CN1191493A - In vivo diabetes test - Google Patents

Abstract

The invention concerns the use of substances which react together to prepare an agent for the diagnosis of cell-transmitted diseases or a predisposition towards such diseases, the agent being applied intradermally and the diagnosis being made on the basis of the appearance or absence of a positive reaction at the point of application.

Description

Diabetes detect in the body

The present invention relates to produce a kind of material that is used to diagnose the body constitution of immune cell-mediated disease of influence or cell-mediated disease with the autoreactivity material.

In recent years, explanation such as the branch subrelation in the production process of rheumatoid arthritis and this class autoimmune disease of juvenile diabete (IDDM) has been had progress rapidly, and concrete application occurred the early diagnosis of this class disease and treatment subsequently.

Verified at present, in the production process of this class disease, except the disease body constitution of heredity, environmental factors has also play a part certain.Aspect the risk factor of heredity, for example in IDDM, have only the more antigenic allele of MHC II type and this class disease closely related.Therefore can by analyze these allele be Susceptible population of IDDM definition (as referring to people such as Thomson, J.Hum.Genet.43 (1988), people such as 799-816 or Todd, Nature 329 (1987), 599-604).

Environmental factors in the IDDM production process may be the immunogenic bioactive peptides sequence of external source.Virus antigen is discussed in this respect, they and endogenous structure have homeologous.Under special circumstances, especially in postnatal a period of time, absorbed antigen such as bovine serum albumin can cause immunoreation by food since with the homology of endogenous structure, this reaction can cause the process of an autoaggression.

In the pathogenic process in IDDM, typical phenomenon is exactly the destruction gradually of the pancreatic beta cell that caused by cytotoxic lymphocyte.In this process becomes obviously early than destroyed this phenomenon of glucose metabolism far away.When the symptom of diabetes can be detected, the β cell more than 80% was destroyed.Therefore, in Susceptible population, detect these autoaggressions T cell in period early and just seem important completely, so just can provide treatment timely for infected people.

Verified at present, in self exempting from the disease disease, the destruction of endogenous tissue is carried out very slowly when beginning.In the incipient stage of this process, autoaggression T cell may only be discerned one or more autoantigens.(Nature 368 (1993) people such as Kaufman, 69-72) and people such as Tisch (Nature 368 (1993), in report 72-78), the animal model of type i diabetes (NOD mice) shows, when this mice spontaneous generation diabetes, the cell-mediated autoimmune response of initial T is at glutamate decarboxylase.In the starting stage of this process, in NOD mice body, only discern 1 to 2 epi-position at the C-of glutamate decarboxylase (GAD) end.Mention above, the change of glucose metabolism can't be detected in this stage, and this moment, perinsulitis can be detected.Have only when disease further develops, the spectrum of the peptide of the GAD that discerns by autoaggression T cell just can become wideer.In case diabetes are obvious, also just can detect activated T cell in advance, they resist other island cellular antigens, for example peripheral protein, heatshock protein HSP65 and carboxypeptidase H.

Evidence suggests in the people, also cause effect relation is arranged with the generation of type i diabetes at the immunoreation of GAD.Therefore for instance, can detect the autoantibody at GAD in about 80% pre-diabetes, be not main cause of disease although people estimate these autoantibodys.And there is the people to infer that the T lymphocyte has progressively destruction to the pancreas beta cell in type i diabetes.Had some research groups (Harrison etc., J.Clin.Invest.89 (1992), 1161; Honeyman etc., J.Exp.Med.177, (1993), 535) detected above-mentioned T lymphocyte at GAD.T lymphocyte that these groups find and the reaction of the fragments of peptides of GAD 67kd molecule, this segment contains aminoacid 208 to 404.

EP-A-0 519 469 discloses the autoimmune response polypeptide that derives from people GAD 65kd molecule.These amino acid sequence of polypeptide are as follows:

X-P-E-V-K-(T or E)-K-Z

Wherein X is an optional sequence of selecting from 1 to 10 aminoacid, and Z is an optional sequence of selecting from 1 to 8 aminoacid.

Id reaction peptide sequence and a kind of pharmaceutical composition that contains these peptides of people GAD 65 have also been proposed to derive from the European Patent Application No. 95 100 764.0.This application has also been described and has been used this pharmaceutical composition to produce a kind of material, and this material is used to diagnosis immune disease of influence or disease body constitution, or is used for the body constitution of diagnosing tumour disease or tumor disease.

One of purpose of the present invention provides a kind of diagnostic method that is used for immune cell-mediated disease, particularly autoimmune disease, and this method reduces patient's sense of discomfort on the one hand as much as possible, can carry out on the other hand fast and reliable diagnostic.

This purpose is to realize that by the material of the body constitution that produces a kind of disease that is used for the diagnosis cell mediation or cell-mediated disease with the autoreactivity material wherein this material is by intradermal administration, and diagnosis basis is positive reaction whether to occur at medicine-feeding part.

Diagnostic method of the present invention is with available method is different in the market, the method of using is used to the generality detection of cell mediated immunity at present, difference is, the contacted exogenous antigen of patient has been used in these detections, for example the protein derivatives of purification (PPD), tetanus toxoid, Candida (Candida) etc.These detections are used to determine detected person's immune general state.Immune cell-mediated disease does not have detected.

Be widely used for determining the detection of atopic reaction at another, during for example puncture (prick) detected, antigen was not at intradermal administration.Not in four hours, to be detected, it is characterized in that swelling appears in the surface, rather than tuberosity occurs by the cell-mediated positive reaction of T.

The invention provides a kind of method that is used for detecting in vivo at the generation of the cell mediated immune response of autoreactivity material.This cell mediated immune response has shown the existence of the body constitution of immune specified disease, particularly autoimmune disease or this class disease.In addition, cell mediated immune response also shows the existence of tumor disease or its body constitution.

The autoreactivity material or the autoantigen that are suitable for the inventive method are natural, synthetic or the reorganization material, for example nucleic acid, lipid, saccharide, polypeptide, protein, peptide class and their complex, for example peptide with such as MHC molecule or the so formed complex of polypeptide of peptide bound derivative.Suitable id reaction material in addition is specific immunogenicity epi-position, segment, analog, plan shape thing or the derivant that for example produces by chemical modification and the mixture of above-mentioned these materials.Can be with the former state administration of autoreactivity material or with carrier combining form administration, for example with the lipopeptid form administration.The autoreactivity material can by independent administration or with additional material, for example additional stimulation component or together administration of adjuvant.By the recombinant DNA technology or the synthetic method of the solid phase synthesis of peptide for example, can make autoantigen by natural origin.

Aspect endogenous antigen, except the id reaction material, are id reaction materials that exogenous antigen and autoantigen have the immunology relation although can also use those.The example of this class exogenous antigen is exactly the peptide that homology is arranged with the autoreactivity peptide sequence that derives from endogenous protein.Concrete example has: have the peptide that derives from cattle beta-casein A of sequence L-V-Y-P-G-P-I-P-N (aminoacid 60-68), it and endogenous glucose transport protein GLUT-2 have the part of homology region in sequence Q-I-G-P-G-P-I-P-W (aminoacid 412-420) zone; The peptide that derives from Coxsackie disease poisonous protein P2-C with sequence K-V-K-I-L-P-E-V-K-E-K-H-E (aminoacid 33-45), it and glutamate decarboxylase have homology in sequence R-F-K-M-F-P-E-V-K-E-K-G-M (amino acid/11 55-167) zone.

Method of the present invention comprises different autoantigens at intradermal administration and determine at medicine-feeding part whether the reaction of partial positive cell to take place.This positive reaction determines in the peculiar moment of the cell-mediated immunoreation of T, and it determines that the time preferably after administration 24 hours, is more preferably within 24 hours to 1 week after the administration at least, most preferably is between the 40-80 after the administration hour.

Positive reaction is preferably incorporated in medicine-feeding part and lump occurs, can be by range estimation or/and palpation it is carried out qualitatively determining, also can carry out quantitative assay to it by method such as ultrasound wave or photograph.

On the other hand, can also determine the product of positive reaction, for example such as the cytokine of gamma interferon, it is by discharging at medicine-feeding part such as leukocytic infiltration cell.

Method of the present invention is particularly suitable for the body constitution of diagnosis of autoimmune disease or autoimmune disease.

For instance, the autoimmune disease of available method diagnosis of the present invention has diabetes, especially type i diabetes (IDDM), multiple sclerosis, rheumatoid arthritis, Basedow's disease, poker back, acute anterior uveitis, Good-pasture syndrome, myasthenia gravis, systemic lupus erythematosus (sle), pemphigus vulgaris, immune thyroiditis, scleroderma, Crohn disease, Sjogren syndrome, Lai Teer syndrome, enteritis or Graves disease.Method of the present invention especially is preferably used for the disease of diagnosing T cell-mediated, for example IDDM, rheumatoid arthritis or multiple sclerosis, and method of the present invention most preferably is used to diagnose IDDM.

For instance, be used to diagnose the id reaction material of IDDM that insulin secretion granule protein matter or its part of glutamate decarboxylase (GAD) 65kd or 67kd, tyrosine phosphatase (antigen of 38kd), carboxypeptidase H, insulin, heatshock protein, 38kd are arranged.People GAD 65kd or its partial peptide sequence more preferably are used as the autoreactivity material.

Yet similarly diagnosis also can be used for other autoimmune disease.The example of this class autoimmune disease has: multiple sclerosis wherein can detect the reaction-ive T cell that resists myelin basic protein matter or PLP etc.; Rheumatoid arthritis wherein can detect antagonism II Collagen Type VI, cytokeratin, the dnaJ albumen that derives from escherichia coli (E.coli) and the reaction-ive T cell of Hsp65; Basedow's disease wherein can detect the reaction-ive T cell that resists thyroid peroxidase.When suffering from myasthenia gravis, can detect reaction-ive T cell to AChR or other relevant id reaction material.When suffering from lupus erythematosus, can detect the reaction-ive T cell of antagonism HSP90, the reaction-ive T cell of antagonism tsh receptor is then arranged in the Graves disease.

In a word, this diagnosis can be used to influence immune all diseases, for example also can be used for arteriosclerosis.It is in this case, verified should disease relevant with the immunoreation of antagonism heatshock protein Hsp65 that (people such as Xu, Lancet 341,8840 (1993), 255-259).

Further using for one is that diagnostic ground detects the T cell that reacts with tumor antigen.For example detect the T cell of the antagonism antigen MAGE1 relevant with melanoma, (people such as van der Bruggen, Science 254 (1991), 1643-1647) from melanoma patient for this T cell separation.Just these T cells can have been detected before can't detecting tumor (because cellular material also very little) with traditional method.In addition, specific reaction T cell also can be used to monitor a kind of antineoplastic vaccination.

In addition, using the derivant or the bonded similarly molecule of peptide, peptide also is possible as the autoreactivity material.In order to diagnose IDDM, preferably use one or more to derive from GAD, especially derive from the peptide of people GAD 65kd, perhaps intend the shape thing by its deutero-peptide derivant or peptide.

In order to diagnose IDDM, very preferably use peptide or the peptide derivant that contains following aminoacid sequence:

(a) aminoacid sequence (I)

D-V-N-Y-A-F-L-H-A-T-D-L-L-P-A-C-D-G-E-R，

(b) aminoacid sequence (II)

S-N-M-Y-A-M-M-I-A-R-F-K-M-F-P-E-V-K-E-K，

(c) aminoacid sequence (III)

N-W-E-L-A-D-Q-P-Q-N-L-E-E-I-L-M-H-C-Q-T，

(d) aminoacid sequence (IV)

T-L-K-Y-A-I-K-T-G-H-P-R-Y-F-N-Q-L-S-T-G，

(e) aminoacid sequence (V)

P-R-Y-F-N-Q-L-S-T-G-L-D-M-V-G-L-A-A-D-W，

(f) aminoacid sequence (VI)

T-Y-F-I-A-P-V-F-V-L-L-E-Y-V-T-L-K-K-M-R，

(g) aminoacid sequence (VII)

F-F-R-M-V-I-S-N-P-A-A-T-H-Q-D-I-D-F-L-I，

(h) aminoacid sequence (VIII)

G-M-A-A-L-P-R-L-I-A-F-T-S-E-H-S-H-F-S-L-K-K-G-A-A，

(i) aminoacid sequence (IX)

E-R-G-K-M-I-P-S-D-L-E-R-R-I-L-E-A-K-Q-K，

(j) be shown in the aminoacid sequence of Fig. 1 or Fig. 2 one,

(k) length has the subregion of the aminoacid sequence in 6 amino acid whose being shown in (a) to (i) at least, or/and

(l) be shown in (a) to (j) in aminoacid sequence substantially the same binding specificity to the MHC molecule is arranged or/and the aminoacid sequence of affinity.

Aminoacid sequence (I) to (VII) is corresponding to amino acid residue 86-105 (I), 246-265 (II), 146-165 (III), 166-186 (IV), 176-195 (V), 206-225 (VI) and the 556-575 (VII) of people GAD 65kd.In sequence SEQ ID NO.1-7, aminoacid sequence I-VII is stated.

Aminoacid sequence (VIII) is corresponding to the amino acid residue 266-290 of people GAD 65kd, and aminoacid sequence (IX) is corresponding to the amino acid residue 306-325 of people GAD 65kd.The aminoacid sequence that is shown among Fig. 1 and 2 also is the partial sequence of people GAD 65kd.In sequence SEQ ID NO.8-28, provided the aminoacid sequence of Fig. 1 and 2.In sequence SEQ ID NO.29 and 30, provided aminoacid sequence (VIII) and (IX).

People are surprised to find, and contain the peptide of people GAD 65 above-mentioned zones and the T cell subsets generation specific reaction of separating from the type i diabetes of recent findings.Thereby the peptide among the present invention is early stage self epi-position, and its application makes the extremely early stage diagnosis to type i diabetes become possibility.

Aminoacid sequence (I) to (IX) is the subregion of isoreagent that derives from the 65kd of human glutamic acid decarboxylase (GAD), and the aminoacid sequence that GAD is complete is described in people such as Bu, and (Proc.Nat.Acad.Sci USA 89 (1992), in article 2115ff.).Aminoacid sequence (I) to (IX) is found like this, promptly set up earlier the T cell line of the peripheral blood that derives from type i diabetes, derive from the GAD of Medulla sus domestica or stimulate them and in propagation detects, use the synthetic peptide sequence that derives from people GAD sequence to detect these T cell line at external use then with the people GAD of reorganization.

Can produce these peptides by the known building-up process of using chemical method, also can produce by genetic engineering, gene engineering method is to clone and express the DNA sequence of these peptides of coding in proper host cell, especially escherichia coli (E.coli).

The peptide that has the aminoacid sequence of specific (I) to (IX) or be shown in the subregion of the aminoacid sequence among Fig. 1 and 2 also is preferred, its length is at least 6 aminoacid, be preferably at least 8 aminoacid, more preferably at least 10 aminoacid most preferably are at least 15 aminoacid.Amino acid whose minimum length of the present invention be by its identification MHC molecule, with this molecular specificity combine and determine with the ability that corresponding TXi Baoshouti reacts.

Derive from GAD and be preferably 100 aminoacid with the greatest length of the part of the bonded peptide of the present invention of MHC, more preferably 50 aminoacid most preferably are 25 aminoacid.

Except above-mentioned peptide, also can use with above-mentioned peptide sequence and essentially identical binding specificity to the MHC molecule be arranged or/and the peptide of affinity, they preferably derive from above-mentioned aminoacid sequence, and method is to replace, lack or insert to different amino acid residues or to the sub-fraction of amino acid residue or to the material after the bonded modification in a similar manner.

Especially, the invention still further relates to the mutation of peptide, their sequence and above-mentioned aminoacid sequence are not identical, but only contain identical or closely-related " anchor position ".Term " anchor position " be meant a kind of for a kind of MHC molecule, requisite amino acid residue especially combines with the MHC molecule of DR3, DR4 or DQ class.For instance, people such as Hammer state in conjunction with the anchor position of primitive being used for DRB10401 among the 197-203 at Cell 74 (1993).This class anchor position is retained in the peptide of the present invention or is had that the amino acid residue of closely-related side chain replaces on chemical property (valine substituted lactamine for example, isoleucine replaces leucine, vice versa).The anchor position that is present in the peptide of the present invention can be measured with a kind of simple mode, and method is to detect the mutation of above-mentioned particular peptide and the binding ability of MHC molecule.The characteristics of peptide of the present invention be they have with the essentially identical binding specificity to the MHC molecule of above-mentioned peptide or/and affinity.Compare with initial peptide or its partial sequence, the peptide that derives from these peptides preferably has at least 30% sequence homology, and more preferably at least 50%, most preferably be at least 60%.

The example of the mutation of specific peptide has the corresponding homeopeptide position that derives from people GAD 67, and people such as Bu also are described its complete aminoacid sequence, together above.

" essentially identical binding specificity to the MHC molecule is or/and affinity " this notion comprises that also a kind of and (I) compare or compare the binding specificity that is improved or/and affinity with the aminoacid sequence in being shown in Fig. 1 and 2 to the aminoacid sequence of (VIII), and this especially is more common in the peptide that length is preferably 8 to 15 amino acid whose shortenings.

The invention still further relates to the derivant of peptide.This term comprise those one of them or several amino acid by chemical reaction institute derivatization peptide.The example of peptide derivant of the present invention especially refers to these molecules, and promptly those main chains are or/and reactive amino acid side group, for example free amino group, free carboxyl group or/and free hydroxyl by derivatization molecule.The object lesson of aminoderivative has sulfonamides or carboxylic acid amides, thioxanthamide derivant and ammonium salt, for example hydrochloride.The example of carboxy derivatives has salt, esters and amide-type.The example of hydroxy derivatives has O-acyl group or O-alkyl derivative.According to the present invention, this notion of the derivant of peptide also comprises some peptides like this, and one of them or several amino acid are replaced by the natural existence of homologue or the aminoacid of non-natural existence with 20 " standard " aminoacid.The example of these homologues has 4-hydroxyproline, 5-oxylysine, 3-Methyl histidine, homoserine, ornithine, Beta-alanine and 4-aminobutyric acid.

Suitable peptide derivant in addition is complex or the covalent bond between peptide and the adjuvant component, they with such as the autoantigen of lipid by together administration.In this case, peptide also can be used as lipopeptid.

The present invention also comprises polypeptide, and wherein the peptide position in conjunction with MHC is a unitary component of bigger polypeptide, wherein preferably has a pre-determined breakpoint in conjunction with the peptide of MHC and the junction between the unitary nubbin of polypeptide, for example a protease cutting site.

The invention still further relates to peptide and intend the shape thing, the derivant of they and above-mentioned peptide or peptide has basic identical binding specificity to the MHC molecule or/and affinity.With regard to the interaction of itself and MHC molecule, it is the chemical compound that can replace peptide that peptide is intended the shape thing, and compares with natural peptide, and they have higher metabolic stability, better biological availability and longer effect persistence.The method for preparing peptide plan shape thing is described in Giannis and Kolter, Angew.Chem.105 (1993), 1303-1326; People such as Lee, Bull.Chem.Soc.Jpn.66 (1993), people such as 2006-2010 and Dorsch, Kontakte (Darmstadt) (1993) (2), 48-56.With regard to the preparation that peptide of the present invention is intended the shape thing, with reference to disclosed content in these documents.

A kind of more suitably autoreactivity material is a kind of complex, and it comprises the derivant of at least a peptide, peptide or the peptide bound derivative that peptide is intended shape thing and at least a MHC molecule or a kind of MHC molecule.In this complex, binding constant is preferably at least 10 ^-7L/mol, more preferably 10 ^-810 ^-9Peptide between the l/mol, the derivant of peptide or peptide are intended the shape thing and are combined with the peptide bound derivative of MHC molecule or MHC molecule.In addition, the derivant of peptide, peptide or peptide are intended the shape thing also can pass through light joint and MHC molecule covalent bond, or the heritability of covalency peptide-MHC fusant.This peptide-MHC fused protein preferably contains a kind of HLA-DR β chain and autoreactivity peptide a kind of and that its heritability ground merges.This complex more preferably contains II type MHC molecule or its peptide bound derivative.

The nucleotide sequence of the gene of coding II type MHC molecule referring to people such as Corell (Mol.Immunol.28 (and 199D, 533-543).The document is included in the content of the present invention.

" the peptide bound derivative of MHC molecule " this notion comprises the segment of MHC molecule, and they are by preparing to the Proteolytic enzyme enzyme action of natural MHC molecule or by recombinant DNA technology, and they have kept their peptide binding characteristic basically.Also can wherein except containing the MHC part of being responsible for binding peptide, also contain other polypeptide fractions with this conceptual understanding for comprising fused protein.

The preparation of the peptide-MHC complex preferably MHC molecule by will not containing peptide or MHC molecule derivant and peptide of the present invention, peptide derivant or peptide is intended the shape thing and is combined and realize.For instance, the preparation of MHC molecule that does not contain peptide is by stretching natural MHC molecule to dissociate bonded peptide and the refolding of sky MHC molecule is realized (referring to Dornmair and McConnell, Proc.Natl.Acad.Sci.USA 87 (1990), 4134-4138 and WO91/14701).Other method that is used for preparing the complex of peptide and MHC molecule is described in european patent application NO.95 100 764.0.The document is included in the content of the present invention.

The invention still further relates to the use of a kind of compositions in a kind of diagnostic method, said composition comprises a kind of autoreactivity material as active component, for example the derivant of protein, polypeptide, peptide, peptide, peptide are intended the shape thing or/and peptide-MHC complex, they are optionally combined with adjuvant or other common medicated premix, and this method comprises this compositions at intradermal administration.Said composition can also contain a kind of additional stimulation component, for example such as IL-2 and IL-4 or/and the cytokine of surface antigen B7 (referring to people such as Wyss-Coray, Eur.J.Immunol.23 (1993), 2175-2180; People such as Freeman, Science 262 (1993), and 909-911), this stimulation component can be combined on the surface molecular CD-28 of T cell.The existence of this additional stimulation component can improve or/and improve the diagnosis effect of said composition.

In addition, has the positive in the method for the invention or/and the additional detection of negative control also is preferred.For example, can use a kind of nonreactive polypeptide, human serum albumin for example perhaps only contains the goods of additive, and for example buffer is as negative control.For example, the protein derivatives (for example available from Statens Serum Institute, Copenhagen Denmark) or the known tetanus toxoid of intensive T cytositimulation that can cause in many people that derive from behind the purification of the culture medium of having cultivated the tumor antibacterial can be used as positive control antigen.

In certain embodiments of the invention, with autoreactivity material and a kind of adjuvant together administration also be preferred.The example of suitable adjuvant has aluminium hydroxide, incomplete Freund's adjuvant, the lipid (lipopeptid) that aluminum phosphate and being suitable for is connected with peptide.

The autoreactivity material can for example use to have the general intradermal syringe of size as the 26G syringe needle by known method administration.The preferred intradermal administration dosage that contains the compositions of autoreactivity material is 20 μ l to 500 μ l, 50 μ l to 200 μ l more preferably, and the suitable position of administration is so that the autoantigen that is provided is convenient to reach immune cell most is as the criterion.For example, this medicine can be injected into forearm (front) for diagnosis IDDM.

The concentration of the autoantigen in compositions can change in a very wide scope according to employed autoantigen under the various situations.The concentration of 1 to 100 μ g/ml, the especially concentration of 5 to 2 μ g/ml are preferred.

In addition, also can carry out administration in a table apparatus, this device comprises one and is used to store the container of the preparation that contains the autoreactivity material, a container and a container that is used to store the negative control preparation that is used to store the positive control preparation.For instance, this device can comprise 2 to 10 containers that are used to store the preparation that contains the autoreactivity material.This device can be used in a simple manner so that detect many autoreactivity materials simultaneously.

Another theme of the present invention is to determine a kind of specific T-cells subgroup, and the sample that wherein contains the T cell contacts with the autoreactivity material, and this sample preferably derives from the tissue sample of medicine-feeding part, measures the reaction of T cell and autoreactivity material then.For instance, the specific reaction of T cell and autoantigen can detect by the increase of T cell proliferation, and the increase of breeding can be measured by radioactive mixing.On the other hand, the reaction of T cell also can directly detect by using a kind of autoantigen that has been labeled, and for example the MHC molecule of this autoantigen and solubility exists with the form of complex.In this embodiment, the autoreactivity material preferably together uses with the fluorescent labeling group that is connected thereon.For instance, can estimate by facs analysis, wherein the T cell contacts with first fluorescent marker that is connected with the T cell specific antibody, contact with the autoantigen that is connected with second fluorescent marker then, determine the existence of double labeling cell then by the fluorography analytic process.Just can determine a T cell subsets by this way, its feature is exactly the reactivity of it and a kind of autoantigen.This method can also optionally comprise the selection of activated T cell in advance, for example by with the cultivation of IL-2 or/and by with the cultivation of IL-2 receptor antibody, for example use immunity-magnetism method antagonist to separate to come optionally enrichment IL-2 receptor-positive T cell in conjunction with cell subsequently.On the other hand, with after autoantigen contacts, can at first select activatory in advance cell at the T cell.

In a correction, can also determine activatory in advance autoreactive T cell (having T cell) and the ratio that does not have activatory autoreactive T cell (the T cell that does not have the IL-2 receptor) as the IL-2 receptor of surface markers to this method.

Another theme of the present invention is to separate the T cell subsets that reacts with a kind of autoantigen.In the method, the tissue sample that contains the T cell of taking from medicine-feeding part contacts with a kind of autoantigen, discerns the T cell of these and autoantigen reaction then and optionally with the T cell separation of they and other.In this case, the T cell with before autoantigen contacts or/and afterwards, also can be to activated T cell in advance, the T cell that promptly contains the IL-2 receptor is selected.In the method, for instance, can use autoantigen with following form: complex, autoantigen that autoantigen and MHC molecule form are immobilized on the carrier, and this has just simplified the T cell mass of positive reaction and the process of other T cell separation.By stimulating again, from setting up T cell line the isolated T cell subsets by this way.Just can use these autoreactive T cell systems to make patient's immunity then.

At the diagnostic method that is used for identification specificity T cell subsets, also can use a kind of anti-idiotype antibody to replace autoantigen, this antibody has imitated the effect of MHC peptide complexes.By producing the immunogenic (for example in mice) that antibody is used a specific antigen there being specific T cell subsets be used as, or by at first producing a kind of first antibody that resists autoantigen, and then produce a kind of anti-idiotype antibody that resists first antibody, just can obtain above-mentioned antibody easily.

Product by discerning these T cells that are activated in vivo rather than separate these T cells also can be discerned special T cell subsets.This can and measure wherein contained cytokine and realize by collection organization's sample (for example meticulous pin aspirate).In addition, also can the in-site detecting cytokine, method is to use a kind of long-acting ointment, and wherein immobilization has the antibody at various cytokines.Detect cytokine by a kind of traditional solid-phase immunoassay method, wherein used zymolyte, it can produce a kind of insoluble coloured product, and this product can directly be read or use scanner to read.

At last, the present invention relates to a kind of test kit, it is used for the disease of diagnosis cell mediation in vivo or the body constitution of this disease, comprising:

A) at least a autoreactivity material that exists with the acceptable drug composition forms,

B) the Ren Xuan at least a contrast material that exists with the acceptable drug composition forms,

C) be used for the device of intradermal administration of autoreactivity material and contrast material, and

D) the optional device that is used for the diagnostic evaluation of testing result.

The compositions that is used for autoreactivity material and contrast material preferably contains adjuvant and optionally contains other common pharmaceutical carrier material, diluent and additive.

The following embodiment that combines with Fig. 1 and 2 and sequence SEQ ID NO.1-30 is in order further to illustrate the present invention.

Fig. 1 shows the autoreactivity aminoacid sequence that derives from people GAD 65kd,

Fig. 2 shows other autoreactivity aminoacid sequence that derives from people GAD 65kd,

SEQ ID NO.1-7 shows the autoreactivity aminoacid sequence that derives from people GAD (I)-(VII),

SEQ ID NO.8-11 shows the autoreactivity aminoacid sequence that derives from the people GAD among Fig. 1,

SEQ ID NO.12-28 shows the autoreactivity aminoacid sequence that derives from the people GAD among Fig. 2,

SEQ ID NO.29 and 30 demonstrations derive from people GAD (VIII) and autoreactivity aminoacid sequence (IX).

Embodiment 1 (Comparative Examples)

In vitro tests

In 8 patient bodies that infect IDDM recently and in 12 healthy human bodies, collect peripheric venous blood.Separate peripheral blood lymphocyte by Ficoll-Hypaque density gradient centrifugation, then itself and following substances one be used from stimulated in vitro:

A) recombined human GAD 65kd is as detecting antigen;

B) protein derivatives behind the purification (PPD) is as positive control antigen;

C) human serum albumin (HSA) is as negative control antigen.

Exist or have only under the condition that culture medium exists at above-mentioned antigen, in 1 hole of round bottom incubator with 96 holes with 1 * 10 ⁵Individual peripheral blood lymphocyte (PBL) is at 37 ℃ and 5%CO ₂Condition under in the culture medium of 100 μ l, cultivate five days (RPMI 1640 culture medium, 5% human serum, 2mmol/l glutamine, 10U/ml penicillin, 10 μ g/ml streptomycins and 50nmol/l 2 mercapto ethanol).

After 5 days, in each hole, add RPMI 1640 culture medium that 20 μ l contain 0.5 μ Ci 3H thymidine.Continue to cultivate 16 hours, then collecting cell.By mixing of liquid scintillation counting metering thymidine.With the result as stimulation index (cpm when SI, the cpm when promptly existing with antigen have only culture medium to exist when not existing divided by antigen).

The lymphocyte that derives from patient IDDM and matched group normal person is listed in table 1 and 2 at different antigenic breeding reaction results.

Table 1

		The PBL that the PBL that SI=is stimulated, cpm/ are not stimulated, cpm
					Patient IDDM	Base value, cpm	PPD 10μg/ml	rhGAD?65 10μg/ml	HSA 10μg/ml
1	730	17.9	2.6	1.1
					2	833	16.7	8.8	1.2
3	639	23	6.6	0.9
					4	328	28.9	0.8	2.9
S	1211	10.6	1.9	0.5
					6	1041	11.9	1.8	2.2
7	520	19.8	7.2	1.0
					8	412	2.7	8.3	0.8

Table 2

		The PBL that the PBL that SI=is stimulated, cpm/ are not stimulated, cpm
					Healthy people	Base value, cpm	PPD 10μg/ml	rhGAD????65 10μg/ml	HSA 10μg/ml
1	1108	25	1.8	0.7
					2	1035	2.1	3.0	0.5
3	390	6.5	2.7	1.1
					4	578	6.2	0.9	1.2
5	243	1.3	0.9	1.9
					6	375	6.3	2.5	2.6
7	648	12.5	4.2	0.8
					8	730	20.6	4.2	0.5
9	542	0.8	0.9	2.0
					10	915	0.5	2.8	2.8
11	567	90.6	3.5	2.3
					12	1247	12.5	1.0	0.6

The statistical analysis of result of the test is shown when using rhGAD 65kd, the PBL propagation that derives from patient IDDM of nearest diagnosis can be considered to male, its SI be higher than 4.7 (matched group normal person's meansigma methods for+2SD).

Therefore, in 50% patient IDDM (patient 2,3,7 and 8), observed external t cell responses at rhGAD 65kd.In patient 1,2,4 and 8, found antibody, do not found and in patient 3 and 5, at GAD.The antibody at GAD to patient 6 and 7 does not detect.The normal person of matched group is reactionless to GAD 65kd.

In the reaction in contrast HSA and PPD, diabetes patient and matched group normal person do not demonstrate the notable difference on the statistics at all.

Embodiment 2

In vivo test

Be in eight patients IDDM of rhGAD 65kd intradermal injection in the embodiment 1 the right forearm of 10 μ g/ml with 100 μ l concentration.

With containing 1%HSA, the normal saline dilution antigen of pH7.3.With the concentration of same volume (100 μ l) is that the HSA of 10 μ g/ml is expelled to right upper arm as negative control.

In 5 in 18 patients IDDM with PPD as positive control antigen.In all patients all with HSA as negative control.

In the diagnosis, promptly be considered to positive reaction in vivo in the nodular formation of medicine-feeding part (specificity of dermis thickness increases).Diagnose after 48 hours in administration.By determining in the ultrasonic measurement of medicine-feeding part and measuring the result.Do not observe side effect.

The results are shown in Table 3 in this test.

Table 3

Patient IDDM	PPD 10μ/ml	rhGAD?65 10μg/ml	HSA 10μg/ml	Detection time
					1	+	++	-	48 hours
2	n.t.	++	-	48 hours
					3	n.t.	+	-	48 hours
4	-	-	-	48 hours
					5	-	-	-	48 hours
6	-	-	-	48 hours
					7	n.t.	+++	-	48 hours
8	+	+	-	48 hours

In eight detected patients IDDM; there are five rhGAD 65 had positive reaction. ( 1 ) ( i ) ( A ) ：Boehringer Mannheim GmbH ( B ) ：Sandhofer Str.112-132 ( C ) ：Mannheim ( E ) ： ( F ) ( ZIP ) ：68305 ( ii ) ： ( iii ) ：30 ( iv ) ( A ) ： ( B ) ：IBM PC ( C ) ：PC-DOS/MS-DOS ( D ) ：PatentIn Release#1.0,version#1.30 ( EPA ) ( vi ) ( A ) ：DE19526561.0 ( B ) ：20-July-1995 ( 2 ) SEQ ID NO：1 ( i ) ： ( A ) ：20 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：1：Asp Val Asn Tyr Ala Phe Leu His Ala Thr Asp Leu Leu Pro Ala Cys Asp1 5 10 15Gly Glu Arg

20 (2) SEQ ID NO:2: information: (i) sequence signature: (A) length: 20 aminoacid (B) type: aminoacid (C) chain: (D) topological structure: line style (xi) sequence description: SEQ ID NO:2:Ser Asn Met Tyr Ala Met Met Ile Ala Arg Phe Lys Met Phe Pro Glu Val1 5 10 15Lys Glu Lys

The information of 20 (2) SEQ ID NO:3: (i) sequence signature (A) length: 20 aminoacid (B) type: aminoacid (C) chain: (D) topological structure: line style (xi) sequence description: SEQ ID NO:3:Asn Trp Glu Leu Ala Asp Gln Pro Gln Asn Leu Glu Glu Ile Leu Met His1 5 10 15Cys Gln Thr

The information of 20 (2) SEQ ID NO:4: (i) sequence signature: (A) length: 20 aminoacid (B) type: aminoacid (C) chain: (D) topological structure: line style (xi) sequence description: SEQ ID NO:4:Thr Leu Lys Tyr Ala Ile Lys Thr Gly His Pro Arg Tyr Phe Asn Gln Leu1 5 10 15Ser Thr Gly

The information of 20 (2) SEQ ID NO:5: (i) sequence signature: (A) length: 20 aminoacid (B) type: aminoacid (C) chain: (D) topological structure: line style (xi) sequence description: SEQ ID NO:5:Pro Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu Asp Met Val Gly Leu Ala1 5 10 15Ala Asp Trp

The information of 20 (2) SEQ ID NO:6: (i) sequence signature: (A) length: 20 aminoacid (B) type: aminoacid (C) chain: (D) topological structure: line style (xi) sequence description: SEQ ID NO:6:Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Val Thr Leu Lys1 5 10 15Lys Met Arg

The information of 20 (2) SEQ ID NO:7: (i) sequence signature: (A) 20 aminoacid (B) type: aminoacid (C) chain: (D) topological structure: line style (xi) sequence description: SEQ ID NO:7:Phe Phe Arg Met Val Ile Ser Asn Pro Ala Ala Thr His Gln Asp Ile Asp1 5 10 15Phe Leu Ile

20 ( 2 ) SEQ ID NO：8： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：8：Ile Leu Ile Lys Cys Asp Glu Arg Gly Lys Met Ile Pro Ser1 5 10 ( 2 ) SEQ ID NO：9： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：9：Leu Gly Ile Gly Thr Asp Ser Val Ile Leu Ile Lys Cys Asp1 5 10 ( 2 ) SEQ ID NO：10： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：10：Leu Ala Phe Leu Gln Asp Val Met Asn Ile Leu Leu Gln Tyr1 5 10 ( 2 ) SEQ ID NO：11： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：11：Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Ala Leu Gln Cys1 5 10 ( 2 ) SEQ ID NO：12： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：12：Val Ser Tyr Gln Pro Leu Gly Asp Lys Val Asn Phe Phe Arg1 5 10 ( 2 ) SEQ ID NO：13： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：13：Leu Ala Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr Asn Met1 5 10 ( 2 ) SEQ ID NO：14： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：14：Leu Leu Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gly Gly1 5 10 ( 2 ) SEQ ID NO：15： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：15：Val Asn Tyr Ala Phe Leu His Ala Thr Asp Leu Leu Pro Ala1 5 10 ( 2 ) SEQ ID NO：16： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：16：Leu Leu Gln Tyr Val Val Lys Ser Phe Asp Arg Ser Thr Lys1 5 10 ( 2 ) SEQ ID NO：17： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：17：Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu Tyr1 5 10 ( 2 ) SEQ ID NO：18： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：18：Leu Glu Tyr Val Thr Leu Lys Lys Met Arg Glu Ile Ile Gly1 5 10 ( 2 ) SEQ ID NO：19： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：19：Asn Met Tyr Ala Met Met Ile Ala Arg Phe Lys Met Phe Pro1 5 10 ( 2 ) SEQ ID NO：20： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：20：Lys Ile Trp Met His Val Asp Ala Ala Trp Gly Gly Gly Leu1 5 10 ( 2 ) SEQ ID NO：21： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：21：Trp Gly Gly Gly Leu Leu Met Ser Arg Lys His Lys Trp Lys1 5 10 ( 2 ) SEQ ID NO：22： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：22：Glu Gly Tyr Glu Met Val Phe Asp Gly Lys Pro Gln His Thr1 5 l0 ( 2 ) SEQ ID NO：23： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：23：Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu Asp Met Val Gly1 5 10 ( 2 ) SEQ ID NO：24： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：24：Trp Leu Thr Ser Thr Ala Asn Thr Asn Met Phe Thr Tyr Glu1 5 10 ( 2 ) SEQ ID NO：25： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：25：Thr Ala Asn Thr Asn Met Phe Thr Tyr Glu Ile Ala Pro Val1 5 10 ( 2 ) SEQ ID NO：26： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：26：Leu Val Ser Ala Thr Ala Gly Thr Thr Val Tyr Gly Ala Phe1 5 10 ( 2 ) SEQ ID NO：27： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：27：Tyr Ile Pro Pro Ser Leu Arg Thr Leu Glu Asp Asn Glu Glu1 5 10 ( 2 ) SEQ ID NO：28： ( i ) ( A ) ：14 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：28：Val Ile Ser Asn Pro Ala Ala Thr His Gln Asp Ile Asp Phe1 5 10 ( 2 ) SEQ ID NO：29： ( i ) ( A ) ：25 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：SEQ ID NO：29：Gly Met Ala Ala Leu Pro Arg Leu Ile Ala Phe Thr Ser Glu His Ser His1 5 10 15Phe Ser Leu Lys Lys Gly Ala Ala

The information of 20 25 (2) SEQ ID NO:30: (i) sequence signature: (A) length: 20 aminoacid (B) type: aminoacid (C) chain: (D) topological structure: line style (xi) sequence description: SEQ ID NO:30:Glu Arg Gly Lys Met Ile Pro Ser Asp Leu Glu Arg Arg Ile Leu Glu Ala1 5 10 15Lys Gln Lys

20

Claims (28)

1. produce the application of material of the body constitution of a kind of disease that is used for the diagnosis cell mediation or cell-mediated disease with the autoreactivity material, wherein this material is by intradermal administration, and the basis of diagnosis is positive reaction whether to occur at medicine-feeding part.

2. the application that is used to diagnose autoimmune disease or autoimmune disease body constitution as claimed in claim 1.

3. the application that is used for diagnosing tumour disease or tumor disease body constitution as claimed in claim 1.

4. as the application of claim 1 to 3,

Wherein

Nucleic acid, lipid, saccharide, protein, polypeptide and their complex, immunogenicity epi-position, segment, analog, plan shape thing or derivant and their mixture optionally are used as the autoreactivity material with a kind of carrier-bound form.

5. application as claimed in claim 4,

Wherein

Peptide, peptide derivant or peptide are intended the shape thing and optionally are used as the autoreactivity material with the form with MHC molecule or the formed complex of its peptide bound derivative.

6. as each the application in the claim 1 to 5, be used for diagnosing diabetes, multiple sclerosis, rheumatic arthritis, Basedow's disease, rheumatoid spondylitis, acute before ommochrome inflammation, Good-pasture syndrome, myasthenia gravis, systemic lupus erythematosus (sle), pemphigus vulgaris, immune thyroiditis, scleroderma, Crohn disease, Siogren syndrome, Lai Teer syndrome, enteritis or Graves disease.

7. be used for diagnosing the application of type i diabetes (IDDM) as claim 6.

8. application as claimed in claim 7,

Wherein

Glutamate decarboxylase (GAD) or its part are used as the autoreactivity material.

9. as the application of claim 7 or 8,

Wherein

People GAD 65kd is used as the autoreactivity material.

10. application as claimed in claim 7,

Wherein

One or more derive from the peptide of GAD or its peptide derivant or peptide intend the shape thing optionally with the form of MHC molecule or the formed complex of its peptide bound derivative as the autoreactivity material.

11. as the application of claim 10,

Wherein

Used peptide or peptide derivant, they comprise:

(a) aminoacid sequence (I)

D-V-N-Y-A-F-L-H-A-T-D-L-L-P-A-C-D-G-E-R，

(b) aminoacid sequence (II)

S-N-M-Y-A-M-M-I-A-R-F-K-M-F-P-E-V-K-E-K，

(c) aminoacid sequence (III)

N-W-E-L-A-D-Q-P-Q-N-L-E-E-I-L-M-H-C-Q-T，

(d) aminoacid sequence (IV)

T-L-K-Y-A-I-K-T-G-H-P-R-Y-F-N-Q-L-S-T-G，

(e) aminoacid sequence (V)

P-R-Y-F-N-Q-L-S-T-G-L-D-M-V-G-L-A-A-D-W，

(f) aminoacid sequence (VI)

T-Y-E-I-A-P-V-F-V-L-L-E-Y-V-T-L-K-K-M-R，

(g) aminoacid sequence (VII)

F-F-R-M-V-I-S-N-P-A-A-T-H-Q-D-I-D-F-L-I，

(h) aminoacid sequence (VIII)

G-M-A-A-L-P-R-L-I-A-F-T-S-E-H-S-H-F-S-L-K-K-G-A-A，

(i) aminoacid sequence (IX)

E-R-G-K-M-I-P-S-D-L-E-R-R-I-L-E-A-K-Q-K，

(j) be shown in a kind of in the aminoacid sequence of Fig. 1 or 2,

(k) length is at least the subregion of the aminoacid sequence in 6 amino acid whose being shown in (a) to (i), or/as

(l) be shown in aminoacid sequence in (a) to (j) and have essentially identical binding specificity to the MHC molecule or/and the aminoacid sequence of affinity.

12. as each application in the claim 5,10 or 11,

Wherein

The length of peptide or peptide derivant is at least 8 aminoacid.

13. as the application in the claim 12,

Wherein

The length of peptide or peptide derivant is at least 10 aminoacid.

14. as each application in the claim 5,10 or 11,

Wherein

The length of peptide or peptide derivant can reach 25 aminoacid.

15. each application in the claim as described above,

Wherein

The mensuration of positive reaction is constantly at least after 24 hours of administration.

16. as the application of claim 15,

Wherein,

Be engraved in during the mensuration of positive reaction within 24 hours to 1 week after the administration.

17. each application in the claim as described above,

Wherein

Positive reaction is measured by tuberosity occurs at medicine-feeding part.

18. as the application of claim 16,

Wherein

Positive reaction by to the visual observation of medicine-feeding part or/and the touch of relevant skin area is measured.

19. as the application of claim 16,

Wherein

Positive reaction is preferably by ultrasound wave or photographic means quantitative assay.

20. each application in the claim as described above,

Wherein

Positive reaction is measured by the material that soaks into cell release by detecting at medicine-feeding part.

21. as the application of claim 20,

Wherein

Detect cytokine.

22. each application in the claim as described above,

Wherein

With the positive or/and negative control carries out additional mensuration.

23. each application of claim as described above,

Wherein

Autoreactivity material and the together administration of a kind of adjuvant.

24. each application in the claim as described above,

Wherein administration is carried out in a table apparatus, and this device comprises at least one container, a container and a container that is used to store the negative control preparation that is used to store the positive control preparation of being used to store the preparation of autoreactivity material.

25. each application in the claim as described above, also comprise and determine or separate a species specificity autoreactive T cell subgroup, the tissue sample that wherein will derive from medicine-feeding part contacts with a kind of autoreactivity material, be identified with the T cell of autoreactivity substance reaction and by optionally with other T cell separation.

26. be used for the method for the body constitution of the disease of the mediation of diagnosis cell in vivo or cell-mediated disease, wherein the autoreactivity material is by intradermal administration, the basis of diagnosis is positive reaction whether to occur at medicine-feeding part.

27. be used for the disease of diagnosis cell mediation in vivo or the test kit of its body constitution, it comprises:

(a) at least a autoreactivity material that exists with the acceptable drug composition forms,

(b) the selectable at least a contrast material that exists with the acceptable drug composition forms,

(c) be used for the device of intradermal administration of autoreactivity material and contrast material, and

(d) be used for the option means that testing result is estimated on diagnostic ground.

28. as the test kit of claim 27,

Wherein

The compositions that is used for autoreactivity material and contrast material contains adjuvant.

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