GB2122343A - Adult t-cell leukemia antigens, methods for their preparation and for assaying antibodies thereto - Google Patents

Abstract

An insolubilized antigen is disclosed which comprises an insoluble support having immobilized thereon an adult T-cell leukemia associated antigen selected from a soluble cytoplasmic protein of an adult T-cell leukemia cell and a solubilized protein of an adult T-cell leukemia virus, together with a process for preparing the insolubilized antigen and a method for assaying an antibody to adult T-cell leukemia associated antigen according to fluorescence immunoassay or enzyme immunoassay using the insolubilized antigen.

Description

SPECIFICATION Adult T-cell leukemia antigens, method for their prepartion and for assaying antibodies thereto Background of the invention This invention relates to an adult T-cell leukemia antigen, a method for its preparation and a method for assaying an antibody associated with adult T-cell leukemia and, more particularly, to a method for assaying an antibody according to indirect fluorescent antibody technique or enzyme-antibody technique using an antigen which specifically reacts with sera of patients with adult T-cell leukemia.

Adult T-cell leukemia (hereinafter referred to as ATL) is a malignant disease which attacks adults, but the cause of the disease has not yet been clarified completely. (See, Takatsuki, K., Uchiyama, T., Sagawa, K. and Yodoi, J., (1977) in "Topics in Hematology", eds. Seno, S., Takaku, F., and Irino, S. (Excepta Medica, Amsterdam), pp.73-77 and Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K. and Uchino, H., Blood 50, 481-492 (1977), etc.) Based on the knowldge that cultured ATL cells or ATL virus-associated cell lines contain an antigen specific to ATL (ATL-associated antigen or ATL-antigen, hereinafter referred to as ATLA) which is not detected in fresh ATL cells in peripheral bloods of ATL patients, and that sera of ATL patients or of certain healthy adults in ATL-endemic areas contain an antibody which specifically reacts with said ATLA (hereinafter referred to as ATLA antibody), there has recently been proposed a method for assaying ATLA antibody using fluoresence immunoassay which is capable of diagnosing ATL. (Proc. Nazi. Acad. Sci. U.S.A., Vol 78, No. 10, pp.

6476-6480 (1981)).

This method comprises placing ATL-positive cells as an antigen on a glass slide, reacting the antigen with a test sample serum and fluorescence labeled anti-human immunoglobulin G, and microscopically observing localization of the fluorescence marker to assay ATLA antibody.

However, this method has various disadvantages since it uses living cells per se as an antigen and it is necessary to conduct microscopical observation for each sample (test sample serum), resulting in that not only operation is complicated but also it is difficult to assay and diagnose a large amount of test samples with ease and rapidly.

Further, the above method is disadvantageous in that it is difficult to avoid the fluctuation in antigen positivity since intact cells are used and, therefore, it is difficult to obtain a desired quality of antigen constantly, which would lead to erroneous assay and diagnosis. Therefore, this method does not meet the requirement that assay can be carried out under fixed conditions, which requirement needs to be satisfied when wide prevalence of the technique concerned is attempted.

Summary of the invention It is an object of the present invention to provide a method for assaying ATLA antibody which obviates the defects of the conventional method for assaying ATLA antibody.

As a result of extensive investigation it has now been found that soluble cytoplasmic protein of cultured ATL cells (hereinafter referred to as SCP) and solubilized protein of ATL virus (hereinafter referred to as VAP) can be used as an antigen which can specifically react with ATLA antibody and that a large amount of test samples can be assayed with ease and rapidly as well as under fixed conditions with high efficiency when an insolubilized antigen is used which is prepared by insolubilizing SCP orVAP on an insoluble support.

The present invention is based on the above findings and provides a method for assaying ATLA antibody according to fluorescence immunoassay or enzyme immunoassay comprising using an insolubilized antigen comprising an insoluble support having insolubilized thereon at least one substance selected from the group consisting of a soluble cytoplasmic protein of ATL cells and a solublized protein of ATL virus.

According to the present invention, a large amount of test samples can be assayed rapidly with simplified operation; for example, sampls more than about 10 times as much as those which can be treated with conventional microscopical observation can be assayed or the same amount of samples can be assayed more than about 10 times as rapidly as the conventional method.

Brief description of the drawing Figure lisa graph plotting against dilution of sera optical density of 492 nm of ATLA antibody determined according-to the method of the present invention (Example 5).

Detailed description of the invention As the culture of ATL cells to be utilized as the source of SCP in the present invention, there are illustrated ATL cells already established as cell line (Miyoshi, I., Kubonishi, I., Sumida, M., Hiraki, S., Tsubota, T., Kimura, l., Miyamoto, K. and Sato, J., Gann 71, 155-156(1980)), culture of ATL cells separated from the peripheral blood or lymph node of patients having ATL in a conventional manner, and ATL virus-associated cells obtained by co-culturing the above-described ATLcells and human T-cells (e.g., Nature 294, p.770-771 (1981)).The human T-cells to be utilized in the above-described co-culturing are not particularly limited, and there are illustrated, for example, various T-cellsfrom peripheral blood, bone marrow, lymph node, spleen, tonsil, thymus, etc.

Culturing of the above-described ATL cells or co-culturing of the ATL cells and the human T-cells can be conducted in an ordinary manner. Medium is not particularly limited, and any of various nutrient media used for ordinary cell culture can be used. Preferred examples of media include, for example, RPMI 1640 medium (Flow Laboratories, Co.) and minimum Eagle essential medium (MEM medium) modified with a supplementary serum such as fetal bovine serum (FCS) or calf serum.

Culturing conditions are not particularly different from those employed in ordinary cell culture, and a temperature of about 36 to about 38"C and a pH of about 6.4 to 7.6 are generally employed. If desired, growth accelerators such as T-cell growth factor (TCGF) or chemicals known as inducers of retrovirus of mammals; such as 5-iodo-2'-deoxyuridine (IdUrd), cyclohexylimide (CH), puromycin, phorbol 12-myristate 13-acetate (TPA), n-butyrate (sodium n-butyrate), etc., can be added. Culturing is advantageously conducted by exchanging the solution every 3 to 5 days, thereby allowing desired cells to grow favorably.

Further, as the SCP a preparation can be used which is obtained by homogenizing the above cultured ATL cells in an appropriate buffer solution such as physiological saline, phosphate buffered saline, etc., and recovering the substance as supernatant by means of centrifugation or a like suitable separating means.

Examples of the ATL virus which can be used as the source of VAP include those isolated from the culture fluid of the above-described ATL cells in a conventional manner. The isolation of the ATL virus can be conducted using a conventional centrifugation technique. It is preferred to use ATL virus further purified by density gradient method.

VAP can be obtained by solubilizing the ATL virus. Solubilization of ATL virus can be carried out with ease using ordinary solubilizing agents.

Examples of solubilizing agents include various surfactants such as nonionic surfactants, e.g., "Triton X-100" (a trade name for a product of Wako Pure Chemical Co., Ltd.), "NP-40" (a trade name for a product of Shell Co.), digitonin, urea, etc., and anionic surfactants, e.g., sodium dodecyl sulfate (SDS), etc.

There is no particular limitation on the method of solubilization. However, it is preferred to conduct solubilization using a solubilizing agent in an amount of 0.01% to that corresponding to a micelleforming point at a temperature of about 0 to about 1 OO"C for several minutes to about 6 hours. Preferably, the VAP thus obtained is further purified by ordinary techniques such as centrifugation, dialysis, etc., and then subjected to an anion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex, etc., to adsorb and remove viral nucleic acid which may remain in VAP.

The insolubilized antigen which is used in the present invention can be prepared by insolubilizing the SCP and/or VAP on an insoluble support. There is no particular limitation on the insoluble support to be used and ordinary ones used in physical adsorption methods, more particularly, porous carriers of polystyrene, glass, polycarbonate, polypropylene, etc., can be used.

Fixation of SCP and/or VAP on the insoluble support can be carried out in a conventional manner. For example, a insoluble support and SCP and/or VAP are added to a solution such as physiological saline, phosphate buffer, etc., and the mixture is reacted at about 0 to about 37"C for about 2 to about 24 hours. It is preferred this reaction be effected under reduced pressure. After completion of the reaction, adsorption sites which remain in the insoluble support are saturated in a conventional manner, for example, with 0.2% gelatin, 0.2% bovine serum albumin (BSA), etc.

The insoluble antigen thus obtained is washed with water. The washed preparation is stored in a dried state or in the buffer solution described above.

The method of the present invention can be carried out by assaying the label activity of a labeled antigen-antibody complex according to ordinary fluorescence immunoassay or enzyme immunoassay using the insoluble antigen. More particularly, it can be carried out by adding a test sample serum optionally diluted to the insoluble antigen to effect immune reaction therebetween, washing the resulting antigen-antibody complex, reacting the complex with a labeling effector to label the complex, and assaying the label activity of the labeled complex in a conventional manner.

Thus, in the present invention, use of the insoluble antigen enables one to assay at a time a large amount of test sample sera rapidly with ease and high precision at fixed conditions.

As the test sample sera that can be used, sera separated from blood collected in a conventional manner from an adult on whom assay of ATLA antibody is desired or diluted preparations of these sera diluted with an appropriate buffer solution. The buffer solution to be used is not limited particularly. Usually, it is preferred to use a phosphate buffer solution (pH 7.4).

The immune reaction between the test sample serum and the insoluble antigen can proceed simpiy by mixing them. Usually, the reaction can proceed at about 4 to about 37"C for about 0.5 to about 16 hours.

Desirably, after completion of the reaction, the reaction product is washed sufficiently with an appropriate buffer solution, preferably physiological saline or the buffer solution which is used for the dilution of the test sample serum.

Labeling of the resulting antigen-antibody complex with a labeling effector which follows the immune reaction can be carried out by mixing the antigen-antibody complex obtained with a labeling effector diluted with the same buffer solution as described above and allowing the mixture to react at about 4" to about 37"C for about 0.5 to about 16 hours. After completion of the reaction, it is preferred to wash the resulting complex sufficiently in a similar manner to the above.

As the labeling efector there can be used any composite material composed of a substance capable of specifically binding to an antibody and various enzyme or fluorescein. Representative examples of enzymes include various ordinary enzyme reagents such as peroxidase (POX), chymotrypsinogen, procarboxypepti dase, glyceroaldehyde-3-phosphoric acid dehydrogenase, amylase, phosphorylase, D-Nase, P-Nase, etc.

The fluorescein is exemplified by ordinary fluorescent dyes such as fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), substituted rhodamine isothiocyanate (XRITC), Rhodamine B isothiocyanate, dichlorotriazine fluorescein (DTAF), etc.

Examples of the substance capable of specifically binding to an antibody include "Protein A" (Pro A, a product of Pharmacia Co.) and anti-human immuno-globulin G (hereinafter referred to as IgG) antibody such as sheep anti-human IgG antibody, rabit anti-human IgG antibody, goat anti-human IgG antibody, mouse anti-human IgG antibody, rat anti-human IgG antibody, etc.

Various complexes of the substance capable of specifically binding to an antibody and the labeling substance are available commercially, and they can be used as a labeling effector in the present invention.

On the other hand, such complexes may be prepared freshly before use according to conventional method as described by B.F. ERLANGER et al.: Acta. Endocrinol. Suppl. 168,206(1972) and M.H. KAROL et al.: Proc.

Nat Acad. Sci. U.S.A. 57,713 (1967).

More specifically, when an enzyme is used, this enzyme and Pro A or anti-human IgG antibody are coupled in a buffer solution (pH 4 to 6) in the presence of an appropriate oxidizing agent such as NalO4 at room temperature for 2 to 5 hours and then reduced with an appropriate reducing agent such as NaBH4.

With respect to the amounts of the reagents used, it is prefered that about 1 to about 3 moles of enzyme and about 100 to 300 moles of oxidizing agent be used per mole of Pro A or anti-human IgG antibody.

Preferably, the reducing agent is used in an amount of about 1 to about 2 moles per mole of the oxidizing agent.

When labeling effectors are prepared using a fluoroescein, the fluorescein is added to water or physiological saline whose pH is 6 to 8 and the mixture is reacted with Pro A or anti-human IgG antibody at about 0 C to room temperature for about 0.5 to about 3 hours (cf. "Keiko Kotai Ho" (Fluorescent Antibody Method)lkagaku Jikkenho Koza, No. 4, 263-70 (1972), 1st Ed., Nakayama Shoten Co., Ltd., Tokyo). It is preferred that the amount of fluorescein to be used be about 1/50 part be weight per part by weight of Pro A or anti-human IgG antibody.

In the present invention, theinsouble antigen-test sample serum (ATLA antibody-labeling effector complex can be assayed in a conventional manner appropriately depending on the labeling substance in the labeling effector used by determining the label activity (i.e., activity of enzyme or fluorescence). In this way, a large amount of test sample serum, ATLA antibody, can be assayed with ease and rapidly.

The present invention will now be described in more detail by the following reference examples and examples which, however, are not to be construed as limiting the present invention in any way.

EXAMPLE 1 Preparation of insoluble antigen Blood (20 m4) collected from a patient having ATL (50-year-old, male, living in Nagasaki city) using heparin was cenrifuged in "Ficoll Pack" (produced by Pharmacia Japan Co.) to obtain 5 x 107 cells of peripheral blood lymphocyte.

These cells were cultured at 37"C for 3 days in RPMI 1640/10% calf serum (Flow Laboratories Co.) at a cell concentration of 3 x 105 cells/me. A portion of the thus-obtained cells (6 x 108 cells) was homogenized in a 0.05 M phosphate buffer solution (containing 0.14 M NaCt, pH 7.4, hereinafterreferred to as PBS) and centrifuged for 1 hour (105,000 x g). The supernatant was collected and its protein content was adjusted with PBS to 120 Fg/mt (the protein content was determined by colorimetric method using "Tonein R -TP", a trademark for reagent for quantitative determination of total protein produced by Otsuka Assay Laboratories, Co., Ltd.) to obtain an SCP solution.

To the SCP solution (140 me) were added 700 pieces of polystyrene beads (diameter: 6.4 mm, produced by Precision Plastic Co., Ltd., U.S.A.) previously washed serially with an aqueous 0.1 N HCt solution, an aqueous 0.1 N NaOH solution and ethanol, and the mixture was allowed to stand for 6 hours at room temperature under suction using an aspirator, followed by filtration to obtain insolubilized antigen. This was stored in a 0.05 M phosphate buffer solution containing 0.2% gelatin (pH 7.4) at 40C.

EXAMPLE 2 Preparation ofinsoluble antigen (1) ATL cells (Kyo-Ya cell obtained from Virus Research Institute of Kyoto University) was cultivated at 37"C for 4 days in RPMI 1640/10% calf serum + 50 ;game IdUrd (Flow Laboratories Co.) at a cell concentration of 3 x 10"C5 cells/m". The culture fluid was centifuged at 1,500 rpm for 10 minutes to separate cells and the culture medium, which were collected separately.

(2) To a portion of cultured ATL cells (5 x 109 cells) obtained in (1) above was added 30 me of physiological saline and the mixture was homogenized and then centrifuged at 105,000 x g for 1 hour. The supernatant was collected and its protein content was adjusted in the same manner as in Example 1 to prepare an SCP solution having a protein content of 120 yglmC, from which an insoluble antigen, i.e., antigen-adsorbing polystyrene bead, was prepared in the same manner as in Example 1. The antigen obtained was allowed to stand for one night in a 0.05 M phosphate buffer solution containing 0.2% gelatin (pH 7.4), washed with water, dried and stored till use.

(3) The culture medium (500 me) obtained in (1) above was centrifuged at 40,000 x g for 1 hour and the pellet formed was collected. The pellet was subjected to density gradient centrifugation on 25 to 60% sugar, and fractions corresponding to density of 1.15 to 1.16 were collected. These fractions were solubilized with 1.5 mt of an aqeous 0.8 M NaCt solution containing 0.5% Triton X-100 at 40C for 60 minutes and then subjected to centrifugation at 35,000 rpm for 1 hour to collect supernatant.

The supernatant thus-obtained was dialyzed against 0.02 M Tris-HCt buffer solution containing 0.3 M Nact (pH 7,5) using a cellophane membrane for 5 hours to remove solubilizing agent and adjust the concentration of salts. The VAP solution thus obtained was passed through DEAE-cellulose column equiiibrated with the same Tris-HCe buffer solution as above to obtain the fraction which passed though the column without stopping. Distilled water was added to this fraction to adjust its protein content to 2.8 Fglme.

To a 40 mt portion of this solution were added pieces of polystyrene beads described in Example 1 previously washed as in Example 1 and allowed to stand for 6 hours at room temperature to obtain insoluble antigen. This was allowed to stand for one night in a 0.05 M phosphate buffer solution containing 0.2% gelating (pH 7.4), washed with water, dried and stored till use.

EXAMPLE 3 Preparation ofsample serum Blood samples were collected from a patient having ATL and a healthy adult using heparin, and allowed to stand for 3 hours at room temperature to collect the supernatants. Each of the supernatants was cenrifuged for 10 minutes at 2,000 rpm to collect the supernatants which were used as test sample sera.

EXAMPLE 4 Assay ofA TLA antibody Aseriesofdilutedsera (80 x, 160 x,320 x,640 x, 1,280 x ) were prepared by double diluting each test sample serum prepared in Example 3 with a 0.05 M phosphate buffer containing 0.2% gelatin (pH 7.4). To 0.5 mt of each of the diluted sample sera was added one piece of the insoluble antigen (antigen-adsorbing polystyrene bead) prepared in Example 1 and allowed to stand at 37"C for 2 hours. The reaction mixture was removed by suction with an aspirator, the bead was washed with 2 mt of physiological saline and the washing was removed by suction with an aspirator. This procedure was repeated three times.

Further, one piece of the bead thus-treated was added to 0.55 me of peroxidase-labeled Protein A (a product of E.Y. Laboratories Co.) diluted with the same phosphate buffer as above to 44,000 x and allowed to stand at 37"C for 2 hours followed by washing sufficiently in the same manner as above.

On the other hand, H202 was added with stirring to 20 mt of 0.2 M McLevin buffer solution containing 60 mg of o-phenylenediamine (pH 5.8) to adjust it to a final concentration of 0.02 VN % to prepare a coloring agent.

In a test tube, 2 me of physiological saline and 0.5 me of the coloring agent were brought and one piece of bead prepared as described above was added thereto. After allowing to stand at room temperature for 30 minutes, 1 me of 3 N hydrochloric acid was added to stop the enzymatic reaction, and optical density at 492 nm of the reaction mixture was measured.

The results obtained are shown in Table 1 below.

TABLE 1 Dilution of test sample sera x80 x 160 x320 x640 x7280 ATL Patient A 0.789 0.748 0.646 0.550 0.410 B 0.701 0.624 0.550 0.423 0.356 C 0.558 0.449 0.367 0.291 0.222 Healthy Person D 0.166 0.114 0.081 0.047 0.028 E 0.114 0.089 0.054 0.037 0.027 F 0.142 0.089 0.063 0.037 0.023 The same procedures as above were repeated except that instead of the insoluble antigen prepared in Example 1,the insolbule antigen (antigen-adsorbing polystyrene beads) prepared in Example 2-(2) and Example 2-(3), respectively, were used for 80-fold or 100-fold diluted sera.

The results obtained are shown in Table 2 and Table 3 below.

TABLE 2 Insoluble antigen prepared in Example 2-(2) Optical density at 492 nm 100-fold diluted serum ATL Patient G 1.154 H 1.012 0.879 Healthy Person J 0.108 K 0.094 L 0.011 TABLE 3 Insoluble antigen prepared in Example 2-(3) Optical density at 492 nm 80-fold diluted serum ATL Patient M 0.826 N 0.722 0 0.611 Healthy Person P 0.234 Q 0.329 EXAMPLE 5 For assaying ATLA antibody, the same procedures as in Example 4 were repeated except that the antigen adsorbing polystyrene beads prepared in Example 2-(2) as an insoluble antigen and 600-fold diluted peroxidase labeled goat anti-human IgG (a product of E.Y. Laboratories Co.) as a labeling effector were used.

Optical density at 492 nm was obtained for each dilution of sera.

The results obtained are shown in Figures 1 in which curve 1 represents ATLA antibody level for ATL patient, and curve 2 that for healthy person.

As will be clear from the results shown in Tables 1,2 and 3 and Figure 1, according to the method of the present invention ATLA antibody can be assayed with ease by measuring optical density, and, therefore, the present invention is useful for diagnosing and screening ATL patients.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims (25)

1. An insolubilized antigen comprising an insoluble support having immobilized thereon an adult T-cell leukemia associated antigen selected from the group consisting of a soluble cytoplasmic protein of an adult T-cell leukemia cell and a solubilized protein of an adult T-cell leukemia virus.

2. The insolubilized antigen as claimed in Claim 1,wherein said adult T-cell leukemia associated antigen is said soluble cytoplasmic protein immobilized on said insoluble support selected from the group consisting of polystyrene, glass, polycarbonate and polypropylene.

3. The insolubilized antigen as claimed in Claim 2, wherein said insoluble support is polystyrene beads.

4. The insolubilized antigen as claimed in Claims 2 or 3, wherein said soluble cytoplasmic protein is centrifugation supernatant of homogenate of said adult T-cell leukemia cell.

5. The insolubilized antigen as claimed in Claim 2, wherein said adult T-cell leukemia cell is that isolated from peripheral blood or lymph node collected from patient adultT-cell leukemia.

6. The insolubilized antigen as claimed in Claim 5, wherein said adult T-cell leukemia cell is an established adult T-cell leukemia cell line.

7. The insolubilized antigen as claimed in Claim 2, wherein said adult T-cell leukemia cell is that transformed by co-cultivating an adult T-cell leukemia cell isolated from peripheral blood of lymph node collected from a patient having adult T-cell leukemia with human T-cell.

8. The insolubilized antigen as claimed in Claim 7, wherein said human T-cell is derived from peripheral blood, bone marrow, lymph node, spleen, tonsil or thymus.

9. The insolubilized antigen as described in Claim 7, wherein said co-cultivation is carried out in an RPMI 1640 medium or a minimum Eagle essential medium modified with a fetal bovine serum or calf serum.

10. The insolubilized antigen as claimed in Claim 7, wherein said adultT-cell leukemia cell is an established adult T-cell leukemia cell line.

11. The insolubilized antigen as claimed in Claim 1, wherein said adult T-cell leukemia associated antigen is said solubilized protein immobilized on said insoluble support selected from the group consisting of polystyrene, glass, polycarbonate and polypropylene.

12. The insolubilized antigen as claimed in Claim 11, wherein said insoluble support is polystyrene beads.

13. The insolubilized antigen as claimed in Claim 11, wherein said adult T-cell leukemia virus is that purified by density gradient centrifugation.

14. The insolubilized antigen as claimed in Claim 13, wherein said solubilized protein is a protein obtained by solubilizing adult T-cell leukemia virus with a solubilizing agent.

15. The insolubilized antigen as claimed in Claim 14, wherein said solubilizing agent is a nonionic surfactant such as Triton X-100, NP-40, digitonin and urea or an anionic surfactant such as sodium dodesyl sulfate.

16. The insolubilized antigen as claimed in Claim 15, wherein said solubilizing agent is Triton X-100.

17. A process for preparing an insolubilized antigen comprising an insoluble support having immobilized thereon an adult T-cell leukemia associated antigen selected from the group consisting of a soluble cytoplasmic protein of an adult T-cell leukemia cell and a solubilized protein of an adult T-cell leukemia virus, which comprises mixing an adult T-cell leukemia associated antigen with an insoluble support in an aqueous medium, reacting them and optionally saturating the adsorption site of said insoluble support and washing the resulting antigen with water.

18. The process as claimed in Claim 17, wherein said adult T-cell leukemia associated antigen is said soluble cytoplasmic protein immobilized on said insolble support selected from the group consisting of polystyrene, glass, polycarbonate and polypropylene.

19. The process as claimed in Claim 17, wherein said adultT-cell leukemia associated antigen is said solubilized protein immobilized on said insoluble support selected from the group consisting of polystyrene, glass, polycarbonate, and polypropylene.

20. A method for assaying an antibody to adult T-cell leukemia associated antigen according to fluoroescence immunoassay or enzyme immunoassay comprising reacting an insolubilized antigen as claimed in any one of Claims 1 to 16 with a test sample serum and determining the resulting antigen-antibody complex.

21. The method as claimed in Claim 20, which further comprises reacting said antigen-antibody complex with a labeling effector capable of specifically binding an antibody.

22. The method as claimed in Claim 21, wherein said labeling effector is an enzyme- or fluorescentlabeled substance capable of specifically binding an antibody selected from the group consisting of Protein A, sheep anti-human immunoglobulin G antibody, rabbit anti-human immunoglobulin G antibody, goat anti-human immunogloulin G antibody, mouse anti-human immunoglobulin G antibody, and rat anti-human immunoglobulin G antibody.

23. The method as claimed in Claim 22, wherein said labeling effector is an enzyme labeling effector, said enzyme being selected from the group consisting of peroxidase, chymotrypsinogen, procarboxypeptidase, glyceroaldehyde-3-phosphoric acid dehydrogenase, amylase, phosphorylase, D-Nase and P-Nase.

24. The method as claimed in Claim 22, wherein said labeling effector is a fluorescein labeling effector, said fluorescein being selected from the group consisting of fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, rhodamine B isothiocyanate, and dichlorotriazine fluorescein.

25. The method as claimed in Claim 23, wherein said labeling effector is peroxidase-labeld Protein A.