JPH08285846A - Paul-bunnell antibody and measuring reagent therefor - Google Patents

Abstract

PURPOSE: To measure Paul-Bunnell antibody with high sensitivity by employing a Paul-Bunnell antibody produced by treating an erythrocyte membrane of mammal with a nonionic surfactant. CONSTITUTION: A Paul-Bunnell antibody being employed in the measuring reagent is obtained by treating an erythrocyte membrane of mammal with a nonionic surfactant. The material erythrocyte membrane is obtained by treating and refining the erythrocyte of mammal, e.g. horse, sheep or goat. More specifically, the erythrocyte of mammal is dispersed into a physiological saline buffered with phosphoric acid, and the operation for collecting the precipitate centrifugally is repeated. Subsequently, it is added with a Tris buffer solution containing ethylene diamine tetraacetate, phosphoric acid buffer solution, etc., in order to hemolyze the erythrocyte and then an erythrocyte membrane is obtained centrifugally. Since the erythrocyte membrane contains hemoglobin and the like, dispersion into the buffer solution and centrifugal operation are repeated to obtain a refined milky white erythrocyte membrane which is finally treated with nonionic surfactant thus obtaining a Paul-Bunnel antibody.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring reagent for measuring Paul Bunnell antibody and a novel Paul Bunnell antigen.

[0002]

2. Description of the Related Art It has been found that Paul-Bunnell antibody contained in the serum of patients with infectious mononucleosis reacts with erythrocytes of animals such as sheep and cattle. Therefore, it has been attempted to isolate an antigen that reacts with this antibody and is present in animal erythrocyte membrane (hereinafter referred to as Paul Bunnell antigen). In this method, (a) a method of fractionating a bovine erythrocyte membrane by column chromatography using DEAE-cellulose as a carrier from the aqueous layer of the residue obtained by extraction with a chloroform-methanol mixture (Merick et al .; J. Supramoleccu.
lar Structure 6: 275-290 (1
977)), (b) the bovine erythrocyte membrane is washed with acetone and ethanol, the residue is dissolved in water and then ethanol is added to crystallize, and further treated with a cation exchanger to obtain a sialic acid-containing fraction. Pole from the aqueous layer extracted with lipid solvent
Method for fractionating vannel antigen (JP-A-57-20669)
No. 4) and the like are known.

[0003]

When a reagent for detecting Paul Bunnell antibody in a sample is produced using Paul Bunnell antigen obtained from a mammalian erythrocyte membrane by a conventional method and antibody detection is carried out, the antibody concentration is increased. When it was low, it could not be said to have sufficient sensitivity because it could not be detected (see Examples below).

Therefore, an object of the present invention is to provide a measuring reagent capable of measuring Paul Bunnell antibody with high sensitivity.

[0005]

Means for Solving the Problems As a result of earnest research, the inventors of the present invention obtained a Paul Bunnell antigen by treating a Paul Bunnell antigen present in a mammalian erythrocyte membrane with a nonionic surfactant, Then, it was found that the Paul-Bunnell antibody in a sample can be measured with high sensitivity by using this antigen bound to a solid phase and completed the present invention.

[0006] That is, the present invention provides a reagent for assaying Paul-Bunnell antibody, which has a Paul-Bunnell antigen obtained by treating a mammalian erythrocyte membrane with a nonionic surfactant as an active ingredient. Further, the present invention is obtained by treating a mammalian erythrocyte membrane with a nonionic surfactant, shows a molecular weight of 100 kd by gel filtration chromatography, and is 50 kd, 35 kd by SDS-polyacrylamide gel electrophoresis.
It provides a molecular weight of 20 kd and 15 kd and provides a Paul Bunnell antigen that reacts with the Paul Bunnell antibody.

The present invention will be described in detail below.

The Paul Vannel antigen used in the assay reagent of the present invention is obtained by treating mammalian red blood cell membranes with a nonionic surfactant.

The mammalian red blood cell membrane, which is a raw material for producing the Paul-Bunnell antigen used in the assay reagent of the present invention, is used by treating and purifying red blood cells of mammals such as cows, horses, sheep and goats. . This erythrocyte membrane is prepared, for example, by first dispersing mammalian erythrocytes in phosphate buffered saline (PBS) or the like, centrifuging and collecting the precipitates, and then repeating 5 to 10 times the amount of ethylenediamine tetrachloride in the erythrocytes. It can be obtained by adding tris buffer containing acetic acid (EDTA), phosphate buffer, etc. to hemolyze erythrocytes and centrifuging. Since this erythrocyte membrane contains hemoglobin and the like, a purified milky white erythrocyte membrane can be obtained by repeating dispersion in a buffer solution and centrifugation.

Examples of the nonionic surfactant used for treating the erythrocyte membrane include alkyl glucoside, n-alkanoyl-N-methylglucanamide, and sucrose monoalkylate. The number of carbon atoms in the alkyl group portion of these nonionic surfactants is 5 to
About 13 is preferable. Specific examples of such nonionic surfactants include alkyl glucosides such as n-dodecyl-β-D-maltoside and n-octyl-β.
-D-glucoside, n-heptyl-β-D-thioglucoside, n-octyl-β-D-thioglucoside, etc., n-
Examples of the alkanoyl-N-methylglucanamide include n-octanoyl-N-methylglucanamide and n-
Menanoyl-N-methylglucanamide, n-decanoyl-N-methylglucanamide, and the like, and examples of the sucrose monoalkylate include β-D-fructopyranosyl-α-D-glucopyranoside monodecanoate, β-D-fructopyranosyl-α-D-glucopyranoside monododecanoate and the like can be mentioned. These nonionic surfactants can be used alone or in combination.

As described above, the Paul Vannel antigen used in the measuring reagent of the present invention can be obtained by treating a mammalian erythrocyte membrane with a nonionic surfactant. This treatment is preferably performed in the presence of a buffer such as a phosphate buffer. Paul Bunnell antigen is a reaction mixture obtained by stirring a mixture of a mammalian erythrocyte membrane, a nonionic surfactant and preferably a buffer solution at 4 to 25 ° C. for about 1 to 18 hours, and then stirring the mixture. Centrifuge (eg 13
000 rpm) to obtain the supernatant.
Here, the concentration of the erythrocyte membrane in the mixture is not particularly limited, but is preferably about 0.1 to 2% by weight, and the concentration of the nonionic surfactant is not particularly limited, but is 0.1.
It is preferably about 5% by weight. In a particularly preferred embodiment, a solution of about 2% by weight nonionic surfactant in a buffer is mixed with approximately equal volumes of red blood cell membranes. In this embodiment, Paul Bonnell antigen can be efficiently obtained, which is economically advantageous.

The Paul-Bunnell antigen obtained in the centrifugation supernatant as described above can be used as a Paul-Bunnell antibody measuring reagent by directly binding to a solid phase or labeling it. It is preferred to purify and use. Purification can be performed by a method commonly used for protein purification, and can be performed, for example, by combining known gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography and the like.
An example of this purification method is detailed in the Examples below.

The measuring reagent of the present invention may be used in any of the methods for measuring the Paul-Bunnell antibody by utilizing the antigen-antibody reaction between the Paul-Bunnell antigen and the Paul-Bunnell antibody. , Reagents for indirect agglutination immunoassay for the measurement of Paul Bonnell antibody, enzyme immunoassay (EIA) reagent, ELISA reagent, radioimmunoassay (R
IA) reagents and the like.

The indirect agglutination immunoassay reagent can be produced by binding the above-mentioned Paul Bunnell antigen to particles for an indirect agglutination immunoassay. As particles for producing this reagent, gelatin particles obtained by insolubilizing a mixture used in the conventional indirect agglutination immunoassay, for example, gelatin, metaphosphate and gum arabic (JP-A-57).
No. 153658), and latex particles such as polystyrene. In particular, gelatin particles have excellent advantages as an indirect agglutination immunoassay reagent for Paul-Bunnell antibody in that they have less non-specific reaction, particles of any size can be obtained, and properties depending on the application can be added.

In order to bind the Paul Vannel antigen obtained by the method of the present invention to the particles for indirect agglutination immunoassay, a publicly known method for binding an antigen may be used.
The reagent used for this binding is, for example, a method using a so-called coupling agent such as tannic acid, glutaraldehyde, bisdiazobenzidine, toluene diisocyanate, difluorodinitrobenzene, carbodiimides, quinones and chromium chloride, or a physical agent for the particles. It can also be carried out by a method of adsorption.

The thus obtained sensitized particles to which the Paul-Bunnell antigen has been bound can be lyophilized and stored by a conventional method. Furthermore, at the time of use, a reconstitution solution (for example, PBS) is added to form a solution, which is combined with a test sample (usually serum), a diluted solution, standard serum, unsensitized particles, etc., as an indirect agglutination immunoassay reagent for detecting Paul-Bunnell antibody It can be used for detection and measurement.

In addition to the indirect agglutination immunoassay reagent, the Paul Vannel antigen of the present invention can be bound to a solid phase to form a latex agglutination reagent, EIA reagent, ELISA reagent, or RIA reagent. As the solid phase used for these reagents,
Examples thereof include beads made of various polymers such as polystyrene or glass, magnetic particles, microplates for ELISA, and known solid phases such as filter paper and glass filters used in immunochromatography. As a method for binding the Paul-Bunnell antigen to this solid phase, a method using the above-mentioned coupling agent or a physical adsorption method can be used.

The antigen-sensitized solid phase obtained by the above method is combined with an antibody labeled with an enzyme, a radioisotope, a chemiluminescent substance, a fluorescent substance or the like, and the known competitive method, 1-step sandwich method, 2-step sandwich method,
It can be used to measure the Paul-Bunnell antibody by a simple method such as immunochromatography.

The Paul-Bunnell antigen obtained by the above method can be used as a Paul-Bunnell antibody measuring reagent without binding to a solid phase. For example, when antibody measurement is performed by a competitive method using a mammalian erythrocyte membrane bound to a solid phase, the Paul-Bunnell antigen obtained by the above method may be used as it is or with an enzyme label or a fluorescent label. After suitable labeling, it can be used as a reagent that competes with the erythrocyte membrane immobilized on the solid phase. In Example 2 below, the calibration curve shown in FIG. 1 was prepared by such a method.

In the following examples, a novel Paul Bunnell antigen was isolated from bovine erythrocyte membrane by the above method. This Paul Bunnell antigen elutes at a molecular weight of about 100 kd by gel filtration chromatography, but SD
When analyzed by S-polyacrylamide gel electrophoresis, bands were observed around the molecular weights of 50 kd, 35 kd, 20 kd and 15 kd. Furthermore, after transferring these antigens to a nitrocellulose membrane by Western blotting, confirmation tests of bands that react with the serum of patients with infectious mononucleosis were performed, and molecular weights were 50 kd, 35 kd, 20 kd and 15 kd.
Four major bands were detected near d. From the results of this analysis, it was confirmed that
The vannel antigen is formed by associating multiple proteins with each other to form an antigen protein having a molecular weight of 100 kd.
-During analysis such as gel electrophoresis, it is expected that these associated proteins will appear as the above-mentioned four main bands due to separation.

[0021]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 Preparation of Paul Bonnell Antigen (1) Preparation of Bovine Erythrocyte Membrane 4.8% trisodium citrate-0.9% sodium chloride was added to bovine fresh blood as an anticoagulant in 1/10 volume of blood. added. The bovine blood was centrifuged at 4 ° C. for 2000 rpm for 10 minutes, and the supernatant was removed with an aspirator to obtain a precipitate composed mainly of red blood cells. A 2.5-fold amount of 50 mM phosphate buffer pH 7-150 mM isotonic sodium chloride buffer was added to the precipitate to suspend it, and the suspension was centrifuged at 4 ° C. for 3000 rpm for 10 minutes. After centrifugation, the supernatant and the yellowish white precipitate layer were removed with an aspirator. This washing with the isotonic buffer solution was repeated 3 times, and the red precipitate from which the yellowish white precipitate had been sufficiently removed was used as a bovine red blood cell preparation.

The bovine red blood cell preparation obtained above was added to 10 times its volume of 10 mM Tris buffer pH 7.4-0.1.
After adding mMEDTA-0.5 mM phenylmethylsulfonyl fluoride and stirring, 0.15% by volume of the total volume of 5% acetic acid was added, and the mixture was slowly stirred overnight at 4 ° C. to hemolyze bovine red blood cells. The next day, this hemolyzed blood was centrifuged at 4 ° C. for 8,000 rotations for 20 minutes, and the supernatant was suctioned off with an aspirator to recover the precipitate. This precipitate was cooled to 4 ° C 1
0 mM Tris buffer (pH 7.4) was added in an amount 5 times the volume of the precipitate, and the suspension was well suspended. After centrifugation under the same conditions as above, the supernatant was removed with an aspirator. The centrifugation and washing operations were repeated until the absorbance at 550 nm of the supernatant became 0.01 or less and the precipitate became milky white. This washed precipitate fraction was used as a bovine erythrocyte membrane preparation in the following studies. By this method, about 600 mg of erythrocyte membrane was recovered from 1 liter of bovine blood and could be stored at -20 ° C for at least 1 month.

(2) Extraction of Paul Bunnell antigen from bovine erythrocyte membrane The bovine erythrocyte membrane preparation obtained in (1) was mixed with an equal volume of 0.1 M sodium carbonate-1 mM EDTA, pH 11, and stirred in ice water. While being left for 1 hour, the precipitate was collected by centrifugation at 4 ° C. for 13,000 rotations for 20 minutes. Next, 0.1 M phosphate buffer (pH 7.0) was added to the precipitate to restore the whole pH to neutral, and then the precipitate was collected by centrifugation at 13,000 rpm for 20 minutes at 4 ° C. The final concentration of 1% n
A solution of -octyl-β-D-thioglucoside-0.1M phosphate buffer (pH 7.0) was added, and the mixture was stirred at 4 ° C overnight for extraction. Then, by centrifugation at 13,000 rpm for 20 minutes at 4 ° C., a crude fraction containing Paul Bunnner as an antigen in the supernatant was obtained.

(3) Purification of Paul Bonnell antigen The crude extract of Paul Bonnell antigen obtained in (2) was added to 0.2
1M phosphate buffer pH 8.0-0.3% n-octyl-
Supe well equilibrated with β-D-thioglucoside
The sample was applied to an rdex200 gel filtration column (Falmatia). Elution was performed with the buffer used for equilibration at a flow rate of 3 ml / min, and a fraction having a molecular weight of about 100,000 was collected. Next, the pH of this fraction was adjusted to 9.0 and then 10 mM ammonium carbonate pH 9.0-0.3% n-octyl-β-D.
-Q-Sepharose FF equilibrated with thioglucoside
(Made by Pharmacia). After washing the column with the same buffer, the antigen was eluted by gradually increasing the sodium chloride concentration in the buffer from 0 to 0.5M. At this time, Paul Bunnell's antigenic activity was eluted mainly in the fraction near the sodium chloride concentration of 0.25 M, but was also partially eluted in the first washed fraction. The fraction eluted with 0.2M to 0.3M sodium chloride was collected, and 10 mM ammonium carbonate p was added.
It was applied to phenyl sepharose CL4B (manufactured by Pharmacia) equilibrated with H9.0-0.3% n-octyl-β-D-thioglucoside. After washing the column with the same buffer as the antigen, adjust the concentration of ethylene glycol in the buffer from 0 to 5
Elution was performed by gradually increasing it to 0%. The Paul Bunnell antigenic activity was mainly recovered mainly in the 20% ethylene glycol fraction, but it was also observed in some washing fractions. 15% to 30
Collect the fraction range eluted with% ethylene glycol,
After concentration by ultrafiltration, 0.1M phosphate buffer pH8.
It was dialyzed against 0-0.3% n-octyl-β-D-thioglucoside and stored at 4 ° C.

Example 2 Comparison with conventional Paul Bunnell antigen (1) Preparation of bovine erythrocyte membrane-sensitized plate Bovine erythrocyte membrane was adjusted to 0.1 mg / ml with 0.1 M citrate buffer pH 3.5. Diluted to 1
Each 00 μl was dispensed into a 96-well ELISA plate (Falcon) and left overnight at 4 ° C. for sensitization. After sensitization, the solution in the plate was removed and 0.05% Tween was added.
20 (trademark), 0.1M phosphate buffer containing 0.15M sodium chloride pH 7.0 (PBST) wash | cleaned 3 times. Then, 0.1M phosphate buffer pH 7.0-2
% Bovine serum albumin-0.02% sodium azide solution: 150 μl was dispensed into the wells and left at least overnight at 4 ° C. The bovine red blood cell membrane sensitized plate could be used in this state for at least one month.

(2) Measurement by enzyme immunoassay The plate prepared in Example 2 (1) was washed 3 times with PBST before use. Paul Bunnell antigen obtained in Example 1 (3) or the method of Merrick et al. (Journal of Supramolecule).
r Structure 6: 275-290, 197.
The antigen obtained according to 7) was added to 0.1 M phosphate buffer pH 7.
After diluting to the same concentration with 0-1% bovine serum albumin,
Dilute 1/3 ³ to 1/3 ¹¹ times with the same buffer to 50μ
l was dispensed into each well of the plate. Sequentially 50 mM 0.1M phosphate buffer pH 7.0-1% bovine serum albumin
Serum 50μ of infectious mononucleosis (IM) patient diluted 0 times
1 was further added to each well of the plate and left at room temperature for 1 hour. After washing 5 times with PBST, the anti-human IgM-peroxidase (POD) -labeled product was added with 0.1 M phosphate buffer p.
100 μl of 1/1000 diluted with H7.0-1% bovine serum albumin was added and left at room temperature for 1 hour.
After that, wash 5 times with PBST and hydrogen peroxide and 2,2 '
-Add 100 μl of a mixed solution of azinodi (3-ethylbenzthiazoline) -6'-sulfonic acid (ABTS) and add 5
After color development for 100 minutes, 100 μl of 1% oxalic acid was added to stop the reaction, and the absorbance at 405 nm was measured. When the results are shown in FIG. 1 and compared with the antigen concentration required to inhibit the color development by 50%, the antigen of the present invention obtained in Example 1 is about 10 times as large as the conventional Paul Bunnell antigen of Merrick et al. Have the same inhibitory activity at low concentrations, ie about 10
It was shown that the activity was twice as high.

Example 3 Preparation of Paul Bunnell's antigen-sensitized gelatin particles Gelatin particles prepared by the method described in Example 1 of JP-A-57-153658 were prepared using 5 ppm tannic acid and 1 μM ferric chloride. Containing 150 mM phosphate buffer p
H7.2-150 mM sodium chloride with a particle concentration of 5%
And dispersed for 10 minutes at 37 ° C. The particles were collected by centrifugation, washed with physiological saline, and then collected again by centrifugation to obtain 150 mM phosphate buffer solution p.
H6.4-150mM sodium chloride with a particle concentration of 5%
Dispersed so that Next, the pole obtained in Example 1
Vannel antigen in 150 mM phosphate buffer, pH 6.4-1
Dilute with 50 mM sodium chloride to 3 μg / ml, mix with the above tannic acid-treated particle dispersion at a ratio of 1: 1 and heat at 37 ° C. for 60 minutes to sensitize the particles with Paul Bunnell antigen. did. The sensitized particles were centrifugally washed with physiological saline and then suspended in a particle suspension (50 mM phosphate buffer pH 6-7.5 mM sodium chloride-0.25% acacia-0.2 M glycine-0.01% thimerosal-).
The suspension was resuspended in 1% normal rabbit serum) to a particle concentration of 1%.

Example 4 Assay of Serum of Infectious Mononucleosis Patient by Indirect Agglutination Immunoassay A U-type 96-well microplate was diluted with a serum diluent (50 m).
M phosphate buffer pH 6-7.5 m sodium chloride-0.
25% acacia-0.15% sodium azide-1
25% of normal rabbit serum) from the first well to the tenth
After dispensing to the well, use the same serum dilution solution in advance.
Double-diluted serum of infectious mononucleosis or Hanga
Twenty-five microliters of nutziu-Deicher (HD) antibody-positive serum or normal human serum was placed in separate first wells and well stirred, and then 25 μl was transferred to the second well. This operation was repeated up to the 10th well to dilute 2 ⁿ . Next, gelatin particles not sensitized with the antigen were added to the first well, and 25 μl of the Paul-Bunnell antigen-sensitized gelatin particles prepared in Example 3 were dropped into the second well to the tenth well, and well mixed. Then, it was allowed to stand at room temperature for 2 hours. Then, the agglutination image of the antigen-sensitized gelatin particles was compared with the image of non-sensitized gelatin particles, that is, a negative control to determine positive and negative. The results are shown in Table 1.

[0030]

[Table 1]

Example 5 ELI of sera from patients with infectious mononucleosis
Assay by SA method The Paul Bonnell antigen obtained in Example 1 (3) was diluted with 0.1 M phosphate buffer pH 7.0 to a protein concentration of 3 ng / ml, and 100 μl of each 96 well ELIS.
The mixture was dispensed to the A plate and left overnight at 4 ° C. Next, after removing the solution in the plate and washing 3 times with PBST,
M phosphate buffer pH 7.0-2% bovine serum albumin-
150 μl of 0.02% sodium azide solution was dispensed into each well and left at 4 ° C. for at least overnight. Less than,
Experiments were performed using this plate.

Wash the plate 3 times with PBST,
5 cases of infectious mononucleosis sera (serums 1 to 5) diluted 1000 times with 0.1 M phosphate buffer pH 7.0-2% bovine serum albumin and 100 cases diluted HD antibody positive sera 2 cases (Serum 6, 7) and 100-fold diluted normal human serum 1
Each 100 μl of the example (serum 8) was dispensed into each well and reacted at room temperature for 2 hours. After washing 5 times with PBST, 0.
100 μl of anti-human IgM-POD labeled substance diluted 1000-fold with 1 M phosphate buffer pH 7.0-2% bovine serum albumin was dispensed into each well and further reacted at room temperature for 1 hour. Then, the plate was washed 5 times with PBST, 100 μl of a mixed solution of hydrogen peroxide and ABTS was added to develop color for 5 minutes, 100 μl of 1% oxalic acid was added to stop the reaction, and the absorbance at 405 nm was measured. Table 2 shows the results.

[0033]

[Table 2] (The substrate blank at this time was 0.02.)

[0034]

INDUSTRIAL APPLICABILITY The Paul Bonnell antigen obtained by the present invention shows high reactivity with the Paul Bonnell antibody. Therefore, the reagent produced using this antigen is also useful for detecting a low concentration of the Paul Bonnell antibody. Is. In addition, when this reagent is used, it is possible to use Paul reagent without cross-reacting with Forssman antibody, HD antibody, etc., which are known as other heterophilic antibodies.
Since it is possible to detect the Vannel antibody, it can be used, for example, for screening patients with infectious mononucleosis.

[Brief description of drawings]

FIG. 1 is a diagram showing an inhibitory activity measurement when the Paul Bunnell antigen of the present invention and the Paul Bunnell antigen produced by a conventional method are used.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kyoko Miwa 2-7-1 Nishishinjuku, Shinjuku-ku, Tokyo Fuji Rebio Co., Ltd. (72) Inventor Yasuhiro Hara 2-7-1 Nishishinjuku, Shinjuku-ku, Tokyo Fuji Rebio Co., Ltd. (72) Inventor Ichiro Matsuda 2-7-1, Nishi-Shinjuku, Shinjuku-ku, Tokyo Fuji-Rebio Co., Ltd. (72) Masahisa Okada 2-7-1, Nishi-Shinjuku, Shinjuku-ku, Tokyo Fuji Rebio Within the corporation

Claims (12)

[Claims] 1. A reagent for assaying Paul-Bunnell antibodies, which comprises a Paul-Bunnell antigen obtained by treating a mammalian erythrocyte membrane with a nonionic surfactant as an active ingredient. 2. The nonionic surfactant is an alkylglucoside, n-alkanoyl-N-methylglucanamide or sucrose monoalkylate.
The measurement reagent described. 3. The nonionic surfactant is n-dodecyl-β-D-maltoside, n-octyl-β-D-glucoxide, n-heptyl-β-D-thioglucoside and n-octyl-β-. The measuring reagent according to claim 2, which is an alkyl glucoside selected from the group consisting of D-thioglucoside. 4. The measuring reagent according to claim 1, wherein the mammal is cow, horse, sheep or goat. 5. The measuring reagent according to claim 1, wherein the Paul Vannel antigen has a molecular weight of 100 kd by gel filtration chromatography. 6. The Paul Vannel antigen is SDS-
50kd, 35k by polyacrylamide gel electrophoresis
The measuring reagent according to any one of claims 1 to 5, which has a molecular weight of d, 20 kd, and 15 kd. 7. The measuring reagent according to claim 1, wherein the Paul-Bunnell antigen is immobilized on particles for indirect agglutination reaction. 8. A reagent for measuring Paul Vannel antibody in blood of a patient with infectious mononucleosis.
The measuring reagent according to any one of 1. 9. Obtained by treating a mammalian erythrocyte membrane with a nonionic surfactant, which is 1 by gel filtration chromatography.
It shows a molecular weight of 00 kd and is 50 kd, 35 kd, 20 kd and 15 by SDS-polyacrylamide gel electrophoresis.
A Paul Bunnell antigen showing a molecular weight of kd and reacting with a Paul Bunnell antibody. 10. The Paul Vannel antigen according to claim 9, wherein the nonionic surfactant is an alkylglucoside, n-alkanoyl-N-methylglucanamide or sucrose monoalkylate. 11. The nonionic surfactant is n-dodecyl-β-D-maltoside, n-octyl-β-D-glucoxide, n-heptyl-β-D-thioglucoside and n-octyl-β-. The Paul Vannel antigen according to claim 9, which is an alkyl glucoside selected from the group consisting of D-thioglucoside. 12. The Paul Bunnell antigen according to claim 9, wherein the mammal is bovine, equine, ovine or goat.