NO154409B - PROCEDURE FOR DETERMINING THE PRESENCE OF A PROTEIN-BONDED CONNECTION IN A PROTEIN CONTAINING TEST. - Google Patents

Description

The invention relates to the detection and determination of the concentration of unbound compounds, e.g. hormones, in a liquid sample, e.g. serum. More particularly, the invention relates to a method which is suitable for the detection of free compounds of the type capable of reversibly binding with protein in samples containing the free substances, the binding protein and a concentration of compound/protein complex.

The so-called "competitive assay" analysis technique for measurement

of the concentration of various biologically active compounds for diagnostic purposes is now well established. The technique involves a reaction system where antibody that is specific for the compounds to be determined is incubated together with the sample and an aliquot of an analogue of the sample that can be distinguished. The analog and the natural compounds compete for sites for attachment to the antibody, and after separating the antibody from the rest of the reaction system, the antibody or supernatant can be examined for the analog. The amount of analogue bound to the antibody is an inverse function of the concentration of the test compounds in the sample.

Two common methods of performing such competitive assays are known as "liquid phase radioimmunoassay" and "solid phase radioimmunoassay". In each system, the immunogenic compound that is bound to an antibody must be removed from the unbound immunogenic compound in order to be able to measure the amount of labeled analog on the antibody. Based on this measurement, the amount of immunogenic compound in the sample is determined.

In "liquid" phase radioimmunoassay systems, the antibody, the labeled analog, and the immunogenic compound to be determined are incubated in solution. A number of antibody binding

seats are available, and the reaction time is short. To sepa-

To separate the antibody-complexed immunogenic compound from the non-complexed immunogenic compound, the antibody/immunogen complex is precipitated, centrifuged and the supernatant decanted.

"Solid phase" radioimmunoassay systems avoid the precipitation step. The antibody binds to a glass "chip" or inner surface in a tube. Centrifugation of the glass pieces or decantation of the coated tubes will separate the antibody-complex

compound from the non-complexed immunogenic compound. However, the reaction time is relatively long because a number of available active sites on the antibody are blocked by the piece of glass ("chip") or tube.

Hormones, e.g. those secreted from the thyroid gland, testes, ovaries, adrenal cortex and other mammalian glands are often transported through the circulatory system in association with hormone-binding proteins or globulins. Often, it is of diagnostic importance to be able to determine the presence and concentration of free hormone, as opposed to protein-bound hormone or total hormone. For example, thyroxine (T4) exists in the bloodstream in a dynamic equilibrium relationship as a compound bound to a transport protein (thyroxine-binding globulin, albumin, or prealbumin) and, in a small fraction (potentially 0.1% or less), as an unbound compound ( "free" or unbound (T^). Free is believed to be the active compound in the thyroid hormone system. Unfortunately, the methodology for determining free T. is cumbersome, complicated, expensive, and associated with sources of error due to the difficulty in standardizing materials and measuring small amounts of unbound hormone in the presence of overwhelming amounts of hormone relatively loosely bound to protein.The competitive determination process discussed above is unable to determine concentrations of free compound.

Of the methods for measuring free T. and other hormones that exist in vivo as protein complexes, the dialysis membrane method provides a high degree of accuracy. A dialysis bag containing known amounts of serum to be tested and labeled hormone is suspended in puffs. The labeled hormone distributes itself between the free and bound states in the same proportions as the serum hormone. The mass of labeled hormone that is added must be extremely small so that serious disturbance in the system is avoided, and yet the counting rate must be so high that detection of small amounts is possible. In the case of radioactive labels, this requires that a labeled hormone with a very high specific activity be used. After an appropriate period (about 24 hours), the labeled and natural hormone that is not bound to protein diffuses through the semipermeable dialysis bag, while hormone that is bound to protein (molecular weight above 20,000) remains in the dialysis bag. To determine the amount of free hormone present in the serum, an aliquot of the buffer for labeled hormone is determined. As a result of the determination, the fraction of labeled hormone that migrated into the buffer can be calculated and the fraction of total hormone that exists in the free state can be deduced. To convert this fraction into mass units (eg ng/dl) of free hormone, a total hormone determination must also be made on the sample.

Although this system can produce significant results

in free T^- and other free hormone determinations, it is poorly suited for routine use. Two tests must be performed, and the accuracy of the final result may be compromised by either method. Fairly large amounts of radioactivity must be used for each test. Interfering contaminants in the labeled hormone can cause special problems, and long incubation times are required. Only serum can be used, and it must

be fresh. Furthermore, the collection and standardization of all the materials necessary for the determination is not a routine matter. Thus, the application of the method has been limited due to the high price and labor intensity and the fact that skilled personnel are necessary.

Another method for determining free hormone involves reaction kinetics and requires two separate tests. Each test measures a kinetic curve that is in relation to how quickly an antibody captures hormone, e.g. T^, away from the counteracting pull of the primary binder, e.g. thyroxine-binding globulin.

In such an analysis system, inaccuracies result from several pathological conditions. If more protein binding sites are present than usual, the effective attraction of the globulin for the hormone will be greater and the rate of binding to antibody will decrease. In the case of thyroxine analysis, this would give the appearance that the sample had little when in fact there could be a high level of 1^, but all in bound form.

The present invention provides an immunoanalytical method for determining the presence of a compound that is not bound to protein, in a protein-containing sample by incubating the sample with an antibody that is complementary to the said compound and a labeled analog of the said compound, and which is contained in microcapsules which have membranes with a permeability sufficient to allow said compound and its labeled analog to travel through, but insufficient to allow pass-

ation of said antibody or protein-bound compounds present in the sample.

The method according to the invention is characterized

by the antibody being separated from unbound compound and unbound analogue by inducing a change in osmolality in the reaction

the reaction system that causes the microcapsules to fold together, and the microcapsules are separated from the rest of the reaction system; and the amount of the labeled analog bound to the antibody or unbound to the antibody is determined.

The method according to the invention can be carried out in

presence of serum proteins and bound hormone. Thus, semipermeable microcapsules containing antibody are incubated, which are com-

complementary to the hormone or other compounds to be applied

is shown, with both a distinguishable analog for the hormone and the sample. To reach-

the log can be introduced into the microcapsules before incubation in such quantities that the antibody is saturated at the hormone binding sites. The micro-capsule walls comprise membranes that separate the sample from the anti-

the substance, and has sufficient permeability to allow the pass-

ation of the free compound and its analog, but insufficient to allow passage of the antibody, native proteins, or protein-bound hormone. If a sample is added to an aliquot of the microcapsules, free hormone diffuses through the membranes and competes with its analog for sites of attachment on the antibody.

Thus, the distribution of analogue between the antibody and the rest becomes

of the reaction system indicative of the amount of free hormone originally present in the sample. This method combines the inherent accuracy of a conventional liquid-phase radioimmunoassay with the simplicity and convenience of a solid-phase

system.

The antibody is then separated together with its bound hor-

mon (and the bound analogue) from the free compound, and either the antibody or the rest of the reaction system is determined by

view of analog. The separation can be carried out by inducing an os-

motic change so that the capsules clap together and unbound hor-

moner migrates out of the capsules. This can e.g. is carried out by adding serum albumin or polyethyleneimine to the system which

causing drainage of intracapsular fluid. Alternatively, free compound may simply be washed out from the microcapsules. The results are interpreted by comparing the analysis of the analogue content with a standard, e.g. a plot of free hormone concentration versus radioactive count, fluorescent intensity, or other marker characteristic used to indicate the presence of the analog.

The method according to the invention can be used for detecting and/or determining the concentration of free thyroxine, triiodothyronine, neonatal thyroxine, testosterone, cortisol, other steroid hormones and other compounds that bind reversibly with protein. The method can also be used for the detection of compounds that do not bind to protein to any significant extent, e.g. drugs, such as digoxin. Essentially any such material can be determined provided that a complementary binding compound and a labeled analog of the compound are available or can be produced.

The method can be performed routinely using a sample kit comprising analog, microcapsules containing antibody standards and blanks containing predetermined concentrations of the compounds of interest, and a reagent for removing unbound compound and analog from the capsules after completion of the incubation. For quality control, the solution in the microcapsule can be colored. A colored supernatant, after the capsules have been precipitated or sedimented via centrifugation, would be indicative of microcapsule breakage and possible leakage of antibody. Unlike conventional determinations, this method avoids loss of clinical correlation with diagnosed conditions over a wide range of concentrations of protein, hormone and interfering compounds.

The present invention thus provides a fast, simple and reproducible method for detecting and/or determining the concentration of a compound that is not bound to protein in a liquid sample. The invention can also be used for the determination of free T. in samples extracted from human blood and which contain protein-bound T.

Furthermore, by means of the method according to the invention, it is possible to determine an immunogenic compound which combines the advantages of the solid-phase radioimmunodetermination system and the advantages of the liquid-phase radioimmunodetermination system. In addition, a very reliable method for determining the amount of digoxin in serum is thereby achieved. These and other objects and features of the invention will be apparent from the following description. Fig. 1 illustrates a standard curve drawn up when carrying out the method according to the invention. It consists of a point for counts per minute (x 10 3 ) on a y-axis in linear scale versus the concentration of free T4i ng/dl on an x-axis in log scale (data, see table 1); Fig. 2 illustrates another standard curve for the determination of free T4, according to the invention, in cpm (linear) ctra. ng/dl free-T^ (log scale). Data for this figure can be found in table 2; 125 Fig. 3 is a diagram of cpm from (I) bound to T^-antibody contained in semipermeable microcapsules immersed in 1 standard solutions of free T. ctra. time. As 125 serum T^ replaces ( I) at T^-antibody binding sites, decreases the cpm remaining in association with the antibody. It is noted that the turnover rate is largely linear up to 2 hours of incubation at 37°C; Fig. 4 graphically illustrates the correlation between results between the dialysis assay technique and the microencapsulation system; Figure 5 graphically illustrates the normal range for free as established by the microencapsulated assay system; Fig. 6 graphically illustrates the correlation between kinetic radioimmunoassay and the microencapsulated type assay system; and Fig. 7 shows a digoxin standard curve in the form of counts per minute (cpm) bound to antibody, plotted as points on the linear scale, ctra. digoxin in nanograms/milliliter on the log scale on 2 cycles of semilog paper (data from Table 5); Fig. 8 shows digoxin as % bound (relative) ctra. the digocin concentration. % bound (relative) is calculated as % bound (relative) = mean cpm bound) mean cpm bound by 0 ng/dl digoxin standard x 100 (data from Table 8); and Fig. 9 is a diagram showing the retention of antibody and free passage of immunogenic substance in microcapsules.

The method according to the invention requires that the sample containing the compound to be detected be incubated with antibody complementary to the compound (or another compound capable of reversibly binding with the compound) and a distinguishable analogue of the compound, and that the sample and the antibody are separated by means of a semipermeable membrane or such membranes which exclude the passage of high molecular weight proteins.

Antibodies to selected compounds can be produced in accordance with well-known techniques which involve injecting the hormone into laboratory animals, possibly together with an auxiliary substance, and then extracting and purifying the produced antibodies. Many such antibodies are commercially available, many distinguishable analogues of compounds which can be determined by the method according to the invention are also commercially available or can be made using known techniques. The term "distinguishable analog" as used herein refers to a molecule that has antibody-binding properties similar to, and preferably identical to, the compound to be detected, and that is characterized by properties that permit a measure of its concentration and are readily obtained. . Preferred analogs include a sample of the compound to be detected labeled with an isotope. For example, the thyroid gland hormone can be labeled with <125>I and can then be quantified by measuring gamma radiation. However, it is understood that other types of analogs can be used, when only the analog has a molecular weight and resulting dimensions that are well below the values for natural proteins and the antibody used. Thus, analogues can be prepared by labeling a sample of the compound to be detected with a relatively low-molecular-weight enzyme, a fluorescent group or another group that enables quantitative measurement of the concentration of the analogue by physical or chemical means.

To carry out the determination method, the antibody and the analogue must be separated from the sample by means of one or more membranes which have a permeability which excludes the passage of antibodies and natural proteins (which uniformly have a molecular weight in excess of 20,000 daltons), but which allows free passage of the compound to be detected and its analogue. It is preferred, when carrying out the method according to the invention, that the membranes have the form of semipermeable microcapsules containing the antibody and the analogue.

It is worth noting that the semipermeable microcapsules used have a permeability similar to that of the dialysis membranes described above. However, the semipermeable microcapsule wall is 100 times as thin as conventional dialysis membranes, and its available surface area is of an order of magnitude much greater per weight unit. Free or other free hormones freely enter the microcapsule and displace, proportionally to the concentration, the label from the T^-antibody. Thus, an equilibrium is approached between free and labeled T.

in the capsule during the incubation period.

Suitable methods for encapsulating biological materials in membranes having the aforementioned permeability properties are disclosed in detail in US Patent Applications 606,166, 931,177, 30,847 and 24,600. The currently preferred method for producing such microcapsules provides semipermeable polyamide membranes by means of an interfacial polycondensation technique. Mutually immiscible solvents or solvent systems are selected, e.g. water and a cyclohexane-based solvent, and one monomer of a complementary pair forming a copolymer is dissolved in the water together with the material to be encapsulated. The aqueous solvent containing the material to be encapsulated is then emulsified in the other solvent to form a plurality of separated droplets. The second complementary monomer is then added to the continuous phase of the emulsion to initiate the polymerization around the droplets at the phase boundary. Membrane permeability and uniformity of polymer deposits are controlled by varying the affinity of the continuous phase of the emulsion for the encapsulated monomer during the polymerization process and by controlling the concentration of the reacting monomers and the duration of the polymerization.

In one embodiment, the continuous phase is initially a solvent or solvent system which has a relatively high affinity for the encapsulated monomer, so that in a first polymerization step a relatively thick polymer network is produced around the droplets. The continuous phase is then changed so that its affinity for the first monomer decreases, e.g. by diluting the continuous phase with another solvent or by replacing the continuous phase with fresh solvent. After addition of the second monomer, further polymerization takes place in a preferred manner within the initially deposited polymer network, which will patch up macroporous defects and result in uniform capsule membranes that allow diffusion of solutes below a certain molecular weight.

In another embodiment, the continuous phase is initially selected so that it has a low affinity for the encapsulated monomer so that thin, relatively dense membranes are formed in a first polymerization step. The affinity of the continuous phase for the encapsulated monomer then increases so that further amounts of monomer are drawn through the membrane and another, outer layer of insoluble polymer is deposited.

When the discontinuous aqueous droplet phase is buffered to provide a compatible environment for labile biological materials such as an antibody, the encapsulation can be performed in a manner that preserves a large percentage of the labile material's biological activity. The operability of the encapsulated material is also preserved by adding another monomer to the continuous phase in portions during the duration of the polymerization so that its concentration at a given time is relatively low and the antibody is not exposed to high concentrations of potentially destructive compounds.

In a preferred reaction system, aqueous droplets containing a diamine, a high molecular weight filler material and the antibody are produced in a continuous phase of cyclohexane whose affinity for the monomer dissolved in the droplet phase is modified by the addition of chloroform as a diluent. The addition of a diacid halide to the system results in the formation of semipermeable polyamide microcapsules. This microencapsulation embodiment is effective for producing membranes that have an upper permeability limit in the molecular weight range of 2000-30,000 daltons. Thus, the capsules can easily be built up so that they allow the diffusion of many hormones.

To carry out the determination, the sample is mixed with microcapsules of the type described and incubated, preferably at approx. 37°C. Prior to incubation, the capsules may be suspended in a solution of the compound analog of sufficient concentration to load

the antibody with a detectable amount of the analog concentration.

however, the determination can be performed by adding the analog to the reaction system during or after the incubation with serum. Protein in the sample and protein/compound complexes cannot go

through the microcapsule wall.

The antibody is then separated from the rest of the reaction

the system (possibly excluding the microcapsule membranes),

and either the antibody or the rest of the system is analyzed with respect to the analogue. In a preferred method of carrying out

After the separation, a hydrophilic material with a high molecular weight is added, e.g. a solution of polyethyleneimine or serum

albumin, to the extracapsular volume. This induces an osmolality increase which results in the folding of microcapsules

lene and migration of intracapsular unbound hormone and its analogue into the supernatant. The result is a packed pellet of encapsulated

light antibody which, after possible centrifuge treatment, can be isolated by aspiration or decantation of the supernatant.

Alternatively, the separation can be carried out by washing free compounds

else and analog from the capsule.

Tables 1 and 2, and the corresponding figures 1 and 2 in teg-

ning, shows the results of provisions that constitute the execution

forms of the invention intended to show the presence of free T.

125

using free T^ antibody, I-labelled thyroxine as analog, and solutions of known free T^ concentration.

Tests of unknown samples run parallel to the applied pro-

procedure so that you get collected data that can be interpreted under

display to the standard curves.

The invention will be illustrated in the following.

Production of microcapsules

Hexanediamine carbonate (pH = 8.5 in 0.1) solution is prepared

by mixing 17.7 ml of 1,6-hexanediamine with 32 ml of water, and bubbling CO 2 through the solution for approx. 1 hour or until pH-ni-

the water level has been reached. Terephthaloyl chloride (TC1) solution is prepared by adding 20 g of TC1 to 200 ml of organic solvent consisting of 4 parts cyclohexane and 1 part chloroform. TC1 is dissolved by vigorous stirring, and the solution is then centrifuged for 10

minutes at 2600 rpm. Any precipitation product is discarded.

750 ml of cyclohexane are mixed with 125 ml of "SPAN-85" (emulsifier, fatty acid ester or sorbitan) in a 2 liter mixer equipped with a magnetic stir bar. While stirring, a mixed solution made of 1 ml antiserum to thyroxine (4% in phosphate-buffered saline), 25 ml polyvinylpyrrolidone - 4% bovine serum albumin, and 30 ml hexane-diamine carbonate solution is added to the cyclohexane. When droplets of the desired size have been produced, 70 ml of TCl solution is added. 30 seconds later, 37.5 ml TC1 is added. 60 seconds later, 25 ml of chloroform is added. Three additional 25 ml aliquots of chloroform are added at 30 second intervals.

The microcapsules are recovered by centrifugation of the two-phase reaction system, decanting the supernatant and mixing the capsules

lene with "TWEEN-20" (polyoxyethylene derivative of fatty acid-partial

ester of sorbitol anhydride - emulsifier buffered with NaHCO^) and phos-

barrel-buffered saline. The capsules preserve the polyvinylpyrrolidone and bovine serum albumin filler materials, as well as the thyroxine anti-

the substance.

Saturation of antibody with analogue

Microcapsules prepared in accordance with the above procedure can be saturated with <125>I-labeled thyroxine by

following steps.

1. Add to each of 100 standard tubes 0.5 ml of microcapsule suspension

125

pension and 1.0 microCurie of T^( I) (high specific activity of 5-6000 micro-Ci per microgram). Allow incubation at 37°C

for at least 30 minutes.

2. Wash the microcapsules with twice their volume of phosphate buffered saline (0.15m NaCl, pH = 7.5, 0.015m phosphate buffer). 3. Centrifuge at 2000 G for 15 minutes and decant the supernatant liquid.

4. Repeat steps 2 and 3 twice.

5. Dilute the microcapsule suspension with 1.6 times its volume of the phosphate buffered saline mentioned above. Total volume then becomes 80 ml. 0.8 ml microcapsule suspension is used per test. Thus, 100 tests can be carried out with the capsules.

Test method

1) Place 25 microliter samples and 5 samples with known concentration of free T^ in separate tubes. In the standard curve from Table 1, concentrations of 0.5, 1.3, 3.0, 5.0 and 7.7 ng% of free T^ were used, but any series of free T^ concentrations can be adapted in according to well-known experimental techniques. A control tube containing 25 µl of saline can also be included as a further check of the accuracy of the determination. 12 5 2) Pipette 800 µl of T4(I) pre-saturated microcapsules (provided as such) into each tube.

3) Vortex each tube and incubate

120 minutes at 37°C.

4) After the incubation, add 1.0 ml of 2.0% polyethyleneimine (M.V. 40-50 thousand) in phosphate-buffered saline to each tube.

5) Incubate for an additional 20 minutes.

6) Decant the supernatant.

7) Count in each tube for 1 minute in a gamma counter.

Calculation of the results

1) Every time a determination is made of unknown free T^ concentration in one or more samples, standards must be measured to set up the standard curve. 2) After the determination is finished, a standard curve is created as shown in fig. 2 or 3 using values obtained from the standards examined simultaneously with unknown samples. 3) Counts per minute (cpm) for each value can be plotted on the linear scale of 2 cycle semilog chart paper ctra. free T^ concentration in nanogram % on the log scale.

An alternative to point deposit of cpm ctra. free T^ concentration is to deposit % bound (relative) ctra. free T^ concentration.

This can be done by calculating the % bound (relative) for each standard, control sample or unknown sample and plotting these values on two-cycle semilog paper in a manner similar to that described earlier for cpm. % bound (relative) is calculated as follows: % bound (relative) = cpm bound/average cpm bound for the standard of lowest free T^ concentration.

Fig. 4 is a curve showing free T^ concentration

in ng% for approx. 200 samples, each of which was examined by the method according to the invention and the dialysis method. As shown, there is a high degree of correlation between the two test methods. Fig. 5 is a curve showing the frequency of a given free T4 concentration ctra. free T4 concentration based on approx. 200 samples examined in accordance with the procedure indicated above. Fig. 6 is a curve showing free T4 concentration in ng% of approx. 100 samples, each of which was examined by the method according to the invention and the kinetic radiodetermination technique. As shown, there is a high degree of correlation between the two test methods.

Table 3 shows the consistency of the results intra-determination.

Table .4 shows the consistency of the results, inter-determination.

The microcapsule and the principle of its operation are shown schematically in fig. 9. As shown in fig. 9, antibody 9 which is specific for digoxin is captured within the wall 10 of the microcapsule 12. However, the walls 10 of the microcapsule 12 have openings 14 which are small enough to allow free passage of digoxin. Thus, digoxin 16 from the sample and labeled digoxin 18 can freely pass through the walls of the microcapsules and compete with each other for the places on antibody 9. Any unbound digoxin 16 or 18 can be washed out of the microcapsule after the incubation is finished.

As noted above, some of the digoxin is labeled, and it is the labeled digoxin that competes with the unlabeled digoxin

from the sample in terms of spots on the antibody that are specific for digoxin. The preferred labeled digoxin is (I) digoxin.

Test method

1. One or more samples of serum to be tested are obtained by a method that is well known in the field. In this example, control samples are used. 2. Pipette 0.1 mL (100 µl) of each standard, control, or unknown patient serum sample into correspondingly labeled tubes containing 0.5 mL of digoxin-antibody microcapsule suspension as prepared above. 125 3. Pipette out 0.5 ml (500 µl) of I-labeled digoxin (in phosphate-buffered saline) into each tube. Shake the Vortex for at least 3-4 seconds.

4. Incubate all reaction tubes in a water bath (37°C) for at least

15 minutes. [Alternatively, incubate all reaction tubes at room temperature (20-25°C) for at least 30 minutes.] 5. Add 0.5 ml (500 µl) wash solution to each tube. Shake the Vortex for at least 3-4 seconds.

6. Incubate all reaction tubes at room temperature (20-25°C) i

5 minutes.

7. Centrifuge all tubes at at least 1600 G, for 10 minutes at room temperature (20-25°C), and decant the supernatant into a suitable waste container, catching the last drop on absorbent paper.

8. Count all tubes in a gamma counter adjusted for <1>25I i

1 minute each.

9. Plot points for standard curve of known amounts of digoxin ctra. cpm and extract unknown amounts of digoxin from this curve.

Reagents used

The digoxin/antibody is encapsulated in semipermeable nylon microcapsules suspended in phosphate buffered saline as prepared above. For quality control, the antibody microcapsules contain blue dye (Comassie Blue R250^ " A ^>1^ supernatant,

after the microcapsules have been precipitated or sedimented via centrifugation, would indicate microcapsule breakage and possible antibody leakage.

^^ I- labeled digoxin

125

Each ml of solution contains 10 µg of I digoxin with less than 4.2 µCi.

Buffer solution

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 from this experiment, a typical test, are shown in table 5 and shown graphically in figures 7 and 8.

In fig. 7, the CPM is plotted on the linear scale on 2-cycle semilog curve paper ctra. the concentration of digoxin in ng/ml (nanograms/milliliter) on the logarithmic scale. An alternative to the provision of CPM ctra. digoxin concentration is to deposit % bound (relative) ctra. digoxin concentration (see Fig. 8). This can be done by calculating the % bound (relative) for each standard, control or unknown sample and plotting these values on 2-cycle semilog paper in a similar manner as described earlier for CPM: % bound (relative) calculated for the following way:

% bound (relative) = (mean CPM bound by 0 ng/ml digoxin standard) x 100.

The analysis system according to the present method has proven to be completely accurate. The intra-assay variation is less than 7% and the inter-assay variation is less than 9%.

Intra-determination variation

The coefficients of variation, CV, for two control samples, each analyzed 2 5 times within one experiment were found to be:

Inter- determination variation

The coefficients of variation for two control samples, each analyzed three times in 18 separate experiments were found to be:

Table 6 shows the extremely high specificity of this assay for digoxin and for the exclusion of other potentially interfering compounds.

TABLE 6

Specificity

% cross-reactivity for different biochemical compounds with microencapsulated antidigoxin antisera:

Quantitative extraction

The present method has been shown to be highly quantitative, as it reflects no less than 96% recovery of the added digoxin samples.

It is of particular importance to note the high degree of correlation between the results of the present method and digoxin determinations made by other analytical methods.

Table 8 expresses comparative test results obtained by the method according to the invention ctra.

results from other analyses.

Systems A and C represent liquid determination systems with precipitation reagent separation, while system B uses solid phase separation. As shown, there is a 0.97-0.98 correlation coefficient.

Claims (3)

1. Iranunoanalytical method for determining the presence of a compound that is not bound to protein, in a protein-containing sample by the sample is incubated with an antibody complementary to said compound and a labeled analog of said compound, and which is contained in microcapsules having membranes of sufficient permeability to allow said compound and its labeled analog to migrate through, but insufficient to allow passage of said antibody or protein-bound compounds present in the sample, characterized in that the antibody is separated from unbound compound and unbound analog by inducing an osmolality change in the reaction system which causes the microcapsules to collapse, and the microcapsules are separated from the rest of the reaction system; and the amount of labeled analog bound to the antibody or unbound to antibody is determined. 2. Method as stated in claim 1, characterized in that the said compound is L-thyroxine. 3. Method as stated in claim 1, characterized in that said compound is digoxin.