CN1154700A

CN1154700A - Novel glyceroglycophospholipid, antidody there against, and method of detecting mhcoplasma

Info

Publication number: CN1154700A
Application number: CN 95194470
Authority: CN
Inventors: 松田和洋; 山本直树
Original assignee: Seikagaku Corp
Current assignee: Seikagaku Corp
Priority date: 1994-06-03
Filing date: 1995-06-02
Publication date: 1997-07-16

Abstract

Mouse is immunized with an antigen of a lipid fraction originating from Mycoplasma fermentans. Its spleen cells are fused with mouse myeloma cells to prepare hybridomas. A hybridoma is selected, which produces a monoclonal antibody having reaction specificity to GGPL-III that is a phosphocholine-containing glycoglycerolipid specific to Mycoplasma fermentans.

Description

The detection method of glycerin glycophospholipin and antibody thereof and mycoplasma

Technical field

The present invention relates to from mycoplasma fermentans the glycerin glycophospholipin, specifically be present in mycoplasma fermentans for the antibody of GCPL-and use this antibody that GCPL-is carried out method for measuring and detect the method for mycoplasma fermentans.

Technical background

Present inventors are once from MT-4 cell (people's helper cell that HTLV-I (human T lymphocyte's taxis retrovirus I) infects (.Gann such as Miyoshi; 71; 155-156 (1980))) find 5 kinds of GCPL-(glyceroglycolipid that contains phosphorylcholine); wherein a kind is 6 '-O-phosphorylcholine-α-glucopyranosyl (1 '-3)-1; 2-diacyl-sn glycerine (the biochemical research of lipid, the 35th the volume, the 111st～114 page, 1993 years).

On the other hand, report that mycoplasma fermentans (Mycoplasma fermentans) is a human body acquired immunodeficiency disease (AIDS; The factor (Lo. S. ,-C etc., 1991, science 251:1074-1076 are disliked in increasing AIDS); United States Patent (USP) the 5th, 242, No. 820) or rheumatoid reason (Williams, M.H. etc., 1970, lancet ii:277-280).

In the past, the known antibody that each mycoplasma species is arranged, and be applied to clinical examination, but be the antibody at mycoplasma pneumoniae (M.Pneumoniae) or mycoplasma genitalium (M.genitalium) mostly.In addition, monoclonal antibody for these mycoplasmas is also arranged, but it is generally acknowledged that these antibody are to discern the protein of mycoplasma or the antibody of protein and lipid simultaneously, no matter any mycoplasma fermentans is not shown that (spy opens clear 63-298, spy and opens that clear 63-184064, spy open clear 63-32496, the spy opens flat 5-304990, No. the 5th, 158,870, United States Patent (USP), United States Patent (USP) the 4th specificity, 945, No. the 5th, 242,820, No. 041, United States Patent (USP)).In addition, United States Patent (USP) the 5th, 242 discloses for mycoplasma fermentans for No. 820 and to have shown specific antibody, but this is the antibody that the full extract with thalline obtains as immunogen, can think the antibody of identification of protein.So, it is generally acknowledged that this antibody carries out the possibility height of non-specific combination, can not expect highly sensitive if when using this antibody test to contain mycoplasma in the body fluid such as serum of range protein.In addition, also to fully take into account antigen when being protein, because the variation of proteinic aminoacid sequence and might lose antigenicity fully.

Known to present inventors, also do not contain the report of the GCPL-of phosphorylcholine about the mycoplasma tool.Nor know that GCPL-matter from mycoplasma as immunogen, obtains this GCPL-is shown specific antibody example.And then, also be entirely ignorant of for this GCPL-and show specific antibody, can demonstrate high specific for mycoplasma fermentans.

Disclosure of an invention

Can quicken the report (1991 of human body acquired immunodeficiency virus infection person's the AIDS state of an illness relevant for mycoplasma infection, science 251:4991), as mentioned above, it is the report of disliking the factor that increases of AIDS that so-called mycoplasma fermentans is also arranged, but also do not have and correctly to detect in organism, the dynamic means of the growth and decline of this mycoplasma fermentans, thus wish that the antibody that can detect biological intravital mycoplasma fermentans on immunology can be arranged.

The present invention sets about from above-mentioned viewpoint exactly, to being present in resolving of GCPL-matter in the mycoplasma fermentans specifically, develop the antibody for this GCPL-, the method that the assay method of the GCPL-of using this antibody finally is provided and detects mycoplasma fermentans is as problem and unfolded.

The present inventor is in order to find out owing to retroviral infection causes bacterial anomaly hyperplasia, cytoclasis and dysimmunity, in the process of resolving these cells infected lipids, confirmed that unexpectedly the GCPL-of extracting from the strain of human body helper cell (the glycerose lipid that contains phosphorylcholine) is exactly GCPL-(glyceroglycolipid that contains phosphorylcholine: be designated hereinafter simply as " GCPL-") from mycoplasma fermentans and the structure that parses this lipid.And then, when confirming the specificity for each mycoplasma species, find that this antibody discerns mycoplasma fermentans specifically, thereby realized purpose of the present invention at the antibody of making for this lipid.

That is, the of the present invention extraction from mycoplasma fermentans obtains GCPL-and has following character:

(A) with orcinol reagent, special horse reagent, Dragendorff's reagent and ninhydrin reaction.

(B) available bases is decomposed.

(C) in by anion ion exchange body classification, obtain as non-absorption fraction with diethylin ethyl.

(D) molecular weight of measuring by mass spectrograph is 1048+28n (n is-1,0,1 or 2).

Above-mentioned GCPL-may be that this possibility that the phosphoric acid ester of 2-diacyl-sn-glycerine, phosphorylcholine and amino-propanediol makes constituent is very high with α-glucopyranosyl-(1 '-3)-1.

In addition, the present application is to contain phosphorylcholine, glucose, lipid acid and glycerine at least, anion ion exchange body with diethylin ethyl is the lipid of non-adsorptivity, is the anti-GCPL-matter antibody that has atopic for alkali labile GCPL-.This GCPL-can be for example to be present in the GCPL-of mycoplasma fermentans specifically.

Specific form as above-mentioned anti-GCPL-antibody; the present application provides can be with 6 '-O-phosphorylcholine-α-glucopyranosyl-(1 '-3)-1; 2-diacyl-sn-glycerine and have following character; extract both anti-GCPL-polyclonal antibodies of reacting of the GCPL-obtain and for the GCPL-matter that following character is arranged, have the anti-GCPL-matter monoclonal antibody of atopic from mycoplasma fermentans:

(B) available bases is decomposed.

In addition, the present application provides the GCPL-assay method, it is characterized in that using above-mentioned anti-GCPL-antibody, with the GCPL-that has above-mentioned character in the determination of immunological methods sample; The method that detects of mycoplasma fermentans also is provided, has it is characterized in that measuring GCPL-in the sample with this method, whether this GCPL-is existed or its amount and sample in mycoplasma fermentans whether exist or its amount is linked to each other.

And then, the invention provides GCPL-and/or with the similar substance-measuring method of the antigenicity of this GCPL-matter, it is characterized in that using above-mentioned anti-GCPL-antibody mediated immunity ground to measure in the sample and have the GCPL-of above-mentioned (A)～(D) character and/or the analogue of similar this GCPL-of antigenicity.

In addition, the present application provides detection kit, it is the test kit by the GCPL-of mycoplasma fermentans in the immunological method detection sample or mycoplasma fermentans, it is characterized in that it contains above-mentioned anti-GCPL-antibody and with the GCPL-of the mycoplasma fermentans of mark substance mark or with the secondary antibodies of mark substance mark for the immune globulin antibody of immune animal.Above-mentioned immune animal sphaeroprotein is to use the immune animal animal in addition of the above-mentioned antibody of preparation to prepare.

And then, the present application provides the detection method of the relevant myelopathic disease that is selected from AIDS, ephritis and HTLV-1, it is characterized in that detecting and be contained in the blood, have GCPL-or the antigenicity and the similar material of this GCPL-matter of above-mentioned (A)～(D) character or have the antibody of atopic for these GCPL-.

In addition, in this specification sheets,, abbreviate " GCPL-" as with the special GCPL-that is present in mycoplasma fermentans.

Below, explain the present invention.

＜1〉GCPL-of the present invention

Antibody of the present invention is the antibody that has atopic for the GCPL-of mycoplasma fermentans (Mycoplasma fermentans), produces as antigen with this GCPL-.At first, the GCPL-to mycoplasma fermentans is illustrated.

In the past, known distinctive lipid, the discovery 5 kinds of GCPL-(GGPLs:GGPL-I, GGPL-II, GGPL-III, GGPL-IV, GGPL-V) from the human body helper cell that HTLV-I infects of in the retroviral infection cell, existing.Determined the structure (western field etc., Japanese agriculture chemistry meeting will, the 39th page of No. 3 (1994) " 1994 conferences lecture main idea collection " of the 68th volume) of GGPL-I wherein by present inventors.Except that GGPL-I, also confirmed the existence of GGPLs, but just do not known its rerum natura, structure etc.

Present inventors use HPTLC (high speed thin-layer chromatography) from the HTLV-I infection human body helper cell (MT-4 (GGPLT)) of affirmation with its iipidomic with MT-4 (GGPL-) generation of mycoplasma agent (Mc201 (big Japanese pharmacy (strain) system)) processing, isolate GGPLs, with orcinol (orcinol) reagent dyeing glycolipid, special horse (Dittmer) reagent dyeing of land used phosphatide the time, detect 2 bands of on T-4 (GGPL+), seeing and on MT-4 (GGPL-), not seeing, (Fig. 4).This 2 band, also can from add can see through micro-pore diameter and be MT-4 (GGPL+) cell culture supernatant of the strainer of 0.22 μ m after, from MT-4 (GGPL-) cell of cultivation, detect.As described in the following Examples, wherein a kind of is GGPL-I, and another kind is GGPL-III.

Also not having the Mycoplasma microorganism is with the report as the cellularity composition of the GCPL-that contains phosphorylcholine, it is generally acknowledged GGPLs from human body cell, but The above results shows it is from mycoplasma fermentans.Therefore, as long as can think the antibody that obtains having atopic, just can detect mycoplasma fermentans for above-mentioned GCPL-.

The GCPL-of mycoplasma fermentans can prepare from the culture of mycoplasma fermentans.Mycoplasma fermentans can obtain with the following method.(" the GGPL positive ") culturing cell of GGPLs will be confirmed to have, the culture supernatant of MT-4 cell [HTLV-I (T lymphocytes in human body taxis retrovirus 1 type) infect human body helper cell] for example, strainer with pore dimension 0.22 μ m filters, and can obtain mycoplasma fermentans in filtrate.In addition, also can use the type strain etc. of mycoplasma fermentans PG18 strain etc.

The mycoplasma fermentans that cultivations such as use PPLO broth (Difco Laboratories) nutrient agar obtain, after the bacterium colony separation, if in containing the liquid nutrient medium of the phenol red PPLO broth (ディ Off コラボラトリ-ズ system) of 10% (V/V) ox tire serum (FBS), 5% (W/V) yeast extract (Off ロ-ラボラトリ-ズ system), 1000 units/ml penicillin, 1% (w/v) dextrose, 0.002% (w/v) etc., cultivate, just can obtain the cultivation thalline of mycoplasma fermentans.Then, in the thalline of mycoplasma fermentans, add methyl alcohol, place a few hours after, add chloroform and carry out ultrasonication, and then, place a few hours after, use portable type tetrafluoroethylene homogenizer homogenize thalline, reclaim supernatant liquor, distill this supernatant liquor, can obtain lipid-soluble extract.The lipid-soluble extract that obtains like this contains GCPL-.

As described in embodiment, this lipid composition is coated on HPLC (high speed thin-layer chromatography) plate, use chloroform: methyl alcohol: the mixed solvent of 0.2% (w/v) calcium chloride water=50: 45: 10 (v/v/v) launches, when carrying out the analysis of phosphatide, (fat i, fat ii, fat iii, fat iv, fat v and fat is (Fig. 3) vi) can to detect 6 bands.Show that by the bands of a spectrum on the TLC wherein fat v and fat vi are equivalent to GGPL-I and GGPL-III respectively.In addition, by the result of FAB mass spectroscopy, can confirm that GGPL-I is identical with fat v, GGPL-III is identical with fat vi.

The GCPL-of mycoplasma fermentans also can be obtained by the MT-4 cell of the mycoplasma fermentans that infects.For example, with the RPMI-1640 culture medium culturing MT-4 cell that adds 10% (v/v) FCS (calf tire serum), for cell, use chloroform with PBS (phosphoric acid buffer normal saline solution) washing: the mixed solution 400ml of methyl alcohol (=2: 1,1: 1 or 1: 2), carry out lipid and extract.

Lipid-soluble extract with above-mentioned mycoplasma fermentans that obtains or MT-4 cell, use has the anionite-exchange resin of diethylin ethyl (DEAE) base, DEAE-sephadex A25 (Pharmacia Corporation's system) etc. for example, be divided into non-absorbed component (neutral component) and absorbed component (acidic components), obtain non-absorbed component.Should non-absorbed component join in the silica column, (concentration gradient of 83: 16: 0.5～20: 80: 8 (v/v) is washed, and GGPL-III can be separated with other phosphatide with chloroform/methanol/water.And then, with the concentration gradient wash-out of 1-propyl alcohol/ammonia/water (80: 5: 15～75: 5: 20), isolate GGPL-I.

Any of GGPL-I and GGPL-III, for orcinol reagent, special horse reagent, Dragendorff's reagent (Dragendorff) (dyeing choline) all be positive, can be handled by irenine and decompose in addition.And then GGPL-I is negative in ninhydrin reaction (dyeing is amino), but GGPL-III is positive.

To the physico-chemical analysis that purified GGPL-III carries out, its result is as follows.

1. infrared absorption spectrum

Detect and correspond respectively to-CH ₂And-CH ₃The absorption peak of base, hydroxyl, ester carbonyl group, phosphate, choline base and primary amino.

2. liquid 2 secondary ion mass analyses (LSIMS)

Detected result shows have 3 kinds of lipid acid at least in molecule.At least have 4 kinds of molecules in addition among the GGPL-III, wherein, the molecular weight of main component is 1048.And show the GGPL-III that also has molecular weight 1020,1076 and 1104.

3. tandem mass spectrum (MS/MS) is measured

Show and in molecule, have phosphorylcholine.The molecular weight of learning the GGPL-III main component in addition is 1048.

4. one-dimensional NMR hydrogen ( ¹H) spectrum

Detect the signal of glycerine, the signal of choline and the signal of glucose.And then, owing to predict and have amino-propanediol, thus with 3-aminopropane-1,2-glycol (1-aminopropane-2,3-glycol) and 2-aminopropane-1, the one-dimensional NMR hydrogen that the standard substance of 3-glycol obtain ( ¹H) spectrum and GGPL-III hydrogen spectrum compares, and the result has shown that GGPL-III contains 2-aminopropane-1 as constituent, the possibility of 3-glycol.

5. two-dimentional NMR hydrogen spectrum (PH-DQF-COSY method)

Confirmed proton signal from DG.Also confirmed the choline proton signal in addition.

6. two-dimentional ¹H- ³²The NMR spectrum of P

Be presented at and contain 2 P (phosphorus) among 1 molecule GGPL-III.Also infer the compound contain phosphorus in addition, be combined on 1 or 6 of glucosyl residue.6 possibility is higher in this position.

And prompting combines the structure of phosphorylcholine of phosphate on choline, infer on 6 of glucosyl residue, and at first in conjunction with the phosphoric acid ester of amino-propanediol, and then, on the phosphoric acid ester of this amino-propanediol, have structure in conjunction with phosphorylcholine.

From above result, GGPL-III is the glycerin glycophospholipin with following character.

(A) can with orcinol reagent, special horse reagent, Dragendorff's reagent and ninhydrin reagent reaction.

(B) can be decomposed by alkali.

(C) in the classification of the anion-exchange column of DEAE base, can be used as non-absorbed component and obtain.

(D) molecular weight of being measured by mass spectrograph is 1048+28n (n is-1,0,1 or 2).

And then The above results shows that GGPL-III is that the phosphoric acid ester of 2-diacyl-sn-glycerine, phosphorylcholine and amino-propanediol is as constituent with α-glucopyranosyl-(1 '-3)-1.The phosphoric acid ester of this amino-propanediol of deducibility is a 2-aminopropane-1, the phosphoric acid ester of 3-glycol, and its combining site is α-glucopyranosyl-(1 '-3)-1,6 of the glucosyl residue of 2-diacyl-Sn-glycerine ' position.And prompting is combined with phosphorylcholine at this 2-aminopropane-1 on the phosphoric acid ester of 3-glycol.In addition; the main component of deducibility GGPL-III has 2 palmitoyl as acyl group; its structural such as following (I) formula; be GGPL-I (6 '-O-phosphorylcholine-α-glucopyranosyl-(1 '-3)-1; 2-two palmitoyl-sn-glycerine) be inserted with 2-aminopropane-1 between glucosyl residue and the phosphorylcholine, the structure of the phosphoric acid ester of 3-glycol.

In addition; the GGPL-III molecule of other that the deducibility molecular weight is different; be α-glucopyranosyl-(1 '-3)-1, the acyl group different sorts material in 2-diacyl-sn-glycerine, for molecular weight 1020; have myristoyl and palmitoyl; for 1076, have palmitoyl and a stearyl-, for 1140; infer to have 2 stearyl-s or palmitoyl and eicosane acyl group, but details is not clear.

＜2〉preparation of antibody of the present invention

Antibody of the present invention can carry out animal immune from the GCPL-of mycoplasma fermentans as antigen, again from this animal separation of serum, or gets the antibody cell that this animal produces, and the cultivation that continues to go down to posterity is reclaimed from its culture and obtained.Below illustrate the preparation method of antibody of the present invention, but do not limit the present invention, as long as do antigen, even also can by the additive method preparation with the GCPL-of mycoplasma fermentans.

(1) Polyclonal Antibody Preparation

In lipid-soluble extract by the above-mentioned mycoplasma fermentans that obtains, add single phosphatide, Freund formula Freund's complete adjuvant and mineral oil, add the PBS (phosphoric acid buffer normal saline solution) that contains 0.1% (v/v) Tween 80 after the mixing again and carry out emulsification.

Then, the emulsification that obtains is carried out subcutaneous injection or injects the abdominal cavity animals such as small white mouse, white mouse, rabbit, cavy, sheep.Behind the initial immunity, after the 2nd～3 week, carry out supplementary immunization, can obtain the high antiserum(antisera) of tiring with well-established law.After final immune 1 week, take blood sample, separation of serum.This serum is heat-treated, make the complement inactivation after, use ammonium sulfate precipitation, use and the refining same procedure of antibody usually such as ion-exchange chromatography, obtain immunoglobulin components.Just after final immunity, preferably confirm the height of antibody titer in the blood by enzyme immunoassay etc.

As the above-mentioned antibody that obtains, the GCPL-for mycoplasma fermentans has atopic, not with other the GCPL-reaction of mycoplasma such as mycoplasma arthritidis and mycoplasma hominis.

If do not use the lipid-soluble extract of mycoplasma fermentans, and use purified GGPL-III, can obtain having the polyclonal antibody of atopic for GGPL-III.

(2) modulation of monoclonal antibody

Can make monoclonal antibody by this smooth method of triumphant happy Mill (nature, 495～492 pages, 1975).Promptly, with the mammiferous antibody produced cell and myelomatosis (myeloma) cytogamy that GCPL-are produced antibody, make the hybrid cell knurl, filter out the hybrid cell knurl that can produce purpose antibody, by cultivating this hybrid cell knurl, can in nutrient solution, obtain monoclonal antibody.Below, be illustrated respectively for each operation.

(i) preparation of immunity of animal and generation antibody cell

Generation is to the cell of GCPL-antibody, can be by use animals such as GCPL-, immune small white mouse, white mouse, rabbit, cavy, sheep, and can be from allotment acquisitions such as the spleen cell of this animal, lymphocytic nodal cell, peripheral bloods.Can use the method identical to carry out above-mentioned animal immune with GCPL-with (1).

In order to obtain having the monoclonal antibody of atopic for GGPL-III, also the GGPL-III of available purifying carries out animal immune, but also can use animal to produce the antibody cell by the immunity of GCPL-matter mixture, preparation hybrid cell knurl is selected to produce the monoclonal antibody strain with atopic for GGPL-III from the hybrid cell knurl that obtains.According to this method, do not need to obtain GGPL-III, as long as the detectable micro-GGPL-III of useful enzyme immunoassay is just passable for animal immune institute necessary amounts.

The (ii) preparation of hybrid cell knurl

Produce the antibody cell from gathering, carry out cytogamy with the myeloma cell with the animal of GCPL-immunity.Though be used for the cytogamy myeloma cell,, produce the used identical zooblast strain of animal of antibody cell but preferably use with preparation can utilize various mammiferous cell strains.In addition, in order to distinguish not fused cell and fused cell after cytogamy, myeloma cell strain preferably can not be survived with the myeloma cell of not merging, and just the hybrid cell knurl can be bred, and has the cell strain of mark.For example, the strain of 8-azaguanine patience is sarkine-guanine-phosphoribosyl transferase (HGPRT) defective type, and its nucleic acid is synthetic to be to rely on the synthetic path of de novo.The fused cell (hybrid cell knurl) of such myeloma cell and generation normal antibody cell is in the substratum that contains sarkine, aminopterin-induced syndrome, thymidine (HAT substratum), even because aminopterin-induced syndrome hinders the synthetic path of de novo, so owing to exist thymidine, sarkine to pass through from lymphocytic remedial loop nucleic acid, so can breed.In contrast, the myeloma cell of 8-azaguanine resistance, because aminopterin-induced syndrome hinders the synthetic path of de novo, thus can not nucleic acid and death, in addition, can not long-term cultivation as Normocellular generation antibody cell.Therefore,, generation antibody cell and myeloma cell can on the HAT substratum, breed, so can select fused cell (709 pages of science the 145th volumes, 1964) from non-fused cell because merging the hybrid cell knurl of generation.In addition, as the myeloma cell, using the cell strain of not secreting the inherent immunoglobulin (Ig) for obtaining purpose antibody from hybrid cell knurl culture supernatant easily, is ideal.

In order to obtain the cytogamy of hybrid cell knurl, can carry out for example following operation: from immune animal, extract spleen, be suspended in the PRMI1640 substratum, the modulation cell suspending liquid.Make this splenocyte and be in for example SP2/0 cell (azaguanine patience, the non-secretion of IgG: ATCC CRL-1581) of logarithmic phase white mouse myeloma cell, by about 10: 1～1: 1, mix, after the centrifugation, in precipitation, add molecular-weight average 1,000～6,000 polyoxyethylene glycol makes ultimate density reach 30～50%, carries out cytogamy.Do not add polyoxyethylene glycol, and in cell mixture, add electricimpulse, can merge yet.

Carry out the cell after the fusion treatment, can on RPMI1640 substratum that contains 10% (v/v) ox tire serum (FCS) etc., cultivate, be suspended in then on the selection substratum of HAT substratum etc., dispensing is cultivated on the minitype plate in 96 holes of tool, makes the growth of hybrid cell knurl.

(iii) screening produces GCPL-is had the hybrid cell knurl of the antibody of atopic

The above-mentioned hybrid cell knurl that obtains, owing to be the mixture that has produced corresponding to the hybrid cell knurl of the various monoclonal antibodies of a plurality of antigens or epitope, so, particularly select to produce the cell strain that has the monoclonal antibody of atopic for GGPL-III from wherein filtering out the monoclonal antibody that has atopic for GCPL-.In addition, with GGPL-III bonded monoclonal antibody in, preferably select the cell strain of monoclonal antibody that the strong epitope of antigenicity is produced.

Generation can be by selecting it to the cell strain of the monoclonal antibody of glycolipid matter as the enzyme immunoassay that antigen uses.Such method, just like the ELISA method, promptly be a kind of antigen to be carried out immobilization on minitype plate etc., add thereon and cultivate after hybrid cell knurl nutrient solution adds the 2nd antibody with marks such as enzyme, fluorescent substance, luminophores again, according to the bonded mark substance, detect the method for antibody.At this moment, also the antibody immobilization therein, can be added antigenic mark the 2nd antibody successively, cultivate.In addition, for the ELISA method, also to describe in detail below.

In the time can not obtaining purified GGPL-III, use high speed thin-layer chromatography (HPTLC) plate, the lipid composition of separate fermentation mycoplasma is on this plate, after adding 2 antibody and cultivation of hybrid cell knurl nutrient solution, mark successively, can detect mark substance bonded position.As long as this position is identical with the position of launching GGPL-III by HPTLC, just can think that the hybrid cell knurl has produced the monoclonal antibody for GGPL-III.As long as obtain monoclonal antibody, just can from lipid composition, be purified into GGPL-III by using affinity chromatography etc. for GGPL-III.

As long as confirm to contain the hybrid cell knurl of generation, just can from the hole of containing this hybrid cell knurl, filter out the purpose clone by limiting dilution assay etc. as the target monoclonal antibody.

As described later shown in the embodiment, the hybrid cell knurl strain that generation has the monoclonal antibody of atopic for GGPL-III that obtains like this, on May 24th, 1994, preserving number collection in life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce with FERMP-14324, May 26 nineteen ninety-five, according to budapest treaty, be transplanted on international preservation, its preserving number is FERMBP-5115.

(iv) MONOCLONAL ANTIBODIES SPECIFIC FOR

If in suitable substratum, cultivate the above-mentioned hybrid cell knurl that obtains, in this culture supernatant, can obtain monoclonal antibody of the present invention.And then, use well-established law, by the affinity chromatography of sulphur ammonium precipitation, ion exchange chromatography, albumin A or Protein G or the immunosorption chromatogram of immobilized antigen etc., can make with extra care monoclonal antibody.

The monoclonal antibody that obtains like this; the glycolipid matter and the phospholipid that contain sialic acid-carrying glycolipid matter (Sphingolipids,sialo), platelet activation factor (1-alkyl-2-acetoglyceride-3-phosphorylcholine) or its part deacylation thing, Phosphorylcholine or its part deacylation thing or sphingophospholipid etc. with being present in the healthy human serum that does not infect mycoplasma fermentans do not show cross reaction.

Monoclonal antibody of the present invention also can directly be used, but also can use separated each moiety.When the antibody classification, because preserving the antigen-binding site (Fab) of antibody is the prerequisite of antigen and antibodies, so can use the proteolytic enzyme (for example Tryptase, stomach en-, papain) that does not decompose antigen-binding site to handle the component that contains antigen-binding site (Fab) that antibody obtains and then use this component.

In addition, as long as the base sequence of the gene of definite coding monoclonal antibody of the present invention or the aminoacid sequence of antibody just can carry out genetically engineered, make the fragment that contains antigen-binding site (Fab).

＜3〉GCPL-of the present invention and antibody thereof utilizes method

Antibody of the present invention is owing to be that distinctive GCPL-to mycoplasma fermentans has atopic, so available this antibody carries out immunology detection to the GCPL-in the sample.In detection,, just can measure GGPL-I and GGPL-III,, just optionally measure GGPL-III as long as use GGPL-III to have the antibody of atopic if use the antibody that has atopic for GGPL-I and GGPL-III both.When measuring GGPL-III, the above-mentioned GGPL-III that obtains uses as standard substance.In addition, as the GCPL-in the above-mentioned detected sample, the lipid composition that extracts from the sample of organism can be arranged.

As immunologic assay method, the common immunologic method that can make ELISA method, immunostaining etc. and use antibody.For example by sample liquid is contacted with the solid phase of anti-GCPL-antibodies, make the GCPL-and the above-mentioned antibodies that are contained in the sample liquid again, remove non-absorbed component from the solid phase separation, then, the GCPL-matter of the mycoplasma fermentans of applying marking material mark contacts with above-mentioned solid phase, make the reaction that is at war with of the GCPL-matter that is contained in the above-mentioned sample liquid and tokenized GCPL-matter, detect the mark substance that is combined in solid phase or be not combined in any in the mark substance of solid phase, can measure the GCPL-matter in the sample liquid.

In addition, by sample liquid is contacted on the solid phase of anti-GCPL-matter antibodies with GCPL-matter with the mark substance mark, and then the GCPL-matter that makes the GCPL-matter that is contained in the above-mentioned sample liquid and mark is at war with above-mentioned antibody and combines, be combined in the mark substance of solid phase or be not combined in any of mark substance by detection, can determine the GCPL-in the sample.At this moment, GCPL-without mark, and with unmarked standard GCPL-, GCPL-in the sample and standard GCPL-are at war with after the reaction, the tokenized anti-GCPL-antibody of mark substance is contacted with solid phase, also can detect the mark substance that is combined in solid phase or not be combined in mark substance any of solid phase.In addition when measuring, but the also secondary antibodies of applying markingization.

And then, the GCPL-in the sample liquid is combined on the solid phase, make mark anti-GCPL-antibody be in contact with it, also can detect any in the mark substance that is combined in the solid phase labelling material or is not combined in solid phase.

In addition, the standard GCPL-of making is combined in solid phase, and sample liquid and tokenized anti-GCPL-matter antibody are in contact with it, and also can detect the mark substance that is combined in solid phase or not be combined in any in the mark substance of solid phase.But also applying marking secondary antibodies at this moment.As the standard GCPL-,, just can determine the GGPL-III content in the sample as using purified GGPL-III.

And then, sample liquid is contacted with the solid phase of GCPL-antibodies, make the anti-GCPL-antibody and the above-mentioned antibodies that are contained in the sample again, remove non-adsorption component from the solid phase separation, then, anti-human immunoglobulin's antibody and the secondary antibodies with the mark substance mark are reacted,, determine the anti-GCPL-antibody in the sample by detecting mark substance.Because GCPL-matter of the present invention is that mycoplasma fermentans is peculiar,, learn the infection that has or not mycoplasma fermentans so can nonreactive GCPL-antibody be arranged by checking in the sample.

In addition, known have the whole bag of tricks to immunoassay, and any method all can be applicable to the present invention.

Except the method for using above-mentioned solid phase, also can adopt be used to measure haptens, antigenic immunoassay for example can adopt liquid phase method, this method is to make the reaction that is at war with of GCPL-in the sample and tokenized GCPL-and above-mentioned antibody, wait antigen and the free antigen that separates with antibodies as polyoxyethylene glycol, dextrose, secondary antibodies, detect the method for the mark substance of free labelled antigen then.

As above-mentioned solid phase, can enumerate common material with holes such as minitype plates such as agarose, latex particle, polystyrene, nylon and form (particle, micropartical, test tube, minitype plate, banding pattern etc.).After making antibody or GCPL-be combined on the solid phase, preferably use BSA (bovine serum albumin) and gelatin etc. to block.In addition, the material that serves as a mark can be enumerated peroxidase, alkaline phosphatase etc., can make the enzyme of pigment color development by enzyme reaction; Radio isotope; The fluorochrome of fluorescein isothiocyanate etc. etc.

Pigment is for peroxidase, can use 4-chloro-1-naphthols, O-Phenylene Diamine (OPD) or 3,3 '-diaminobenzidine etc., for alkaline phosphatase, use p-nitrophenyl phosphoric acid ester etc.Because mycoplasma fermentans has GCPL-of the present invention, so can use anti-GCPL-antibody of the present invention to detect mycoplasma fermentans.For example extract lipid composition from sample, this lipid-soluble extract is contacted with solid phase, the absorption lipid, with the solid state reaction of antibody of the present invention with the absorption lipid, at the same time or thereafter, again with can by the certification mark material, can detect the mycoplasma fermentans in the sample to the AIA reaction of this antibody response of mark substance mark.

In addition, because GCPL-of the present invention is that mycoplasma fermentans is peculiar, so use aforesaid method, measure the GCPL-in the sample, whether GCPL-existed or its amount and sample in mycoplasma fermentans whether exist or its amount connects, also can detect mycoplasma fermentans.

In the detection method of the assay method of above-mentioned GCPL-or mycoplasma fermentans,, can be blood, serum, blood plasma, celiolymph, urine, synovial fluid or cell culture fluid (supernatant) etc. as sample.

Except aforesaid method, also can pass through bio-tissue or cell, after fix processing directly or with GCPL-, make itself and the anti-GCPL-antibody response of using the marker mark, tokenized antibody is combined with bio-tissue or the cell that mycoplasma fermentans infects, by measuring mark substance, also can detect mycoplasma fermentans.As the fixing method of handling, can enumerate the method for for example using formaldehyde, glutaraldehyde, Paraformaldehyde 96 etc.In addition, the anti-GCPL-of applying marking substance markers not, also can make unmarked anti-GCPL-antibody and fixing bio-tissue or the cell response of handling, at the same time or thereafter, the secondary antibodies at the immunoglobulin (Ig) of above-mentioned immune animal of applying marking substance markers (this immune globulin antibody is to use animal preparation beyond the above-mentioned antibody mediated immunity animal of preparation) is reacted, again with mark secondary antibodies and the mycoplasma fermentans bio-tissue or the cell that infect combine, measure mark substance then and carry out.

When GCPL-in the employing immunological method detection test sample or mycoplasma fermentans, contain if be prepared in advance that GCPL-for mycoplasma fermentans has the antibody of atopic and with the test kit of mark substance mark at the secondary antibodies of the immune globulin antibody of above-mentioned immune animal, (above-mentioned immune globulin antibody is to use the animal beyond the above-mentioned antibody mediated immunity animal of preparation to prepare) just can be measured easily.

As concrete test kit example, the test kit that can form with damping fluid by the blocking-up reagent of minitype plate, BSA (bovine serum albumin) etc., the GCPL-(standard substance) of mycoplasma fermentans, antibody of the present invention, the anti-white mouse IgG antibody of peroxidase labelling, aquae hydrogenii dioxidi, OPD, washing.The GCPL-of antibody class, mycoplasma fermentans etc. are the lyophilize product preferably, or it is dissolved in the solvent that can stablize preservation.

The detection of mycoplasma fermentans can be applicable to following diagnosis.

1. the morbidity of retroviral infection disease prediction

Relevant for mycoplasma fermentans be the hyperplasia factor of AIDS (acquired character acquired immunodeficiency syndrome) report (Lo, S.-C. etc., 1993. microbiological researches 144,489-493).AIDS is that HIV (HIV (human immunodeficiency virus)) infects, also not necessarily morbidity, and common latent period is long, and it is relevant with the multiplicity of infection of mycoplasma fermentans to fall ill.Equally, can think that other retroviral infection disease morbidity also may be relevant with mycoplasma fermentans.

In the cell culture fluid of HIV latent infection, with the lipid concentration (ultimate density) of table 1 expression, add the lipid that extracts by each mycoplasma species, cultivate with the ruined cell count of light microscopy, be used as the index of the induction frequency of HIV.Its result is as shown in table 1.

Table 1

The kind of mycoplasma	Lipid concentration (μ g/ml)	Induced activity
The kind of mycoplasma	Lipid concentration (μ g/ml)	Induced activity	Mycoplasma fermentans mycoplasma hyorhinis Mycoplasma orale mycoplasma salivarium mycoplasma hominis mycoplasma arthritidis	????550 ????310 ????1090 ????1090 ????490 ????600	????+?+?+ ??????± ????????????- ??????- ??????+ ??????-

Show that by this result and report in the past mycoplasma fermentans has the activity of inducing retrovirus propagation.Therefore,, see the multiplicity of infection that has or not mycoplasma fermentans by inspections such as blood to the retroviral infection person, the prediction that can fall ill, and then take the treatment measures that suit.

2. rheumatismal diagnosis

Existing mycoplasma fermentans is the report (Williams, M.H. etc., lancet ii:277-280 (1970)) of rheumatismal reason.Therefore, the application of the invention antibody by detecting mycoplasma fermentans, might be diagnosed rheumatosis.

3. for the application of hitting the target therapy

With anti-HIV medicament such as nitrine deoxythymidine, dideoxyinosine or/and mycoplasma agent bonded antibody administration of the present invention in retroviral infection person and rheumatisant, can expect to be applied in the prevention or rheumatismal treatment of retrovirus morbidity.

4. use in treatment as neutralizing antibody

Antibody of the present invention with can expect after mycoplasma fermentans combines to have as stoping by in its infection and the activity of body, this antibody is injected in organism, can be used for the treatment and the prevention of the disease of the high virus infection of mycoplasma fermentans multiplicity of infection possibility.

Shown in the embodiment, think in the blood of nephritis patient that as described later the combination rate height of anti-GCPL-antibody of the present invention and lipid has shown and can carry out the diagnosis of ephritis by detecting this lipid.This lipid, the position from HPTLC can be distinguished with GGPL-III, but from combining with the monoclonal antibody of identification GGPL-III, can think aspect antigenicity to be and the similar lipid of GGPL-III.Therefore use the anti-GCPL-of the present invention can measure the material that antigenicity is similar to GGPL-III immunely.

The simple declaration of figure

Fig. 1 is the electrophoresis result figure of amplified production of the fs PCR of interval region between the 16S-23S ribosomal RNA gene of each mycoplasma species of expression.1. the mycoplasma fermentans GGPL strain 2. mycoplasma fermentans PG183. mycoplasma hyorhinis DBS10504. mycoplasma arginini G2305. Mycoplasma orale CH192996. mycoplasma salivarium PG207. from the MT-4 cellular segregation see through mycoplasma GTV-54-6A18. negative control (negative control)

Fig. 2 is the fragment electrophoresis result figure of expression with the amplified production of the subordinate phase PCR in the 16S-23S ribosomal RNA gene interbody spacer zone of various restriction enzyme treatment mycoplasma fermentanses.

1.VspI，??2.Hind?III??3.ClaI，??4.HincII，??5.HaeIII，

6. non-processor

Fig. 3 is TLC band shape (densitometry) figure of the expression phosphatide that is contained in mycoplasma fermentans lipid component.

Fig. 4 is the phospholipid of the expression lipid component that is contained in mycoplasma fermentans MT-4 cell that infect or non-infection and the TLC band chart of glycolipid matter.

PC is the expression Phosphorylcholine, and SPM represents sphingophospholipid,

Fig. 5 is the infrared absorpting light spectra of expression GGPL-III

Fig. 6 is the liquid 2 secondary ion mass spectrums of expression with (+) method of GGPL-III.

Fig. 7 is the liquid 2 secondary ion mass spectrums of expression with (-) method of GGPL-III.

Fig. 8 is the tandem mass spectrum figure of expression with (+) method of GGPL-III.

Fig. 9 is the tandem mass spectrum figure of expression with (-) method of GGPL-III.

Figure 10 be expression one-dimensional NMR hydrogen ( ¹H) spectrogram

Figure 11 is expression 3-ammonia its propane-1,2-glycol (1-aminopropane-2,3-glycol) (A) and 2-aminopropane-1, the one-dimensional NMR hydrogen of 3-glycol ( ¹H) spectrum (B) figure.

Figure 12 be expression with the two-dimentional NMR hydrogen of the PH-DQF-DOSY method of GGPL-III ( ¹H) figure.

Figure 13 be the expression GGPL-III two-dimentional NMR ( ¹H- ³¹P) spectrogram.

Figure 14 is TLC result's (land used special horse reagent dyeing) figure of the phosphatide of the expression lipid component that is contained in each mycoplasma species.1. mycoplasma fermentans GGPL strain 2. recessive mycoplasma fermentans ィ Application コゲニス 3. mycoplasma fermentans PG18 strains 4. mycoplasma fermentans F17 strains 5. mycoplasma fermentans F1 strains 6. mycoplasma fermentans F7 strains 7. mycoplasma arthritidises 8. mycoplasma hominis

Figure 15 is immunostaining is carried out in expression to the lipid component with HPTLC isolated M T-4 cell with the straight antibody of polyclone of embodiment 2 a photo as a result.

Figure 16 is the monoclonal antibody that embodiment 3 is used in expression, carries out with the lipid component of isolating each mycoplasma species of HPTLC photo as a result.(numeral is identical with Figure 14.)

Figure 17 is that monoclonal antibody of the present invention is used in expression, the MT-4 cell result's that the immunostaining mycoplasma fermentans infects photo.

Figure 18 is the monoclonal antibody that embodiment 3 is used in expression, and immunostaining is with the figure as a result of the lipid that extracts in isolating nephritis patient of HPTLC and the normal human blood." nephritis patient " is the lipid that expression is extracted from nephritis patient blood, and " normal individual " is the lipid result who extracts from normal human blood.

In order to implement top condition of the present invention

Below, further specifically describe the present invention with embodiment.

The separation and the structure elucidation thereof of embodiment 1 glycerin glycophospholipin

＜1〉preparation of the cultivation of mycoplasma fermentans and lipid

Strainer with pore dimension 0.22 μ m, filtration contain GGPLs the MT-4 cell (the human body helper cell strain that T lymphocytes in human body taxis retrovirus I (HTLV-I) infects (and culture supernatant, this filtrate is seeded on PPLO broth (the ディ Off コラボラトリ-ズ system) nutrient agar cultivates, isolate falls behind, and is containing 10% (v/v) ox tire serum (FBS), 5% (w/v) yeast extract (Off ロラボラトリ-ズ system), penicillin 1000 unit/ml, 1% (w/v) dextrose, cultivate in the liquid nutrient medium of the PPLO broth (ディ Off コラボラトリ-ズ system) that 0.002% (w/v) is phenol red.The bacterium colony that forms presents broken (Off ラィ De) egg shape, can be confirmed to be mycoplasma.

And then extract the DNA of above-mentioned mycoplasma, with 2 step PCR (Harasawa, R. etc., 1993 microbiological researches, 144:489-493) interval region to 16～23S ribosomal RNA gene increases, and with mycoplasma fermentans type strain PG18, mycoplasma hyorhinis (M.hyorhinis) DBS1050 (ATCC17981), mycoplasma arginini (M.arginini) G230 (ATCC23838), Mycoplasma orale (M.orale) CH19299, mycoplasma salivarium (M.salivarium) PG20 (ATCC23064), each bacterial classification that sees through mycoplasma (M.penetrans) GTU-54-6A1 compares.Its result, the 1st stage PCR product and the PG18 strain of the type strain of mycoplasma fermentans consistent (Fig. 1).And then, the amplified production that the PCR that uses this PCR product to carry out for the 2nd stage obtains can be cut off by VspI and HindIII, and ClaI, HincII and HaeIII can not cut off (Fig. 2), so this microorganism is confirmed to be mycoplasma fermentans, be named as GGPL strain (M.fermentans GGPL strain).

In the mycoplasma fermentans thalline 1g that cultivates with aforesaid method, add 100ml methyl alcohol, place a few hours after, add the chloroform of 200ml, carry out ultrasonication, and then place a few hours.With its with the homogenate of portable type tetrafluoroethylene homogenizer after, with 10,000rpm carries out centrifugal, reclaims its supernatant liquor with the suspension that obtains.By evaporating its supernatant liquor, obtain the lipid-soluble extract of 50mg.

＜2〉analysis of mycoplasma fermentans lipid composition

To be coated in by the lipid composition of the above-mentioned mycoplasma fermentans that obtains on HPTLC (high speed thin-layer chromatography) plate (Merck ﹠ Co., Inc.'s system), use chloroform: methyl alcohol: the mixed solvent of 0.2% (w/v) potassium chloride solution=50: 45: 10 (v/v/v) launches, and makes the special horse reagent dyeing of land used phosphatide.With TLC photodensitometer (the system CS910 of Shimadzu Seisakusho Ltd.), detect absorption at 580nm.Its result, (fat i, fat ii, fat iii, fat iv, fat v and fat is (Fig. 3) vi) to detect 6 bands.

On the other hand, with currently known methods (Bligh and Dyer, Canada's Physiology and biochemistry magazine, 37,911-917 (1959) extracts from the MT-4 cell of mycoplasma fermentans infection and with it and handles the lipid composition of the MT-4 cell that obtains with mycoplasma agent (MC201 (big Japanese pharmacy (strain) system)).Each lipid composition extract is coated on HPTLC (high speed thin-layer chromatography) plate (Merck ﹠ Co., Inc.'s system), use chloroform: methyl alcohol: the mixed solvent of 0.2% (v/v) calcium chloride water=50: 45: 10 (v/v/v) launches, use orcinol (orcinol) reagent dyeing glycolipid matter, make special horse reagent (Dittmer) reagent of land used, during the dyeing phospholipid, detect at MT-4 (GGPL+) and see, and 2 bands of not seeing at MT-4 (GGPL-) (Fig. 4).One in these bands is the GGPL-I that has known structure, and another is GGPL-III.In addition, the band that other special over the ground horse reagent is positive is GM2, GM1a, GD1a by identification, and the band that orcinol reagent is positive is Phosphorylcholine and lipid sphyngomyelin (Matsuda, Biochem.Biophys.Acta. such as K. by identification, 1168,123-129 (1993)).

In addition, after adding the culture supernatant that can see through the MT-4 cell that micro-pore diameter is the strainer of 0.22 μ m (mycoplasma fermentans infects), lipid composition from MT-4 (handling) cell extraction of cultivating with mycoplasma agent PC201, during with HPTLC analysis same as described above, detect above-mentioned 2 bands.Therefore, show these bands, promptly GGPL-I and GGPL-III are from mycoplasma fermentans.

These GGPL-I and GGPL-III from the mobility on TLC, are equivalent to fat v and fat vi from above-mentioned mycoplasma fermentans respectively.Result from the FAB spectrum can confirm that GGPL-I is identical with fat v in addition, and GGPL-III is identical with fat vi.

In the MT-4 cell 60ml (humid volume) that mycoplasma fermentans infects, add the chloroform of 400ml: methyl alcohol=2: 1,1: 1 and 1: 2, extract, obtain the TL of 993mg.With DEAE Sephadex A-25 post on the TL, be divided into non-absorbed component (neutral component) and absorbed component (acidic components) obtains non-absorbed component 775mg.On should non-absorbed component according in the Aunar post (according to the Aunar corporate system), with chloroform/methanol/water (83: 16: 0.5～20: 80: 8, v/v/v) concentration gradient wash-out 3 times, at last with 1-propyl alcohol/ammonia/water (80: 5: 15～75: 5: 20, v/v/v) concentration gradient stripping obtains GGPL-III3mg.

＜3〉structure elucidation of GGPL-III

GGPL-III for orcinol reagent, special horse reagent, Dragendorff's reagent be positive, in addition, decompose during with gentle alkaline purification.And then GGPL-I is negative for ninhydrin reaction, but GGPL-III is positive.Show that from these results GGPL-III is the glycophospholipin that contains choline.

And then method has as shown below been carried out the structure elucidation of GGPL-III.

(1) infrared absorption spectrum is measured

Infrared spectrophotometer (the FTIR-8100M: Shimadzu Seisakusho Ltd.'s system) infrared absorption spectrum of mensuration GGPL-III of infra-red microscope (IMS-8000, Shimadzu Seisakusho Ltd.'s system) is equipped with in use.Its result as shown in Figure 5.

Its result detects and corresponds respectively to-CH ₂And-CH ₃The base (2957,2920,2852,1467,1419,1378cm ^-1), hydroxyl (3271cm ^-1), ester carbonyl group (1740,1165cm ^-1), phosphate (1091cm ^-1), choline base (970cm ^-1), and primary amino (1560-1670cm ^-1) absorption.

(2) liquid 2 secondary ion mass analyses (LSIMS)

The above-mentioned pure about 1 μ g of GGPL-III is dissolved in chloroform: methyl alcohol (1 volume: among the mixing solutions 1 μ l 1 volume), when (+) method, add 0.5mL 3-nitrobenzyl alcohol; When (-) method, add the 0.5ml triethylamine and mix as matrix., use TSQ 70 3 to connect four layers of polar form mass analyzer (Finnegan MAT system) and carry out liquid 2 secondary ion mass analyses as sample with these mixing solutionss.Use the cesium ion (Cs that accelerates to 20KeV as primary ions stream ⁺).Speed with 250 atomic mass units (amu)/sec obtains spectrum.The spectrum of (+) method as shown in Figure 6, the spectrum of (-) method is as shown in Figure 7.

Its result confirms M/Z1021, M/Z1049 and M/Z1077 ion with (+) method.Prompting thus, lipid acid is formed different GGPL-III molecules and is had 3 kinds at least.In addition, its conclusion, the main component of GGPL-III is the peak of M/Z1049, its molecular weight is to deduct 1048 of protonatomic mass 1 from 1049.The molecular weight of other compositions equally also is 1020 and 1076.

In addition,, can confirm M/Z1047, M/Z1076 and M/Z1103 ion, merge, illustrate that the GGPL-III molecule exists 4 kinds of lipid acid to form different molecules at least as if spectrum resolution with (+) method for (-) method.Its conclusion, the main component of the GGPL-III of (-) method is the peak of M/Z1047, its molecular weight is to add 1048 of protonatomic mass 1 on 1047.The molecular weight of other compositions equally also is 1104.The molecular weight of inferring from the M/Z1076 ion is 1077, but is 1076 from the molecular weight that the result of (+) method infers since with 2 methylene radical that are equivalent to of the difference of the molecular weight of other compositions, so be 1076.

(3) tandem mass spectrum analysis (MS/MS)

Will with the sample of the identical preparation of sample of LSIMS (with the sample of (+) method and with the sample of (-) method), use TSQ70 three to connect four layers of utmost point shape spectrometry mass (Finnegan MAT system) and carry out tandem mass spectrum and measure.As primary ions stream, use the cesium ion (Cs that accelerates to 20KeV ⁺), as CAD (low energy conflict sensitization cracking) gas, use the argon gas that maintains 0.26 pascal (2.0m Torr).Speed with 250 atomic mass units (amu)/sec obtains spectrum.The spectrum of (+) method as shown in Figure 8, the spectrum of (-) method is as shown in Figure 9.

Its result for (+) method, can confirm M/Z184 ion and M/Z1049 ion.Pointed out ion, had phosphorylcholine in the molecule of GGPL-III from M/Z184.In addition, reach a conclusion, the molecular weight of GGPL-III is to deduct 1048 of protonatomic mass 1 from 1049.For (-) liquid, can confirm the M/Z1047 ion.Reach a conclusion thus, the molecule of GGPL-III is to add 1048 of protonatomic mass 1 on 1047.In addition, among the multiple GGPL-III by the LSIMS prompting, the molecule of molecular weight 1048 is main components.

(4) one dimension ¹H NMR spectrum

Will be with diazonium (H) metathetical GGPL-III (500 μ g), Phosphorylcholine (2mg), D-glucose 6-phosphoric acid 2 sodium salts (east yeast system) (about 200 μ g), be dissolved in respectively 0.5mL ( ²H) dimethyl sulfoxide (DMSO) ((C ²H ₃) ₂SO: ²H ₂Use GX-400 spectrograph (NEC system) among the O (98 volumes: 2 volumes)), under 60 ℃, obtain under the 400MHz ¹The H-NMR spectrum.As standard model is tetramethylsilane.

Integrated value with the signal of glucose (GlcH1) is carried out stdn as 1.00, quantizes other strength of signal.Its result as shown in figure 10.

Its result locates to detect the signal of glucose (Gro) at 4.319ppm (GroH1b), 4.145ppm (GroH1a), 5.118ppm (GroH2), 3.677ppm (GroH3b), 3.562ppm (GroH3a).In addition, the signal of choline is at 4.074ppm (POCH ₂-), 3.529ppm (CH ₂N ≡), 3.139ppm (N (CH ₃) ₃) locate detected.In addition, use 3-aminopropane-1,2-glycol (1-aminopropane-2,3-glycol) and 2-aminopropane-1, the standard substance of 3-glycol are with the one dimension that obtains with aforesaid method respectively ¹HNMR spectrum (being respectively Figure 11 A, Figure 11 B) and the spectrum of GGPL-III compare the result, contain 2-aminopropane-1 among the GGPL-III, the possibility height of 3-glycol.And then, as the signal of glucose (Glc), detect 4.647ppm (GlcH1) significantly.

(5) two dimension ¹H NMR spectrum

Use and one dimension ¹The sample of the identical preparation of H NMR, with FX-400 spectrometer (NEC system), under 60 ℃, with 400MHz, it is wide that each ties up 2000Hz, uses the two dimension of PH-DQF-COSY method ¹H NMR analyzes.Its result as shown in figure 12.

Its result can confirm from the proton signal of diacyl propylene glycol (GroH2/H1b, GroH2/H1a, GroH2/H3b, GroH2/H3a among Figure 12).In addition, can confirm choline (among the figure ,-CH ₂-N-/-CH ₂OP-) proton signal.

(6) two dimension ¹H～ ³¹P NMR spectrum

Use and one dimension ¹The sample of the identical preparation of H NMR with FX-400 spectrometer (NEC system), under 60 ℃, obtains two dimension with 400MHz ¹H- ³¹P HMQC spectrum.Its result as shown in figure 13.

Its as a result one dimension ( ¹H NMR) can obtain with above-mentioned ¹The spectrum that H NMR is identical, and two dimension ( ³¹P NMR) can obtain 2 barss.This is illustrated among the GGPL-III of 1 molecule and contains 2 P (phosphorus).

In addition, in one dimension, because near the position (2.9～3.0ppm) of 2,3,4,5 the proton signal that glucosyl residue occurs with the peak of intersection in two dimension is tested, occurs not detecting on the position (0.9ppm and 1.1ppm) of phosphorus signal, so deducibility phosphorated compound is combined on 1 or 6 of glucosyl residue.And then the signal that detects the phosphorus of near the proton signal (unidimensional is the 4.0ppm) of the 6a position of glucosyl residue and two dimension respectively (0.9ppm) and the peak of the signal cross of the signal (0.9ppm and 1.1ppm) of near the proton signal (unidimensional is 3.6ppm) of the 6b position of glucosyl residue and two-dimentional phosphorus.

In addition, (intersection peak 0.9ppm) is so can find phosphorylcholine structure in conjunction with phosphate on choline owing to detect the signal of unidimensional choline signal (about 4.05～about 4.1ppm) and two-dimentional phosphorus.And then, because near the proton signal (first yuan the 4.0ppm) of the 6a position of the intersection peak glucosyl residue of this phosphorylcholine staggers, so deducibility on 6 of glucosyl residue at first in conjunction with the nitric ether of amino-propanediol, and then on the nitric ether of this amino-propanediol in conjunction with the structure of phosphorylcholine.

Conclude above result, GGPL-III is the new GCPL-with following character.

(B) available bases is decomposed.

(C) can in the classification of anion-exchange column, obtain as non-absorbed component with DEAE base.

And then having pointed out GGPL-III by The above results is that the phosphoric acid ester of 2-diacyl-sn-glycerine, phosphorylcholine and amino-propanediol is as constituent with α-glucopyranosyl (1 '-3)-1.The phosphoric acid ester of this amino-propanediol is a 2-aminopropane-1, the phosphoric acid ester of 3-glycol, and its combining site of deducibility is α-pyranoglucose-(1 '-3)-1,6 of the glucose residue of 2-diacyl-sn-glycerine ' position.And then, the prompting at this 2-aminopropane-1, on the phosphoric acid ester of 3-glycol in conjunction with phosphorylcholine.

Each GGPL-III molecule that molecular weight is different; it is α-pyranoglucose-(1 '-3) 1; the molecule that has different types of acyl group in 2-diacyl-Sn-glycerine, deducibility all is a hexadecanoyl at the acyl group of main component (molecular weight 1048), total deduction structure is shown in above-mentioned (I) formula.In addition; GGPL-III molecule with other molecular weight; the length difference of acyl group; for molecular weight 1020; deducibility has tetradecanoyl and hexadecanoyl; for 1076, have hexadecanoyl and stearyl-, for 1104, have 2 stearyl-s or hexadecanoyl and an eicosane acyl group.But details is unclear.

＜4〉parsing of the lipid composition of each mycoplasma species

Containing 10% (v/v) FBS (ox tire serum), 5% (w/v) yeast extract (Off ロラボラトリ-ズ system), 100 unit/ml penicillin, 1% (w/v) dextrose, cultivate (the mycoplasma fermentans GGPL strain of each mycoplasma species in the liquid nutrient medium of the PPLO broth (ディ Off コラボラトリ-ズ system) that 0.002% (w/v) is phenol red, recessive mycoplasma fermentans (incognitus) (ATCC53949), mycoplasma fermentans PG18 strain, mycoplasma fermentans F17 strain, mycoplasma fermentans F1 strain, mycoplasma fermentans F7 strain, mycoplasma arthritidis (M.arthritidis), mycoplasma hominis (M.hominis) (ATCC15488)).From this nutrient solution, with currently known methods (Bligh and Dyer, Canada's Physiology and biochemistry magazine, 37,911-917 (1959) extracts lipid, this lipid-soluble extract is coated on the HPTLC plate (Merck ﹠ Co., Inc.'s system), uses chloroform: methyl alcohol: the mixed solvent of 0.2% (w/v) calcium chloride water=50: 45: 10 (v/v/v) launches.This plate is immersed in is dissolved with in the 0.4% poly-isobutyl-methacrylic acid salts solution dipping time 30 seconds in the hexane.With this plate, according to well-established law, the special horse reagent dyeing of land used.Its result as shown in figure 14.By this result,, on other mycoplasma bacterial classification, do not see just to seeing the band that is equivalent to GGPL-I and GGPL-III on the various mycoplasma fermentanses.Conclusion thus, GGPL-I and GGPL-III are the GCPL-of feature for mycoplasma fermentans.

Embodiment 2 preparation mycoplasma glycolipid polyclonal antibodies

In the lipid-soluble extract 50mg of the above-mentioned mycoplasma fermentans that obtains, add monophosphate lipoid A (リ PVC ィ system ノケ system リサ-チ company) 50 μ g and Freund's complete adjuvant (Na カラィチス Network (strain)) 50 μ g, add mineral oil 250 μ l again, ground 2 minutes with 400rpm.Thereafter the PBS that contains 0.1% (v/v) Tween 80 that adds 250 μ l ground 2 minutes with 400rpm again, obtained containing the emulsion 0.5ml of lipid-soluble extract.

Will be with the female BALB/c white mouse in 7 ages in week of the emulsion of method for preparing injection subcutaneous, each only injects 0.5ml.Behind the initial immunity the 2nd week and the 3rd is when all, will be with the synthetic emulsion that contains lipid-soluble extract of aforesaid method, and every is injected at intraperitoneal with 0.5ml.

Use well-established law, separation of serum from the white mouse blood of above-mentioned immunity.Use this serum, carry out following immunostaining.From GGPL male MT-4 cell, with currently known methods (Bligh and Dyer, Canada's Physiology and biochemistry magazine, 37,911-917 (1959)) extracts lipid composition, go up coating lipid fraction extract at high speed thin-layer chromatography (HPTLC) plate (Merck ﹠ Co., Inc.'s system), use chloroform: methyl alcohol: the mixed solvent of 0.2% (w/v) calcium chloride water=50: 45: 10 (v/v/v) launches.This plate is immersed in the hexane solution after 30 seconds that is dissolved with 0.4% poly-isobutyl-methacrylate, carries out air-dry.

As 1 antibody, above-mentioned serum or non-immune white mouse serum are added on the HPTLC plate, cultivated for 1 night down at 4 ℃.Behind the clean HPTLC plate of PBS, as 2 antibody, peroxidase mark rabbit IgG antibody (カペ Le corporate system) is added on the HPTLC plate, at room temperature, cultivated 4 hours.Behind the clean HPTLC plate of pure water, use antiperoxidase color development test kit (KONICA immunostaining HRPKit: the Konica corporate system) detect antigen zone.Its result as shown in figure 15.From this result, can confirm that above-mentioned serum contains the polyclonal antibody for GGPL-I and GGPL-III.

The anti-GGPL-III monoclonal antibody of embodiment 3 preparations

(1) preparation of hybrid cell knurl

With embodiment 2 in the same manner, will contain the lipid-soluble extract of mycoplasma fermentans, female BALB/c white mouse subcutaneous in 7 ages in week, every injection 0.5ml.In the 2nd week and the 3rd week behind initial immunity, will be expelled to intraperitoneal with every 0.5ml with the emulsion of the lipid-soluble extract of method for preparing.

After 4 days, the spleen of extraction white mouse is with RPMI 1640 medium preparation cell suspending liquids in final immunity.With this 2 * 10 ⁸The mouse myeloma SP2/0 cell of individual splenocyte and logarithmic phase (azaguanine patience, the non-secretion of IgG; ATCC CRL-1581) (2 * 10 ⁷Individual) mix, after the centrifugation, in precipitation, on one side slowly vibration added 45% polyoxyethylene glycol in 1 minute (PEG 4000 (with the pure medicine of light (strain) system) 1ml, then, under 37 ℃, cultivated 2 minutes on the one side that slowly vibrates on one side.Therein, add RPMI 1640 substratum 1ml in 1 minute after, slowly vibration adds 1ml in then same 1 minute again, slowly after the vibration, and then adds 8ml again in 3 minutes.

After above-mentioned cell mixture centrifugation, in the RPMI 1640 substratum 50ml that contain 10% N of tire serum (FCS), on 4 96 hole minitype plates, 100 μ l are respectively injected in each hole with cell suspension, in carbonic acid gas incubator (5% carbonic acid gas, 37 ℃), cultivate.After 24 hours, change substratum into HAT substratum (containing sarkine, aminopterin-induced syndrome, thymidine, 10% (v/v) FCS substratum), continue in carbonic acid gas incubator (5% carbonic anhydride, 37 ℃), to cultivate.Cultivate the 4th day after beginning in the HAT substratum, the new HAT substratum of 100 μ l is appended in per 1 hole.After the HAT culture medium culturing begins the 7th day changes HT substratum (removing aminopterin-induced syndrome from the HAT substratum) into, at its 2nd day, change RPMI 1640 substratum that contain 10% (v/v) FCS into after, confirm to have or not the bacterium colony form.

(2) selection of generation antibody hybrid cell knurl

After the lipid composition 20 μ g with mycoplasma fermentans of adding 50 μ l are dissolved in 10ml alcoholic acid solution in per 1 hole of 96 hole minitype plates, used moisture eliminator dry 30 minutes.In per 1 hole, add the PBS that contains 1% (w/v) bovine serum albumin (BSA) of 100 μ l, at room temperature left standstill 1 hour.After washing per 1 hole 5 times with the 0.3M sucrose solution of 100 μ l, in each hole, respectively add the above-mentioned culture supernatant of 100 μ l, at room temperature vibrated 60 minutes.Behind each hole of 0.05%Tween 20 solution washings, in each hole, add the anti-white mouse IgG antibody (カペ Le corporate system) of 50 μ l with the superoxide mark of 500 times of PBS dilutions, at room temperature vibrated 60 minutes.

Behind each hole of 0.05%Tween 20 solution washings, in per 1 hole, add the 10ml citrate buffer solution of 30% (v/v) aquae hydrogenii dioxidi that contains 10mg O-Phenylene Diamine and 50 μ l of 100ul, at room temperature cultivated 15 minutes.The 0.5M sulfuric acid that in per 1 hole, adds 100 μ l, the enzyme reaction of peroxidase is stopped after, measure the absorbancy of 490nm with the microplate reader.

For the hybrid cell knurl that produces antibody, pass through limiting dilution assay, screen repeatedly, be used for antigenic ELISA by lipid composition and carry out the screening first time mycoplasma fermentans, for ELISA male bacterium colony, further the immunostaining of the HPTLC of the lipid composition by mycoplasma fermentans carries out postsearch screening, obtains producing the hybrid cell knurl strain MF-III-1 strain that has the monoclonal antibody of atopic for GGPL-III.This strain in life engineering Industrial Technology Research Institute of Govement Industrial Research Inst., Ministry of Commerce with the preserving number preservation of FERM P-14324, in May 26 nineteen ninety-five according to budapest treaty, move to international preservation, deposit number is FERM BP-5115.

(3) MONOCLONAL ANTIBODIES SPECIFIC FOR

Then, in RPMI 1640 substratum that contain 10% (v/v) FCS, cultivate the MF-III-1 strain,, obtain monoclonal antibody for GGPL-III by reclaiming its culture supernatant.

The immunostaining evaluation of 4 pairs of monoclonal antibodies of embodiment

The anti-GGPL-III monoclonal antibody that use obtains is carried out following immunostaining.With currently known methods (Bligh and Dyer, Canada's Physiology and biochemistry magazine, 37,911-917 (1959) extracts (the mycoplasma fermentans GGPL strain of each mycoplasma species, recessive mycoplasma fermentans (incognitus) (ATCC 53949), mycoplasma fermentans PG18 strain, mycoplasma fermentans F17 strain, mycoplasma fermentans F1 strain, mycoplasma fermentans, the F7 strain, mycoplasma arthritidis (M.arthritidis), mycoplasma hominis (M.hominis) (ATCC 15488)) lipid composition, (Merck ﹠ Co., Inc.'s system) coating lipid composition extract on high speed thin-layer chromatography (HPTLC) plate, use chloroform: methyl alcohol: the mixed solvent of 0.2% (w/v) calcium chloride water=50: 45: 10 (v/v/v) launches.

With above-mentioned plate, be immersed in the hexane liquid that is dissolved with the poly-isobutyl-methacrylate of 0.4% (w/v) after 30 seconds, carry out air-dry.As 1 antibody, above-mentioned anti-GGPL-III monoclonal antibody is added on the HPTLC plate, cultivate a night down at 4 ℃.Behind PBS washing HPTLC plate, as 2 antibody, the anti-white mouse IgG of peroxidase labelling antibody (カペ Le corporate system) is added on the HPTLC plate, at room temperature cultivated 4 hours.Behind pure water washing HPTLC plate, with peroxidase color development test kit (KONICA immunostaining HRPKit: the Konica corporate system) detect antigen zone.Its result as shown in figure 16.

This result shows, monoclonal antibody of the present invention combines with the GCPL-of mycoplasma fermentans specifically, and do not combine with the phosphatide and the glycolipid of mycoplasma arthritidis and mycoplasma hominis.And then, on HPTLC, in special horse reagent of land used and the orcinol reagent colored zone, in immunostaining, do not see the band of GM2, GM1a, GDIa, phosphatidylcholine and lipid sphyngomyelin and the band of GGPL-I, monoclonal antibody of the present invention only combines specifically with GGPL-III.

Then, use anti-GGPL-III monoclonal antibody, use antibody as 1 antibody with fluorescein isothiocyanate (FITC) mark white mouse IgG as 2 antibody, the MT-4 cell that mycoplasma fermentans is infected carries out immunostaining, and the result of usefulness fluorescence microscope as shown in figure 17.

This result shows, uses monoclonal antibody of the present invention can detect mycoplasma fermentans.

GGPL-III in the blood of embodiment 5 nephritis patients or be similar to GGPL-

The detection of the antigenic material of III

From the blood of nephritis patient and normal people's blood, according to currently known methods (Bligh and Dyer, Canada's Physiology and biochemistry magazine, 37,911-917 (1959)), for the serum 100 μ l with well-established law preparation, use the chloroform of 100 μ l respectively: the mixed solvent of methyl alcohol (2: 1,1: 1 and 1: 2 (v/v)) extracts the TL in the serum.Extraction solvent is divided into 3 layers, reclaim orlop wherein, with its for water dialyse, freeze, drying, the sample that obtains like this is coated on high speed thin-layer chromatography (HPTLC) plate (Merck ﹠ Co., Inc.'s system), uses chloroform: methyl alcohol: the mixed solvent of 0.2% (w/v) calcium chloride water (50: 45: 10 (v/v/v)) launches.In addition,, use purified GGPL-III, be coated in equally on the HPTLC plate, launch as reference material.

Above-mentioned plate is immersed in the hexane solution after 30 seconds that is dissolved with the poly-isobutyl-methacrylate of 0.4% (w/v), air-dry.As 1 antibody, above-mentioned anti-GGPL-III monoclonal antibody is added on the HPTLC plate, cultivate a night down at 4 ℃.Behind PBS washing HPTLC plate, as 2 antibody, the anti-mouse IgG antibody of peroxidase labelling (カペ Le corporate system) is added on the HPTLC plate, at room temperature cultivated 4 hours.Behind pure water washing HPTLC plate, with peroxidase color development box (KONICA immunostaining HRPKit: the Konica corporate system) detect this antigen zone.

Use aforesaid method, 9 samples of serum of nephritis patient and 9 samples of serum of normal people are detected, in 9 samples of nephritis patient 6 samples are arranged, near GGPL-III, confirm to have signal (Figure 18).Sample for the normal people does not detect this signal (Figure 18).This signal of obviously seeing in the nephritis patient sample, the position from the HPTLC, different with GGPL-III, this can be considered to the similar lipid of antigenicity.

In addition, after extracting lipid like this, also can be with each lyophilize sample dissolution at chloroform: methyl alcohol: water be (in the mixed solvent of 83: 16: 0.5 (v/v/v), (with chloroform: methyl alcohol: the composition of water, the material that stage ground wash-out is gone out is added on the HPTLC to be added in ィァトロ PVC-ズ post that ィァトロ PVC-ズ 6RS-8060 (ィァトロ Application corporate system) is housed.

The refining GGPL-III of embodiment 6 usefulness detects blood as antigenic ELISA method

In anti-GGPL-III antibody

To be dissolved in solution in the ethanol of 10ml with embodiment 1 purified GGPL-III40 μ g, and join in the 96 hole microwell plates, per 1 hole adds 50 μ l, uses moisture eliminator then dry 30 minutes.The PBS that contains 1% (w/v) bovine serum albumin (BSA) that adds 100 μ l more at room temperature left standstill 1 hour in each hole.Each hole is after the 0.3M sucrose solution of 100 μ l washing 5 times, with from normal people, AIDS VICTIMS, ephritis (nephritis) patient, the relevant myelopathy (HAM of HTLV-I; HTLV-1 associated myelopathy) patient's blood joins in each hole with each 100 μ l of serum of well-established law preparation, at room temperature vibrates 60 minutes.With 0.05%Tween 20 solution.After washing each hole, the anti-human body IgG of the peroxidase labelling antibody (カペ Le corporate system) with 2000 times of PBS dilutions of 50 μ l is added in each hole, at room temperature vibrated 60 minutes.

Behind each hole of 0.05%Tween 20 solution washings, add the citrate buffer solution of 10ml of 30% (v/v) aqueous hydrogen peroxide solution that contains 10mg O-Phenylene Diamine and 50 μ l of 100 μ l in each hole, at room temperature cultivated 15 minutes.In each hole, add the 0.5M sulfuric acid of 100 μ l, the enzyme reaction of peroxidase is stopped after, measure the absorbancy of 450nm with the microplate reader.

For 46 samples of normal human serum, 10 samples of AIDS (AIDS) patients serum, 22 samples of ephritis (nephritis) patients serum, HTLV-I myelopathy (HAM; HTLV-1 associated myelopathy) 9 samples of patients serum, with the absorbancy of aforesaid method mensuration 450nm, with the normal people in contrast, serum sample that will be bigger than normal people 450nm absorbancy is as anti-GGPL-III antibody positive.Its result is as shown in table 2.

Table 2

The title of disease	Anti-GGPL-III antibody positive rate
The title of disease	Anti-GGPL-III antibody positive rate	Normal people AIDS ephritis HAM	????1/46(2.2％) ????8/10(80.0％) ????3/22(13.8％) ????2/9(22.2％)

As shown in table 2,2 samples (22.2%) in 3 samples (13.8%) in 22 samples of 8 samples (80.0%), nephritis patient in 1 sample (2.2%) in 46 samples of normal people, AIDS VICTIMS's 10 samples, HTLV-I myelopathy patient's 9 samples are the anti-GGPL-III antibody positives in each serum.In addition, replace 2 times above-mentioned antibody (the anti-people's of peroxidase labelling IgG antibody), and the IgM antibody (カペ Le corporate system) that uses the antiperoxidase labelling human is when preparing equally, and the anti-GGPL-III antibody positive rate in the nephritis patient serum further increases.

Usability on the industry

Can obtain glycerin glycophospholipin from mycoplasma fermentans, GGPL-III by the present invention. In addition, can obtain having for the GCPL-Ⅲ from mycoplasma fermentans the antibody of atopic, particularly for GGPL-I and GGPL-III have atopic polyclonal antibody, and only have the monoclonal antibody of atopic for GGPL-III.

The antibody of the application of the invention can be expected to detect specifically mycoplasma fermentans, the morbidity of the retroviral infection disease of prediction AIDS, and the application of the diagnosis of rheumatismal diagnosis, ephritis etc.

And then, can be applicable to treat or prevent the high viral infectious disease of mycoplasma fermentans MOI possibility.

Claims

1. the GCPL-that extracts from mycoplasma fermentans with following character:

(A) can with orcinol reagent, special horse reagent, De Leigendaolefu reagent and ninhydrin reaction;

(B) can be decomposed by alkali;

(C) when separating, obtain with non-adsorption stage branch form with diethylin ethyl anion-exchange column;

(D) molecular weight of measuring with mass spectrograph is 1048+28n (n is-1,0,1 or 2).

2. GCPL-according to claim 1, it is with α-glucopyranosyl-(1 '-3)-1, the phosphoric acid ester of 2-diacyl-sn-glycerine, phosphorylcholine and amino-propanediol is as constituent.

3. GCPL-is had the anti-GCPL-antibody of atopic, this GCPL-contains phosphorylcholine, glucose, lipid acid and glycerine at least, is non-adsorptivity lipid to diethylin ethyl ion exchange column, to the alkali instability.

4. anti-GCPL-antibody according to claim 3, wherein said GCPL-is the GCPL-that is present in specifically in the mycoplasma fermentans.

5. according to claim 3 or 4 described anti-GCPL-antibody, it and 6 '-O-phosphorylcholine-α-glucopyranosyl-(1 '-3)-1, the described GCPL-reaction of 2-diacyl-sn-glycerine and claim 1.

6. according to any one described anti-GCPL-antibody of claim 3-5, it is a polyclonal antibody.

7. according to claim 3 or 4 described anti-GCPL-antibody, it just has atopic to the described GCPL-of claim 1.

8. according to claim 3,4, any one described anti-GCPL-antibody of 5 or 7, it is monoclonal antibody or its fragment.

9. anti-GCPL-antibody according to claim 3, it discerns mycoplasma fermentans specifically, to other mycoplasma nonrecognition.

10. anti-GCPL-antibody according to claim 9, wherein said other mycoplasma is mycoplasma arthritidis and mycoplasma hominis.

11. anti-GCPL-antibody according to claim 4, it does not show cross reaction for containing sialic acid-carrying glycolipid (Sphingolipids,sialo), the biologically active pdgf factor (1-alkyl-2-acetoglyceride base-3-phosphorylcholine) or its part deacylation thing, phosphatidylcholine or its deacylation thing or lipid sphyngomyelin.

12. the measuring method of GCPL-is characterized in that using any one described anti-GCPL-antibody mediated immunity ground of claim 3-11 to measure the GCPL-that has following character in the sample:

It is to contain phosphorylcholine, glucose, lipid acid and glycerine at least, is non-adsorptivity lipid for diethylin ethyl anion-exchange column, to alkali labile GCPL-.

13. the measuring method of GCPL-antigenicity similar substance is characterized in that using any one described anti-GCPL-antibody mediated immunity ground of claim 3-11 to measure the antigenicity similar substance that is contained in the described GCPL-of claim 1 in the sample.

14. the measuring method of GCPL-is characterized in that using any one described anti-GCPL-antibody mediated immunity ground of claim 3-11 to measure and is present in GCPL-in the mycoplasma fermentans specifically in sample.

15. the measuring method of GCPL-in the sample, it is characterized in that making among sample liquid and the claim 3-11 any one described anti-GCPL-antibody institute bonded solid phase to contact, make the GCPL-and the above-mentioned antibodies that are contained in the sample liquid, remove non-adsorption component from the solid phase separation, then will contact with above-mentioned solid phase with the GCPL-of the mycoplasma fermentans of mark substance mark, make the GCPL-that is contained in the above-mentioned sample liquid and the GCPL-competing reaction of mark, detect and be combined in the mark substance on the solid phase or be not combined in the mark substance on the solid phase any one.

16. the measuring method of anti-GCPL-antibody in the sample, it is characterized in that making sample liquid to contact with the solid phase that combines the described anti-GCPL-of claim 1, the anti-GCPL-antibody that is contained in the sample liquid is combined with above-mentioned GCPL-, remove non-adsorption component from the solid phase separation, then react with secondary antibodies again, detect mark substance with anti-human immunoglobulin's antibody of mark substance mark.

17. the detection method of mycoplasma fermentans, it is characterized in that using claim 14 or 15 described assay methods to measure GCPL-in the samples, make this GCPL-existence whether or the existence of the mycoplasma fermentans in its amount and the sample whether or its amount connect.

18. the detection method of mycoplasma fermentans according to claim 17, the GCPL-in the wherein above-mentioned sample is the lipid composition that extracts from biological sample.

19. the detection method of mycoplasma fermentans according to claim 18, wherein biological sample is blood, serum, blood plasma, celiolymph, urine, synovial fluid or cell culture fluid.

20. the detection method of mycoplasma fermentans in the sample, it is characterized in that extracting lipid composition from sample, after making this lipid-soluble extract and solid phase contacting, the absorption lipid, solid phase and any one described anti-GCPL-antibody response of claim 3-11 of having adsorbed lipid, simultaneously or thereafter, with secondary antibodies reaction at above-mentioned immune animal globulin antibody with the mark substance mark, measure mark substance, wherein above-mentioned immune animal globulin antibody is to use the animal beyond the above-mentioned antibody of preparation to prepare.

21. the detection method of mycoplasma fermentans, it is characterized in that biological tissue or cell, directly or after the fixedly processing of implementing GCPL-, any one described anti-GCPL-antibody response of claim 3～11 with the mark substance mark, bonding mark antibody on biological tissue that mycoplasma fermentans infects or cell is measured mark substance.

22. the detection method of mycoplasma fermentans, it is characterized in that biological tissue or cell, directly or after the fixedly processing of implementing GCPL-, with any one described anti-GCPL-antibody response of claim 3-11, simultaneously or thereafter, react with secondary antibodies again at above-mentioned immune animal globulin antibody with the mark substance mark, bonding mark secondary antibodies on biological tissue that mycoplasma fermentans infects or cell then, measure mark substance, wherein above-mentioned immune animal globulin antibody is to use the animal beyond the above-mentioned antibody of preparation to prepare.

23. detect the test kit of the GCPL-of mycoplasma fermentans in the sample or mycoplasma fermentans by immunological method, it is characterized in that containing any one described anti-GCPL-antibody of claim 3-11 and with the GCPL-of the mycoplasma fermentans of mark substance mark.

24. detect the test kit of the GCPL-of mycoplasma fermentans in the sample or mycoplasma fermentans by immunological method, it is characterized in that containing any one described anti-GCPL-antibody of claim 3～11 and use animal preparation beyond the immune animal of the above-mentioned antibody of preparation, at the antibody of the immunoglobulin (Ig) of above-mentioned immune animal secondary antibodies with the mark substance mark.

25. to being selected from the detection method of AIDS, ephritis and the myelopathic disease relevant with HTL-I, it is characterized in that detecting the similar material of antigenicity of the described GCPL-of the claim 1 that is contained in the blood or these GCPL-, perhaps these GCPL-are had the antibody of atopic.