JPS6128942B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for adsorbing and separating a ligand (herein, a ligand means a ligand capable of binding to a protein) from an analysis medium containing a protein.

When examining the binding state of a ligand to proteins, it is often necessary to separate the ligand into a protein-binding portion and a non-binding (no protein) portion in order to examine or measure the amount of a specific portion. . This separation can be accomplished by ultrafiltration or gel filtration; selective precipitation of the protein with bound ligands such as precipitation with ammonium sulfate, polyethylene glycol, or a second antibody; by adsorbents such as activated carbon or suspensions of ion exchange resins. Selective adsorption of free ligands or solid support phases to which binding proteins or adsorbents are immobilized, such as antibody-coated tubes and resin sponges, can be used.

Various adsorption methods and applications are illustrated by the following references:

The activated carbon of Herbert's US Pat. No. 3,442,819 is coated with a molecular sieve that prevents large molecules or complex salts from reaching and adhering to the carbon, and can be used to separate components in biochemical analysis by adsorbing specific molecules. . The selection of the coating material is such that it can selectively adsorb one component over another; in general, molecules smaller than the pore size of the molecular sieve will be adsorbed through the sieve, but will not pass through a sieve of the same size or larger size and will therefore be absorbed into the analytical medium. remain.

US Pat. No. 3,947,564 to Shannon et al. describes the separation of thyroxine from serum proteins by adsorbing thyroxine onto montmorillonite clay in an acid solution followed by centrifugation.

US Pat. No. 3,937,799 to Lewin et al. describes the adsorption of unbound vitamin B-12 from analytical media using pre-measured bentonite tablets.

Applicant is aware of a commercially available superagent called Isolab that uses activated carbon bonded to a polyethylene support. This adsorbent is particulate and amorphous. As a result, the amount of carbon on each particle is changing.
When using this adsorbent, it is necessary to measure the weight or volume of the desired dispersion amount each time it is used. This adsorbent is usually packed into a column used for overload or chromatography. The purported advantage of the plastic support is that it prevents the activated carbon from clogging and improves the liquid flow rate through the column.

Applicants have developed a state-of-the-art activated carbon coated solid supported adsorbent with a regular geometry useful for the separation of ligands by adsorption from proteinaceous materials in analytical media. This adsorbent is a homogeneous, convenient, pre-measured and easily separated form of activated carbon, which eliminates the inconvenience of using activated carbon columns and slurries and completely eliminates the need for separation media measurement, filtration and centrifugation. It is something to do.

The present invention relates to a method for separating ligands using the above adsorbent.

That is, the present invention involves contacting a protein-containing analysis medium containing a ligand with a solid support for solid-phase immunoassay that has a regular geometric shape and is larger than suspended particles and is coated uniformly with activated carbon. This invention relates to a method for separating unbound ligands from an analytical medium, which is characterized by selectively adsorbing ligands that are not bound to proteins onto a coated solid support.

Most easily handled solid structures of uniform size and regular geometry can be used as supports for activated carbon coatings for accurate testing. Also, since the support is coated with activated carbon, its composition is not very important. However, the applicant has proposed a sphere of a convenient size to function easily in a small test tube containing the analytical medium.
It has been found preferable to use lightweight plastic structures that can be processed, molded or extruded into regular geometric shapes such as grains, cylinders or cubes. Commonly commercially available polymeric plastics such as polystyrene, polyurethane, polyethylene, etc. would be suitable as the support structure. This plastic can be molded or extruded into any shape, with a diameter of approximately
It has been found that 0.6 to 0.8 cm spherules are most convenient to use in the test medium. The surface of the globules needs to be ground or roughened. This can be chemical or physical. Although a rough surface is not required, it has been found to improve char adhesion.

The actual coating operation for the spherules involves creating a slurry of activated carbon in water, adding the slurry to a certain amount of spherules or a solid support, and stirring for a certain period of time to uniformly coat all the spherules with an appropriate amount of carbon particles. be. Finally, excess unattached charcoal particles are removed from the surface of the bulb and the wet bulb is dried with warm air.

Although commercially available activated carbon has a very wide range of surface areas and particle sizes, the finest particles can be used as a satisfactory source of coating material for this adsorbent. Of course, activated carbon selection depends on the chemical properties of the ligand and the composition of the analytical medium. However, best results have been obtained using activated carbon in which at least 80% of the carbon particles pass through a #325 mesh sieve screen.

The amount of activated carbon used in the coating operation depends on the number of globules to be coated. Applicants have discovered that there is no benefit to coating the globules with a thick coating of activated carbon. A sufficient effect can be obtained with a length that covers the surface of the small ball.

Industrially, coating operations are carried out in rotating coating pans or forced air columns commonly used for tablet coating. As is self-evident, the term "ligand" used in the present invention means a ligand for a protein, that is, a ligand capable of binding to a protein. In order to investigate the binding state of ligands in biological analysis samples that contain proteins that coexist with ligands, attempts have been made to selectively adsorb only ligands that are not bound to proteins. , such underlying technology is well known. Proteins and proteins bound with ligands have extremely large molecular weights and are not adsorbed by adsorbents. On the other hand, free ligands have smaller molecular weights than proteins and are adsorbed by adsorbents. Such a phenomenon itself is well known, and the present invention is characterized in that a specific adsorbent is used in this well-known technology. The adsorbent of the present invention is easier to operate than conventional adsorbents (activated carbon itself is one of them) in the adsorption separation of free ligands from proteins, and is essential for analysis using it. This method has the effect of eliminating the need for quantitative determination of the medium and complicated separation operations after adsorption, such as filtration and centrifugation.

The following examples illustrate typical industrial coating operations.

Example Activated carbon coated pellets Polyethylene pellets with a diameter of 0.7 cm in a rotating coating pan
I put 27.5Kg in it. 6 kg of activated carbon was added to another container containing 40 g of water for irrigation while stirring gently to avoid scattering the fine powder. This slurry was mixed well for about 1.5 hours and then transferred to a rotating plate. 16 at a speed of 22-26 rpm
Rotated time. The pellets are then heated to a temperature of about 45°C.
Air dried for 30 minutes. Finally, the coated pellets were removed from the rotating dish and stored in a plastic-bagged cylinder.

As mentioned above, the activated carbon coated globules are particularly useful for adsorbing unbound ligands from analytical media. Next, a method for analyzing liothyronine bound to serum proteins using activated carbon-coated globules as claimed in this patent will be described.

Thyroxine is bound by at least three heterologous proteins in serum. These are thyroxine-binding globulin (TBG), which has the strongest binding strength, prealbumin, which has an intermediate binding strength, and albumin, which has a relatively weak binding strength. Liothyronine (triiodothyronine) is similar but has weaker binding to thyroxine-binding globulin and albumin. In thyroid hyperactivity, the first thyroxine binding site is almost saturated. Therefore, the added liothyronine ¹²⁵ is a secondary
taken by the bond position. In thyroid dysfunction, added liothyronine ¹²⁵ assumes the first binding position (relatively unsaturated). In summary, binding or adsorption by globules increases in hyperthyroidism and decreases in hypothyroidism. Adsorption also coincides with the increase in thyroxine-binding proteins that normally occurs in pregnancy.

Although percent globule adsorption cannot be converted into definitive units of thyroxine or liothyronine weight, it is an indication of thyroid function because it indicates how long serum TBG has available excess binding capacity. The greater the binding capacity, the greater the amount of labeled liothyronine taken up, and the smaller the amount of radioactive liothyronine available for globule adsorption. In thyroid insufficiency, serum thyroxine or liothyronine is lower than normal, and serum TBG binds more to the added radioactive material, resulting in less globule adsorption. If the patient is hyperthyroid, the opposite is true; the serum TBG is already more saturated with thyroxine and liothyronine from the serum, so globule adsorption is greater.

Reagent 1 Liothyronine ¹²⁵ is a reagent solution: gelatin 0.1
2. radioactive liothyronine in Tris-maleate buffer at pH 6.9 stabilized at 2.0%, 2. polystyrene globules coated with activated charcoal, 3. comparative control serum: T4 with normal concentration of thyroxine as determined by RIA-PEG and TBG. Human serum with normal saturation of .

Method After all serum samples and reagents were brought to room temperature, 25μ of patient serum was pipetted into properly labeled test tubes. An additional 25 μl of control serum was added to two other normally labeled test tubes. Using a calibrated pipette, 250μ of the liothyronine ¹²⁵ reagent solution was added to each tube. There is no need to stir or mix immediately. Each tube was placed on a shaker and incubated at room temperature for approximately 20 minutes with shaking. Each tube was then removed and the liquid drained into the radioactive waste reservoir. The edges of the pellets and test tubes were blotted dry with paper towels. No need to wash. Place the test tube in a suitable scintillation calculation tube.
Measured for minutes. (or up to a total of 10,000 counts), results were recorded in net counts per minute and patient serum values were calculated.

A radioimmunoassay using an activated carbon coated solid support is demonstrated in the following example in which the ligand T3 is tested.

Example T3, 25 μ of each of the 6 standard sera were added to each of 12 test tubes labeled as 0.05, 1.0, 2.0 and 8.0 ng per 100 ml. 25μ of the test sample was also added to an appropriately labeled test tube. 50μ of reagents containing ¹²⁵ -labeled liothyronine and appropriate protective agents such as ANS were added to both standard and unknown serum solutions. 200μ of liothyronine antiserum (rabbit) was also added to each test tube, and the contents were thoroughly stirred. Each tube was incubated for 2 hours at room temperature. One activated carbon coated pellet was then placed in each tube. The contents of each tube were shaken for 20 minutes in a shaker. The liquid was drained from each tube, and the radioactivity of the remaining activated carbon-coated globules was measured using a scintillation counter. From this the percent bound antigen of each globule was calculated and the average percent bound antigen of each standard serum versus the corresponding liothyronine concentration of the patient sample could be plotted.

An enzyme immunoassay using an activated carbon-coated solid support for separation of protein-bound and unbound ligands is presented below.

EXAMPLE To each labeled test tube containing 25μ of standard serum or unknown sample was added 50μ of reagent solution containing 0-dianisidine-cortisol and ANS. Further, 200 μl of cortisol antiserum was added to each tube, the contents were mixed well, and the tubes were incubated at room temperature for 2 hours. Then, one activated carbon-coated pellet was placed in each tube and the contents were placed in a shaker.
Stir for 20 minutes. The liquid in each tube was aspirated and 0.2 ml of hydrogen peroxide and 0.1 ml of horseradish peroxidase reagent were added to each pellet. Measure enzyme activity at 460 nm using a spectrophotometer at 15 second intervals.
Measured for 10 minutes. The cortisol concentration in the sample was determined by comparing the change in absorbance between the standard serum and the sample.

Although various specific examples and embodiments of the invention have been described, they are not intended to limit the invention, but rather to illustrate the invention within the scope of the claims.

Claims (1)

[Claims] 1. A protein-containing analytical medium containing a ligand is brought into contact with a solid support for solid-phase immunoassay that has a regular geometric shape and is larger than suspended particles and is uniformly coated with activated carbon. A method for separating unbound ligands from an analytical medium, which comprises selectively adsorbing ligands that are not bound to proteins onto a solid support coated with activated carbon. 2 Claim 1 in which the solid support is spherical
The method described in section. 3. The method of claim 1, wherein the solid support is a plastic sphere having a diameter of 0.6 to 0.8 cm.