JPS63154700A - Antigen peptide - Google Patents

Abstract

NEW MATERIAL:An antigen peptide of formula I (R1 is Glu, Thr or Pro; R2 is Ile, Lys or Ala; R3 is Thr, met or Leu; R4 is His or Tyr; R5 is Gln, Glu or Trp). USE:An agent for the examination or diagnosis of autoantibody titer of autoimmune neuropathy such as myasthenia gravis, etc., and reagent for adsorption and removal of autoantibody for the cleaning treatment of blood. PREPARATION:For example, a straight-chain peptide of formula II is synthesized by a solid-phase synthesis, dissolved in a carbonate buffer solution of 9.6pH and cyclized by forming a disulfide bond in N2 gas stream to obtain the peptide of formula I. It has been found that the level of autoantibody is high in a patient of myasthenia gravis, etc., and low in a healthy person, by fixing said antigen peptide to a 96-hole U-shaped polystyrene plate, blocking with a semi- serum albumin solution and reacting with various serums.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention is useful for the diagnosis of autoantibody titers in body fluids of autoimmune neurological diseases and myasthenia gravis, and for the detection of autoantibodies in blood vessel purification therapy. This invention relates to antigenic peptides useful for adsorption and removal.

More specifically, the present invention relates to an acetylcholine receptor molecule that exists on the postsynaptic membrane side of the neuromuscular junction and is effective in acetylcholine-mediated neurotransmission, and a peptide that has antigenic activity and consists of a very small number of amino acid residues.

Various peptides of the present invention have antigenic activity that allows them to specifically bind to anti-acetylcholine receptor antibodies, which are autoantibodies responsible for myasthenia gravis.

(Prior art) As a method for detecting and quantifying anti-acetylcholine receptor antibodies in body fluids in myasthenia gravis, concanavalin A method, New England Journal op.
Volume 294, page 691.

f? 1975) and the immunorecipitation method (Vol. 26, p. 1054, 1976) are known.

In both methods, acetylcholine receptor (molecular weight approximately 250,000,
Since it uses a crude surface containing four types of subunits, α, β, r, and δ, it has the disadvantage that the purity of the specimen is low and it is unstable. Second, the acetylcholine receptor molecule is a bioactive polymer with a complex quaternary structure.

There is a problem with the stability of its antigenic activity. In fact, the current problem has been pointed out that anti-acetylcholine receptor antibody titers measured by these methods do not necessarily correlate with clinical symptoms. That is, there are patients whose antibodies are not detected by these methods but who have severe symptoms, while there are patients who have high antibody levels but whose symptoms are mild.

(Problems to be Solved by the Invention) The present invention has been made in order to provide a method for easily measuring anti-acetylcholine receptor antibodies with good reproducibility, and which is also closely compatible with the clinical symptoms of myasthenia gravis. The present invention relates to an antigenic peptide that can specifically bind to an anti-acetylcholine receptor antibody.

(Means for Solving the Problems) The present inventors have discovered that the antigen acetylcholine receptor extracted from animals can be replaced with the conventional antigen acetylcholine receptor, which is highly pure and stable as an antigen, and can be obtained in large quantities. As a result of extensive research into synthetic peptides with specific antigenic activity, we have completed the present invention.

That is, the present invention relates to the formula: (wherein R1 is QIu, Thr or Pro, and bird is He.

Lys or Ala, R, is Thr, Met or Leu.

R1 is Hisi or Tyr, R, is Qln, Glu
Or Trp. ) or a chain antigen peptide thereof, and furthermore, RI e k , Rs *
R41 birds each have Glu, I Ie, Thr, Hi
s, Gl n te certain antigenic peptide, and Rlt
R1*Rs*R4+ Rs are respectively Thr and Lys
, Met, Tyr, and Trp. Incidentally, the chain antigen peptide refers to one in which the disulfide bond between Cys-Cys in the above formula is broken.

The terminology used to describe the peptides of the invention follows the convention of using the first three letters of the common group. The term also refers to the L form of the contemplated amino acids.

Also included within the scope of the invention are acid addition salts that are acceptable as peptide antigens. Examples of acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate. , tartrate, etc., but are not limited to these.

The antigenic peptide of the present invention includes one having a protecting group that is usually used in peptide synthesis, but the natural antigenic peptide without any protecting group exhibits stronger antigenic activity.

The antigenic peptide of the present invention is a Merrifield (Merr)
Journal of the American Chemical Society, Volume 85.

2149, 1964, using a commercially available peptide synthesizer. The synthesized peptide can be confirmed by a conventional method using a commercially available peptide sequencer.

Hereinafter, the experimental facts that led to the present invention will be explained in detail.

The present inventors discovered that H-Cys-RI-11e-Bq-
Val -R3-R, -Phe-Pro-Phe-As
For a peptide having the amino acid sequence p-Rs-Gin-Asn-Cys -OH, R1m R2
Various peptides in which IRl lR4lR11 was substituted with various amino acids were synthesized, and analyzed by enzyme-linked immunosorbent assay (ELISA) using a 96-well U-shaped polystyrene plate (Falcon ■, Becton Dickinson, USA).
The antibody titer in the patient's serum against the peptide was measured. For some peptides, comparisons of chain and cyclic structures were performed under reducing and oxidizing conditions. As a result, it was revealed that antibodies in the serum of patients with myasthenia gravis have strong binding properties with the following peptides.

That is, R1 is GIu, Thr or Pro and is 1L.
- is He, Lys or Ala), R8 is Thr
lMet or Leu, and melon is His or Tyr.
In addition, Rs is a peptide consisting of Gin, Glu, or Trp. In addition, both the chain-like and m-like structure peptides showed approximately the same binding activity.

When we evaluated the positive rate of autoantibodies (anti-acetylcholine receptor antibodies) using serum from many patients with myasthenia gravis and serum from healthy people, we found that R1eRs eRs * Ra
*Rs are Glu, Ile, Thr, respectively
His, Gl n″T: a certain peptide and each T
The peptides hr, Lys, Met, Tyr, and Trp showed particularly favorable positive rates.

When we looked at the correlation between the antibody titers against these two peptides, we found a high correlation (r=o, 88).

When the measurement results using this antigen peptide were compared with the measurement results using the conventional immunoprecipitation method, it was found that patient serum that was negative in antibody titer using the conventional method showed positive results, which corresponded well with the clinical symptoms. Ta.

(Effect of the invention) Anti-acetylcholine receptor antibodies can be easily measured using the antigen peptide of the present invention with good reproducibility.
It is now possible to perform measurements that correlate well with the clinical symptoms of myasthenia gravis. The antigen peptide of the present invention can also be used for antibody testing and diagnosis of neuroimmune diseases such as Alzheimer's disease.

Furthermore, this antigenic peptide can be immobilized on an insoluble carrier to create a 5-antigen peptide-immobilized immunoadsorbent, which can be effectively used for adsorption and removal of autoantibodies in blood purification therapy.

(Example) Hereinafter, embodiments of the present invention will be explained in more detail using examples.

Example 1 H-Cys-Glu-I 1 e-I 1 e-Va
1-Thr-Hi 5-Phe-Pro-Phe-A
sp-Gln-Gln-Asn-Cys-OH was synthesized by solid phase synthesis. The structure of the synthesized peptide was confirmed using a peptide sequencer using a conventional method. The peptide was dissolved in carbonate buffer (pH 9,6) for 1 hour.
After dissolving at a concentration of μ2/−, the mixture was stirred with a N2 gas stream to form disulfide bonds and cyclize. Pour this solution into 96 wells.
type flexible polystyrene plate (Falcon,
Becton and Dickinson, CA
, USA) in units of 100μ and left overnight at 4t:'. After washing the plate 6 times with phosphate buffer/Tween (PBS/Tween),
m bovine serum albumin/phosphate buffer (1 tiBSA/
200μ of PBS) was added, and the mixture was left standing at 4C overnight for blocking. Furthermore, PBS/T111/
After washing three times with een, dispense 100μ of each serum,
After leaving it overnight at 4C, it was washed, and an anti-human IgG antibody conjugated with alkaline 7-osphatase (Sigma, St.
, Lewis, MO, USA) was added,
After reacting for 2 hours, it was washed and the substrate solution (paranitroph z = Rufuos 7 x −) was added to Sigma, St. Lewis.

MO, USA) was added at 1a attl, reacted for 1 hour, and measured the optical density (OD value) at 405 nm.

The results are shown in Figure 1.

In Figure 1, the mean +2 S of the OD value of a healthy person
If D is the cut-off value, the positive rate will be 63 CH for patients with 5 myasthenia gravis. Furthermore, anti-acetylcholine receptor antibody was negative by conventional immunoprecipitation method, and 3 out of 4 serum samples from fC,@ were positive.

Example 2 H-Cy5-Thr-Ile-Lys-Va
I-Me t-Tyr-Phe-Pro-Phe-A
sp-Trp-Gin-Asn-Cys-OH was synthesized by solid phase synthesis. The structure of the synthesized peptide was confirmed using a peptide sequencer using a conventional method. Other than that, the amounts of antibodies in various serums were measured in the same manner as in Example 1. The results are shown in Figure 2. Approximately 75 in the serum of patients with myasthenia gravis
In addition, 5 out of 4 serum samples from patients with anti-inflammatory properties tested positive using the conventional method.

Example 3 In Example I, mercaptoethanol was added to the necessary reagent solution and measurements were performed under reducing conditions, but the results were almost the same as in Example 1. Chain peptides also effectively determined the antigen structure. It was shown that this expression occurs.

[Brief explanation of the drawing]

Figure 1 is a chart showing the results of measuring antibodies in various serums that bind to the antigenic peptide obtained in Example 1, and Figure 2 shows the results of measuring antibodies in various serums that bind to the antigenic peptide obtained in Example 2. This is a chart showing the results. Figure 1

Claims (3)

[Claims] (1) Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R_1 is Glu, Thr or Pro, R_2
is Ile, Lys or Ala, R_3 is Thr, Me
t or Leu, R_4 is His or Tyr, R_5
is Gln, Glu or Trp. ) or its chain antigen peptide. (2) The antigenic peptide according to claim 1, wherein R_1, R_2, R_3, R_4, and R_5 are each Glu, He, Thr, His, and Gln. (3) The antigenic peptide according to claim 1, wherein R_1, R_2, R_3, R_4, and R_5 are respectively Thr, Lys, Met, Tyr, and Trp.