JPH06213887A - Method for determining content of thyroglobulin and kit for method thereof - Google Patents

Abstract

PURPOSE: To provide a determining method for thyroglobulin content and a kit used for this method. CONSTITUTION: A thyroid gland cytological sample collected by suction is used in this determining method for a thyroglobulin content, this method is useful for the quantitative measurement of thyroglobulin in the quantity of about several mg, and the sample can be easily and quickly collected in a clinical laboratory specialized not so extremely. The immunodiffusion method is the suitable technique for this quantitative measurement.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for determining the content of thyroglobulin and a kit for use in this method. More particularly, the present invention is suitable for the determination of thyroglobulin levels present in the thyroid gland, and can be used inexpensively, even in a highly unprofessional, clinical laboratory, which is quick and simple, The present invention also relates to a method for determining the thyroglobulin content and an accurate method for determining by the kit.

[0002] The publication index numbers cited herein refer to the publications in the list at the end of the specification.

[0003]

Human thyroglobulin (hTg) is an organ-specific protein of the thyroid gland, which is secreted by vesicle cells and deposited in colloids. Its function in synthesis, metabolism, primary immunochemical structure and hormone formation has all been extensively studied (1-5).

The hTg concentration per gram of fresh tissue is 20-
It is 40 mg. This number may change due to various changes in thyroid function. Concentrations of hTg are decreased in goiter caused by Basedow's disease, treatment with antithyroid drugs, hyperthyroid nodules, degeneration in hormone formation, whereas increased after treatment with colloid goiter and iodine. Yes (6, 7).

In thyroid tumors, there is a gradual decrease in this protein, depending on the extent of the resulting tumor differences; this protein is present in adenomas, endemic goiter, sporadic multinodular goiter and It is markedly reduced in the case of cystic and papillary carcinomas and is also absent in anaplastic, thyroid lymphoma and abnormal vesicular tumors (8).

Thyroid tumor disease is very common in the general population, but cannot be accurately estimated to single digit percentages. This is because there is wide regional variation and diversity in the manifestation of these diseases, and in particular goiter disease is regional variation and diversity. However, it can be roughly estimated that 5 to 15% of the general population suffer from thyroid tumors and / or nodule formation.

Therefore, cytological examinations of thyroid specimens aspirated by needles, at their different stages of differentiation, determine whether they are colloid goiters or tumors and are suitable for therapeutic treatment of thyroid nodules. It has recently become widely used clinically as an index for selecting a treatment (medical treatment or surgical treatment).

However, cytological examinations performed on needle-sucked vesicle cells may raise doubts about whether the thyroid hypertrophy is malignant or benign. Quantitative measurement of protein hTg, which is the major specific component of colloid, can greatly assist in the development and completion of cytological diagnostic methods, thereby supporting cytological criteria by quantitative measurement of cell differentiation markers be able to.

[0009] At present, the determination of the thyroglobulin content in a thyroid sample aspirated by a needle is not performed.
This is a method available (RIA, IRMA, developed for quantitative determination of hTg concentration in human serum.
This is because the ELISA) can measure the amount of hTg in the unit of ng and is not suitable for quantitative measurement of the intrathyroid hTg concentration in the unit of mg.

Therefore, there is no need for a highly specialized laboratory and therefore it can be used for clinical and diagnostic purposes and quantitates thyroglobulin in the order of mg in thyrocyte samples aspirated by needle. Clearly, there is a need for a simple, accurate, and also rapid method for measuring.

[0011]

DISCLOSURE OF THE INVENTION The inventors of the present invention have found that the immunodiffusion method satisfies this feature, and that thyroid globulin in a thyroid sample aspirated by a needle can be treated rapidly without the use of radioisotopes. It was found that a method for quantitative measurement can be developed by using an accurate method.

[0012] Therefore, an object of the present invention is to provide a method for quantitatively measuring thyroglobulin in a thyroid sample sucked and collected by a needle. Another object of the present invention is to provide a method for quantitatively measuring thyroglobulin by an immunodiffusion technique.

In a preferred embodiment, the immunodiffusion technique is an immunoelectrodiffusion method, which preferably comprises preparing an electrophoretic gel containing a ligand capable of specifically binding thyroglobulin; loading a sample. The step of performing the electrophoresis described above; the ligand contained in the sample reaction
Measuring thyroglobulin;

Alternatively, the immunodiffusion technique is a simple radial immunodiffusion method, more preferably the method comprises the steps of: preparing an inactive gel containing a ligand capable of binding thyroglobulin; Contacting the gel with a sample; incubating it; measuring the ligand-thyroglobulin contained in the sample reaction.

Further, in the present invention, the above-mentioned ligand comprises anti-thyroglobulin serum or purified anti-thyroglobulin antibody. Furthermore, in the present invention, the inert gel preferably comprises agarose at a concentration of 0.8% to 2.0% dissolved in a solution containing NaCl and a phosphate buffer, and more preferably, This solution is 0.1
It has a NaCl concentration of 5% M and a phosphate buffer concentration of 0.05 M, and also this solution contains sodium glutamate and chlorhexidine.

In a particular embodiment of the invention, the inert gel is injected in the liquid state into the space between two appropriately sealed glass plates and then the gel is allowed to solidify.
Furthermore, preferably one of the two glass plates is coated with a substance which facilitates the adhesion of the gel to the plates themselves.

Furthermore, in the present invention, the contact is performed by suctioning a part of the inert gel by a needle,
The sample is introduced into the circular recess created. The incubation is carried out in a moist container at a temperature of 18 ° C to 30 ° C, preferably 20 ° C to 27 ° C for 2 to 60 hours,
It is preferably carried out for 24 to 50 hours.

Furthermore, in the present invention, the reaction between the ligand and thyroglobulin is a precipitation reaction, and the quantitative measurement is performed by measuring at least one diameter of the cyclic precipitate. Alternatively, the cyclic precipitate is
After drying of the inert gel, it is stained with a protein dye, the sample contains thyroglobulin at a concentration of 0.5-4.0 mg / ml or is diluted until such a concentration is obtained.

Another object of the invention is a kit for use in said method.

[0020]

The present invention is illustrated below by some examples, which are intended to be illustrative, not limiting.

Example 1 Method for quantitative determination of thyroglobulin by simple radial immunodiffusion method
Law immunodiffusion technique is described in (12). 20 ml syringe and 21G × 1-1 / 2,
Performed using a device (Cameco, Sweden) for aspiration of cytological samples, equipped with a 0.8 x 40 mm needle.

The aspirated sample may consist of: a) a few drops of nematic.
c) liquid; b) various amounts (20 ml or more) of cystic fluid of brown or yellow or nematic color.
equal). In the case of a), an aliquot (30 μl) is diluted 1 + 1 in 0.15 M NaCl, which is then centrifuged twice in a polyethylene tube (29 × 6 mm) at 1,000 × g. The supernatant is used to quantitatively measure hTg. The sedimented part is carefully suspended and then smeared for cytological examination.

In the case of b), the cyst fluid is centrifuged and
(1500 × g, 10 times), then treated as in a), the supernatant is used for quantitative determination of hTg, while the sedimented part is used for cytological examination.

1.5 g of agarose was dissolved by heating in 100 ml of saline solution (0.15M NaCl) buffered with 0.05M phosphate buffer containing 0.24M sodium glutamate and 0.005% chlorhexidine. Dispense an aliquot into 10 ml tubes, then +4
Store at ℃.

Rabbit human anti-thyroglobulin serum is prepared by emulsifying 5 mg of hTg in 1 ml Freund's complete adjuvant and injecting this emulsion into the oily cushion of the paw of an animal. One month later, booster injection is performed according to the same operation. After 15 days, the animals were bled by cardiac puncture, the antibody was adsorbed to normal human serum and its concentration was determined by plotting a sedimentation curve. The titer of 10 mg of precipitated hTg is obtained from the sedimentation curve in a numerical relationship per ml of serum.

From the crude extract of colloid goiter, hTg is prepared by salt treatment and subsequent chromatography on an ultrafine Sephadex G200 column (0.005M phosphate buffer; pH 7.4). The purity test shows a single band by SDS electrophoresis on polyacrylamide gel (8-10).

10 ml of agar are dissolved on a water bath at 80 ° C. and then the solution is cooled;
At some point, 1 ml of rabbit anti-hTg is added, then the mixture is subjected to rapid magnetic stirring and thoroughly mixed, then the agar-antiserum is melted and pipetted into the mold.

This mold has two glass plates (7 × 1).
6 cm) are cleaned thoroughly and then the glass plates are brought into close proximity to each other. The two glass plates are separated by a U-shaped intermediate part made of a plastic material and held together by metal claws. One of these two plates is pre-coated with a coating by Gel Bond Lamina (Pharmacia).

The gel-antiserum mixture was allowed to cool to room temperature for 1 hour and then removed by gently sliding the non-GelBond treated plate. Also remove the U-shaped plastic intermediate parts. The gel is punctured by aspiration with a 2.5 mm needle equipped with spectacles. By gentle suction (water pressure reduction), two rows of 5 depressions are obtained. The diameter of each depression is 2.5 mm. Increasing amount (0.005-
0.01 ml of a solution containing hTg at 0.04 mg)
Are loaded into 5 wells with an Eppendorf pipette or similar means. Note the change in the tip of the pipette during each charge. Each test sample is placed in a separate recess. The plates are then left for 48 hours at room temperature in a moist container. At the end of this incubation period, the diameter of the circular precipitate is read according to the appropriate plate reading standard. Measure the diameter twice at right angles and take the average value. The two diameters do not differ from each other by more than 5%.

This reading was also made by coloring the ring, in addition to the reading made on the cooled material,
It can be done more accurately. For this purpose, the plates are washed in water for several hours and then at 100 ° C.
Dry in oven for 30 minutes. Proteins are conventional protein dyes (Promophenol Blue, Comm
Assie, Ponceau Red). A standard curve is prepared by taking the hTg concentration on the horizontal axis and the ring diameter in mm on the vertical axis.

Amounts of hTg of 0.5-4 mg per ml of sample can be measured by this method. For thicker samples, dilute appropriately to make it suitable.

[0032]

[Table 1]

Example 2 Results Obtained Using the Method for Quantitative Determination of Thyroglobulin The results of seven standard curves are shown in Table 1. The change between different experiments is 13% maximum at the lowest point of this curve.
Reached On the other hand, in the sample with the higher concentration, the maximum value in the sample reached 8%, and the minimum value was 3%. R performed using a commercial kit on a limited number of samples
Comparative evaluation between the IA method and the simple radial immunodiffusion method (RID) showed an optimal correlation (r = 0.93, p0.0.
Less than 2). T measured by RID method in a sample of cytological material aspirated from 54 hypertrophic thyroid
The thyroid concentration of hTg is shown in Table 2.

[0034]

[Table 2]

In the case of nodular proliferation, the average concentration of ThTg showed 1.30 ± 0.5 mg / ml, but in 3 out of 8 samples (these are not included in the calculation of the average value). ) The ThTg concentration was clearly high and required dilution of the sample to read it from the standard curve. Concentrations similar to those of growing nodules were found in degenerated pseudocysts (1.68 ± 0.69 mg / ml). Such pseudocysts can be considered to be those in which proliferative nodules progressed to necrosis and fluidization. This group also gives some very high values separately. In such a case, colloid cyst degeneration occurred.

No typical thyroid component was found in 6 of the cytological samples collected by aspiration (oval vesicle cells or thyroid vesicle stumps),
It is extremely important that these samples could be quantitatively measured for ThTg concentrations comparable to those obtained with proliferating nodules. This result clearly demonstrates another advantage of the method of the invention, as it provides a judgment about the nature of organ diagnostic elements and hypertrophy even in the absence of typical cell members. ing.

In another group of thyroid diseases classified by cytological critical signs (chronic lymphocytic thyroiditis, follicular tumors) or by clinical laboratory critical signs (hyperthermic nodules, Graves' disease): It can be judged by whether or not the ThTg concentration is present at a concentration of 0.5 mg / ml. The result is Graves
Disease, hyperthermia nodules, adenomas and colloid goiters (in which case ThTg precipitation is very low), and also lymphoid thyroiditis (in which case the vesicle tissue is lymphoid). Replaced by sphere wetness)
It is in good agreement with the already known results from the histological and biochemical studies found in.

Therefore, in the presence of cytological findings associated with "vesicle proliferation" or suspected "lymphocytic thyroiditis", the quantitative measurement of ThTg can provide a useful element for confirming the diagnosis. For example, in cases where "chronic lymphocytic thyroiditis" and "thyroiditis caused by nodule proliferation" are suspected, this quantitative measurement of ThTg can be used for diagnosis of chronic lymphocytic thyroiditis (in this case, Can be instructed for low ThTg concentrations or no ThTg present, or for the diagnosis of nodular proliferation, in which case ThTg concentrations are high. Similarly, in the case of cell-rich aspirated cytological samples, if suspected to be proliferative or nodular tumor,
The measurement of ThTg can provide a diagnostic tool, Th
A high Tg value allows diagnosis of proliferation, or ThTg
If is not present or is present in low concentrations, a tumor can be diagnosed.

Example 3 Immunoelectrodiffusion (EID) 1% in 80 mM Tris-borate buffer, pH 8.6.
Gel plates are prepared by mixing the agarose gel solution with 2% dextrin 70 kd and 1% rabbit anti-Tg serum at 56 ° C. 8 ml of this solution is poured on the hydrophilic side of the gel band (7 × 8 cm). The plates are left at 4 ° C for 2 hours. Eyewear device (Behr)
With a 2.5 mm suction needle with ng), 11 depressions are made from the bottom to a point 2 cm. Immediately after,
Precision EPPendorf pipette (inaccuracy <2.8
%) Load standard and sample (5 μl). These gels were treated with Tris-borate buffer 80 mM, pH
Process at 8.6 constant voltage for 12 hours in 8.6. The height of the rocket is measured by the EID reader.

Table 3 below shows the linear relationship between the 0 g Tg content and the height of the rocket. The sensitivity of the EID method is higher than that of the RID method.

[0041]

[Table 3]

[0042]

[0043]

[0044]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Helgarottos Rome, Italy Palenzo, Number 2 in Italy

Claims (19)

[Claims] 1. A method for determining thyroglobulin content from an aspirated thyroid sample. 2. A method for determining the thyroglobulin content by an immunodiffusion technique. 3. The method for determining the thyroglobulin content according to claim 2, wherein the immunodiffusion technique comprises immunoelectrodiffusion. 4. The method comprises: a step of preparing an electrophoretic gel containing a ligand capable of specifically binding to thyroglobulin; a step of loading a sample; a step of performing the electrophoresis; a step of the sample reaction. The method for determining the thyroglobulin content according to claim 3, comprising the step of measuring ligand-thyroglobulin. 5. The method for determining the thyroglobulin content according to claim 2, wherein the immunodiffusion technique comprises a simple radial immunodiffusion method. 6. The method comprises the steps of preparing an inactive gel containing a ligand capable of binding thyroglobulin; contacting the gel with a sample; incubating it; ligand-thyroglobulin involved in the sample reaction. The method for determining the thyroglobulin content according to claim 5, which comprises the step of quantitatively measuring 7. The method for determining the thyroglobulin content according to claim 4 or 6, wherein the ligand comprises an anti-thyroglobulin-introduced serum or an anti-thyroglobulin purified antibody. 8. The inert gel comprises 0.8% to 2.0%.
6. Agarose having a concentration in the range of 6 or 7.
The method for determining the thyroglobulin content according to 1. 9. The agarose is dissolved in a solution containing NaCl and a phosphate buffer.
The method for determining the thyroglobulin content according to 1. 10. The method for determining thyroglobulin content according to claim 9, wherein the NaCl is at a concentration of 0.05M, and the solution contains sodium glutamate and chlorhexidine. 11. Injecting the inert gel in solution into the space between two appropriately sealed glass plates,
The method for determining the thyroglobulin content according to any one of claims 6 to 10, wherein the method is followed by solidification. 12. One of the two glass plates is
The method for determining the thyroglobulin content according to claim 11, which is coated with a substance that improves the adhesion of the gel to the glass plate. 13. The method according to any one of claims 6 to 12, wherein the contact is generated by aspirating a part of the inert gel with a needle and introducing the sample into the resulting depression. Method for determining thyroglobulin content. 14. The method for determining the thyroglobulin content according to claim 6, wherein the incubation is performed in a moist container for 2 to 60 hours at a temperature of 18 ° C. to 30 ° C. . 15. The method for determining thyroglobulin content according to claim 14, wherein the temperature is in the range of 20 ° C. to 27 ° C., and the time is 24 hours to 50 hours. 16. The reaction between the ligand and thyroglobulin is a precipitation reaction, and the quantitative measurement is performed by measuring the diameter of at least one of the cyclic precipitates.
The method for determining the thyroglobulin content according to any one of claims 6 to 15. 17. The cyclic precipitate is colored with a protein dye after drying the inert gel.
The method for determining the thyroglobulin content according to 1. 18. The sample is 0.5 to 4.0 mg / ml.
The method for determining the thyroglobulin content according to any one of claims 1 to 17, which comprises thyroglobulin at a concentration of or is diluted until this concentration is obtained. 19. A kit for use in the method for determining the thyroglobulin content according to any one of the preceding claims.