvinylpyrrolidone 40 was charged to a glass base. gare (50 ml) with magnetic rod. 3. Approximately 20 ml of PBS was added. One put one glass cover plate on the beaker. 7905101-7 10 15 20 25 30 35 24 4. Stir until everything is dissolved. 5. 0.1 g of Comassie Blue and 2 g of BSA were added to another glass beaker (50 ml). 6. Approximately 20 ml of PBS was added. One put one glass plate on the beaker. 7. Stir and heat gently with a magnetic hot plate / stirrer no. 10 with the heating set to 2.

Stir until everything is dissolved (about 10 minutes). 8. The entire contents of the beaker were carefully added. containing (polyvinylpyrrolidone.40% solution and (Comassie Blueï solution in a glass bottle (50 ml) with stirrer. 9. The combined solutions were mixed for 10 minutes with magnetic mixer. The stirrer was removed. 10. Make up to 50 ml with PBS. ll. The pH was adjusted to 7.5 in 0.5 with 1 N sodium hydroxide. Orig. pM ___ end ~ pH ___ amount used l N NaOH___. 12. The solution was filtered with a membrane filter (Nalgene, 0.45 p). l3. It was stored tightly in a 60 ml polyethylene bottle with screw cap at approximately 25 ° C.

The process for the preparation of the phosphate buffer The saline solution is as follows: Procedure: Can be done the day before use. l. In a graduated glass cylinder (1000 ml) was measured carefully 1000 ml of phosphate buffered saline solution. 2. It was poured into a 20 L polyethylene lady jeans. 3. Measured in graduated glass cylinders (1000 ml) 9000 ml of deionized water and added to the ladies' jeans containing the phosphate buffered saline solution water. 4. Stir with a magnetic rod. '5. The pH was checked. If necessary, set pH to 7.5 in 0.05. Final pH =. 6. The phosphate buffered saline solution was stored tightly closed 20-liter polyethylene lady jeans. Man lag- at about 25 ° C until use. 10 15 20 25 30 35 'Preferably labeled digoxin is ( 7905101-7 25 The process for the preparation of 1,6-hexanediamine carbonate is as follows: Procedure: Can be done the day before use. l. A bottle of 1,6-hexenediamine was placed in a 3 liter beaker. Sufficient cranes were added. water to the beaker to reach the water level to the hexanediamine level in the bottle. 2. The lid of the bottle was removed with hexanediamine. 3. Place the beaker on a magnetic stirrer / - hot plate with heat setting 2 until the hexanediamine completely melted. 4. Exactly 17.7 ml of hexanediamine was measured in one graduated glass cylinder (25 ml). You poured carefully in a yellow bottle (500 ml). 5. Measure exactly 32 ml of purified water in one graduated glass cylinder (50 ml). Hexane was added the diamine in the yellow bottle. 6. CO 2 was bubbled through the solution for about 1 hour until pH = 8.5 in 0.1. Final pH. 7. The yellow bottle was closed and stored about 25 ° C.

The microcapsule and its working principle are shown in Fig. 9.

As can be seen from the figure, an antibody 9 is captured, which is specific for digoxin, within the wall 10 of the microcapsule 12.

However, the wall 10 of the microcapsule 12 has openings, which are 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 places on the antibody 9.

Unbound digoxin 16 or 18 can be washed from the microcapsule after incubation is complete.

As stated above, a certain portion of the digoxin is labeled and it is the labeled digoxin that competes with it unlabeled digoxin from the sample with respect to the sites on the antibody specific for digoxin. One l25I) ~ digoxin, which can be held by New England Nuclear Corporation, Billerica, Massachusetts. 10 l5 20 25 30 35 7905101-7 26 Test procedure l. One or more samples of the serum, which to be tested, obtained in a well-known manner. In this example, control samples were used. 2. Pipette (Ll ml (100 nl) of each standard standard, control or unknown patient serum test in labeled tubes containing 0.5 ml of microcapsule suspension pension with digoxin antibody. 3. 0.5 (500 μl) 125 I-labeled digoxin was pipetted (in phosphate buffered saline) in each tube. They shook for at least 3-4 s. 4. All reaction tubes were incubated in water. bath (37 ° C) for at least 15 minutes. [Alternatively incubate all reaction tubes at room temperature (20-2500) in at least 30 min]. 5. 0.5 ml (500 μl) of washing solution was added each tube. Shake for at least 3-4 s. 6. All tubes were incubated at room temperature (zo-2s ° c) 1 5 min. 7. All tubes were centrifuged at least 1600 xg for 10 minutes, room temperature (20-25 ° C) and poured over the liquid in a suitable waste container and captured the last straw on a writing pad. 8. All the tubes were counted in a Y-counter, which included set for 125 9. A standard curve for known quantities was established. I under l min var. digoxin against cpm and read the unknown amount of digoxin the one from this curve.

Use reagents Digoxin antibody encapsulated isemipermeable nylon microcapsules suspended in phosphate buffered saline solution, prepared as above. To be able to control the quality contains antibody the microcapsules blue dye (Comassie Blue R25O).

A blue supernatant is after the microcapsules have sunk or sedimented by centrifugation a sign of microcapsule rupture and possible leakage of antibody. 10 15 20 25 30 35 7905101-7 27 1251-labeled digoxin Each milliliter of solution contains l0 pg l25I digoxin with less than 4.2 p Ci.

Buffer solution In 0.015 M phosphate buffer, pH 7.5, containing 0.15 M sodium chloride 0.5% bovine serum albumin (BSA), 0.1% sodium azide.

Washing solution Polyethylene glycol 6000, 20% solution in 0.5 M barbital buffer, pH 8.9, containing 0.15 M sodium chloride.

Calculation of results The results of this experiment are shown in Table 5 and is shown graphically in Figures 7 and 8.

In Fig. 7, the CPM is plotted on the linear scale a 2-cyclic semi-logarithmic paper against digoxin the concentration in ng / ml (nanogram / milliliter) on the log scale. An alternative to setting aside CPM vs. digoxin- the concentration is to set aside a percentage bound (relatively) vs. the digoxin concentration (see Fig. 8). This can be done by calculating the percentage bound (relatively) for each standard, control or unknown sample, and set aside these values on a two-cycle semi-logarithmic paper in a similar way as previously described for CPM.

The percentage bound (relative) is calculated as follows: Percent bound (relative) = (average value for CPM bound in 0 ng / m1 digoxin standard) x 100.

'TABLE 5 ng / ml CPM bound Digoxin l 2 Default 0 16 888 17 136 0.5 14 464 14 473 1.0 12 279 12 192 2.0 9 690 9 717 4.0 7 344 7 445 Control 1 10 360 10 486 2 8 226 8 380 Total CPM = 26,780 ° '\ lO 15 20 25 30 35 7905101-7 for Relatively% bound (CPM Digoxin content Mean bound / cpm bunaet) * x 1oo ng / m1 17 o12 1oo, o 14,469 85.0 12,236 72.0 9,704 57.0 7 395 43.0 1o 423 61.3 1.7 8,303 43.8 3.1 xßundet = CPM bound at 0 ng / ml digoxin The analysis system according to the invention has been shown be very accurate. The variation within the analysis is smaller than 7% and the variation between analyzes is less than 9%.

Variation in the analysis The coefficients of variation, CV, for two control samples, each of which was analyzed 25 times during an ment turned out to be: Mean value for digoxin Q! Controls 1 1.54 4.9% Kontro11 2 2.95 6.2% Variation between analyzes The coefficients of variation for two control samples, each analyzed 3 times in 18 separate experiments, proved: Mean ng / ml * QX Control l 1.50 7.3% Control 2 3.04 8.8% Table 6 shows this analysis extremely high specific digoxin and the potential for exclusion of others disturbing substances. 10 15 20 25 30 35 7905101-7 29 TABLE 6 Specificity % Cross-reactivity for different biochemical compounds with microencapsulated intidigoxin antiserum Compound% cross-reactivity Digoxin 100.00 Digitoxin 0.80 Progesterone 0.16 Cortisol 0.10 Testosterone 0.08 Dehydroandrosterone sulfate 0.08 Cholesterol 0.06 Quabain 0.02 Quantitative uptake The present method has been found to be quantitatively and reflects no less than 96% uptake of added digoxin samples.

TABLE 7 Uggtagningstudie Initial content Added Total content Measured% digoxin digoxin digoxin digoxin Uptake- ng / ml ng / ml ng / ml ning 0.64 0.5 1.14 1.13 99 0.64 1.0 1.64 1.72 105 0.64 2.0 2.64 2.70 102 0.64 4.0 4.64 4.45 96 It is particularly important to note the high correlation the degree of lation between the results according to the present procedure with digoxin determinations, made according to other methods of analysis.

Table 8 shows comparative results obtained according to the invention in relation to results according to other analyzes.

Systems A and C relate to liquid analysis systems 7905101-7 lO 15 20 25 30 35 30 with separation using precipitating reagents, while system B uses solid phase separation. As shown the correlation coefficient is 0.97-0.98.

TABLE 8 concordance between different methods of analysis 5Correlation coefficients for 135 samples, which analyzed according to system A, system B, system C and the present system was: n rä y System A 135 0.98 1.12 x -0.1 System B 135 0.97 0.90 x -0.02 System C 135 0.97 0.96 x +0 Present system 135 0.97 0.95 x + 0.14 kr = correlation coefficient In the light of what has been stated above, it is obvious that the method of analysis shown can be used to mood of presence and concentration of any free substances provided that you have access to a complementary substance, which has specific binding ability of the substance, and a discernible analogue of the substance. An additional requirement is that the substance and its analog has a molecular weight which is low enough for that it should be possible to provide microcapsules membranes, which selectively allow diffusion thereof substances but prevents the passage of more high molecular weight materials, such as natural proteins. Fortunately, steroid hormones, thyroid hormones, many drugs agents and other types of substances of clinical importance of molecular dimensions, which are far smaller than those dimensions of natural proteins.

The invention may be expressed in other specific terms forms without departing from the spirit of the invention or its essential characteristics. The specified embodiments and the examples shall therefore be considered as illustrative in all respects. and non-limiting and the scope of the invention is set forth by 7905101-7 31 attached requirements and not of the previous description.

All changes, which fall within the meaning of the requirements and equivalence meaning is therefore intended to be encompassed by the invention ningen.

Claims (10)

7905101-7 10 l5 20 25 30 32 PATENT REQUIREMENTS Immunoassay method for determining the presence of a substance not bound to protein in a proteinaceous sample, characterized in that the sample is incubated with a complement antibody to the substance, and a distinguishable analogue to the substance which has membranes passage of present in microcapsules, with sufficient porosity to allow the substance and its discernible analog, but insufficient porosity to allow the passage of the antibody and protein-bound substance contained in the sample, to allow unbound substance in the sample to pass through the membrane and compete with its separable analog with respect to binding sites on the antibody, separating the antibody from unbound substance and unbound analog by removing unbound substance and the analog from the microcapsules and separating the microcapsules from the rest of the reaction system, and determining the amount of separable analog , which is bound to the antibody, or which is antibody-free , which amount is a measure of the amount of substance that is not bound to protein in the sample. 2. A method according to claim 1, characterized in that the amount of the analog bound to the antibody is determined. 3. A method according to claim 1, characterized in that the discernible analog is constituted by a substance labeled with a radioactive atom. 4. A process according to claim 1, 2 or 3, characterized in that the substance is L-thyroxine. 5. A method according to claim 1, 2 or 3, characterized in that the separation step is effected by inducing a change in osmolality in the reaction system, which change causes the microcapsules to collapse. Process according to Claim 1, 2 or 3, characterized in that the substance is digoxin. Test equipment for use in the method according to claim 1, characterized in that it comprises in combination: microcapsules containing complement antibody to the substance and a separable analogue to the substance, which microcapsules comprise permeable membranes which allow diffusion of the substance and its separable analog, but which does not allow diffusion of the antibody or other proteins, the microcapsules being intended for absorbing unbound substance from the sample, a reagent capable of removing unbound substance and its unbound distinguishable analogue from the microcapsules, and a standard , which contains a predetermined amount of the substance, which standard is intended for comparison with a sample. Test equipment according to claim 7, characterized in that the substance consists of thyroxine, that the discernible analog consists of radiolabeled thyroxine, that the standard comprises aliquot amounts of materials containing known quantities of free thyroxine which when tested in parallel with the sample, makes it possible to establish a standard curve. 9. Test equipment according to claim 7, characterized in that the reagent comprises a material selected from the group consisting of serum albumin and polyethyleneimine. Test equipment according to claim 7, characterized in that the microcapsules contain antibody to digoxin and that the distinguishable analog is 1251-labeled digoxin.