CN1133192A - Monoclonal antibody with idiocrasy to pneumonia chlamydia and preparation process - Google Patents

Abstract

This invention relates to a monoclonal antibody which has a specific reactivity to the major outer membrane protein and the elementary body that originate from Chlamydiaceae pneumoniae but which does not have any reactivity to the major outer membrane protein of Chlamydiaceae trachomatis; a monoclonal antibody which has a reactivity to the 53K dalton antigen protein of Chlamydiaceae pneumoniae but which does not have any reactivity to the inclusion body of Chlamydiaceae trachomatis nor Chlamydiaceae psittaci; methods for preparing these monoclonal antibodies; cell lines producing the same; methods for detecting and determining Chlamydiaceae pneumoniae using the same; reagents for detecting and determining Chlamydiaceae pneumoniae containing the same; methods for diagnosis of Chlamydiaceae pneumoniae infections using the same; and diagnostic reagents for Chlamydiaceae pneumoniae infections comprising the same as an active ingredient.

Description

Chlamydia pneumoniae had atopic monoclonal antibody and manufacture method thereof

The present invention relates to that Chlamydia pneumoniae is had atopic monoclonal antibody, make this monoclonal antibody method, produce the cell of this antibody, make the method for this cell, check and/or measure the method for Chlamydia pneumoniae, this method agents useful for same, the method and the reagent of diagnosis infection involving chlamydia pneumoniae.The present invention can be used for pharmaceuticals industry effectively, especially for the reagent of making in order to the diagnosis infection involving chlamydia pneumoniae.

Known have four kinds of chlamydia, and they are conjunctivitis chlamydia, Chlamydia pneumoniae, ornithosis virus and eye conjunctiva chlamydia.It is believed that Chlamydia pneumoniae can cause respiratory tract infection, atypical pneumonia or the like.

Because the symptom that the respiratory apparatus that Chlamydia pneumoniae causes infects is similar to the symptom of the infection that mycoplasma pneumoniae or influenza virus cause, the judgement that the doctor often makes mistake to this work.So, just need a kind of straightforward procedure of diagnosing the infection that Chlamydia pneumoniae causes of exploitation.

Generally, the pathogenic cell by detecting infection site or by detecting body fluid as the antibody of this pathogenic cell in the serum etc., just can be diagnosed out infection reliably.Preceding a kind of method is called as antigen test, and the latter is called as antibody test.The two is all very important clinically.

Antigen test comprises indirect fluorescent antibody technique and direct fluorescent antibody technique, in indirect fluorescent antibody technique, detected antigenic antibody is added in the sample, add the another kind of antibody that is served as a mark by fluorescent agent and analog then and have or not this antigen to measure in the sample, in direct fluorescent antibody technique, need not another kind of fluorescent antibody, but the antigenic antibody to be checked of will be in advance having been made labelling by fluorescent agent and analog joins in the sample, has or not antigen thereby measure in the sample.

As the antigen of Chlamydia pneumoniae, known have lipopolysaccharide (LPSs) and protein.LPSs chlamydia accessory has general antigenicity.As proteantigen, 39.5KDa, 60KDa, 75KDa, 98KDa protein and analog thereof are known.39.5KDa albumen is called as major outer membrane albumen (Major Outer Membrane Protein) (hereinafter slightly " MOMP ").

As the chlamydia genus specific antigen is arranged, GLXA (species specificity glycolipid antigen genus), lipopolysaccharide (LPSs), MOMP and analog thereof are known.People such as Elizabeth S.Stuart have reported the monoclonal antibody (CurrentMicrobiology, 28,85-90 (1994)) of the GLXA of anti-chlamydiaceae.People such as Harland D.Caldwell have reported the monoclonal antibody (Infection and Immunity, 44 (2), 306-314 (1984)) of the LPs of anti-chlamydiaceae.They give the occlusion body dyeing of oakmoss substance by direct fluorescent antibody technique with monoclonal antibody.People (Infectionand Immunity such as Ellena M.Peterson, 59 (11), people (Infection and Immunity, 53 (3), 530-533 (1986)) such as 4147-4153 (1991) and Byron E.Batteiger have reported the monoclonal antibody of the MOMP of anti-chlamydiaceae.

Begin from people's (J.Immunology, 128 (3), 1083-1089 (1982)) such as Richard S.Stephens report, delivered many relevant chlamydial monoclonal antibodies of anti-conjunctivitis.People such as H.Puy have reported the monoclonal antibody of anti-ornithosis virus, comprising in indirect fluorescent antibody test, ornithosis virus and Chlamydia pneumoniae being had same reactive monoclonal antibody (Immunology Letters, 23,217-222 (1989/1990).But, still do not have the antigenic description that monoclonal antibody identifies.People such as Kuroda have reported the anti-eye chlamydial monoclonal antibody of conjunctiva (Am.J.Vet.Res, 54 (5), 709-712 (1993)).

In the immunity inoculation of the MOMP of elementary body of general usefulness or Chlamydia pneumoniae, very difficult acquisition can produce with MOMP in the low antigen site of antigenicity the cell of the monoclonal antibody of specific reaction is arranged.No matter be the monoclonal antibody that specific reaction is arranged with the low antigen site of MOMP, still produce the cell of this antibody, all do not obtain at present.

People such as Iijima have reported the proteic monoclonal antibody of 53 KDa (Y.Iijima, J.clinical Microbiology, 32 (3), the 583-588 (1994) as the monoclonal antibody of anti-Chlamydia pneumoniae.

People such as Cho-chou Kuo have reported in indirect fluorescent antibody technique, the physical ability that comprises of Chlamydia pneumoniae is had specific monoclonal antibody (RR402) dyeing (J.Clinical Microbiology by Chlamydia pneumoniae, 24 (6), 1034-1037 (1986) and the clear 64-500083 of Japanese unexamined patent number).But, the not antigenic description of being discerned by monoclonal antibody.

People such as Izutsu have reported the monoclonal antibody (the flat 7-16098 of Japanese unexamined patent number) with the Chlamydia pneumoniae reaction.They disclose by indirect fluorescent antibody technique, make occlusion body dyeing with the monoclonal anti physical ability.

Harland, D, the anti-LPS monoclonal antibody of people such as Caldwell report has the reactivity to the chlamydiaceae kind except that Chlamydia pneumoniae, so be difficult to this monoclonal antibody special detection Chlamydia pneumoniae.

Ellena M, the monoclonal antibody of the anti-MOMP of people such as people such as Peterson and Byron E.Batteiger report has the reactivity to the chlamydiaceae kind except that Chlamydia pneumoniae, so be difficult to these monoclonal antibody special detection Chlamydia pneumoniae.In addition, they are not labeled.

Whether the antibody that it be unclear that people such as Iijima report can the special detection Chlamydia pneumoniae.And, still do not have the chlamydial physical ability that comprises of pneumonia by the painted report of this monoclonal antibody.

People such as Cho-chou and the used indirect fluorescent antibody of Izutsu prop up the method for art need carry out antigen test for a long time, and therefore, it is unfavorable that the short time in the clinical examination is handled a large amount of samples.

The MOMP that an object of the present invention is to provide Chlamydia pneumoniae has atopic monoclonal antibody.These monoclonal anti physical abilitys are used for the special detection Chlamydia pneumoniae, and can be in clinical examination the short time handle a large amount of samples.

Theme of the present invention is following material:

(1) a kind of major outer membrane albumen to Chlamydia pneumoniae has atopic monoclonal antibody.

(2) as (1) described monoclonal antibody, it does not have reactivity to the chlamydial major outer membrane albumen of conjunctivitis.

(3) as (1) described monoclonal antibody, its elementary body to Chlamydia pneumoniae has reactivity.

(4) as (3) described monoclonal antibody, it does not have reactivity to the chlamydial major outer membrane albumen of conjunctivitis.

(5) as (1) described monoclonal antibody, antibody wherein is labeled.

(6) as (1) described monoclonal antibody, antibody wherein is that hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156) produce.

(7) as (6) described monoclonal antibody, antibody wherein is that hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156) produce, and is labeled subsequently.

(8) a kind of monoclonal antibody has reactivity and is labeled the 53KDa antigen protein of Chlamydia pneumoniae.

(9) as (8) described monoclonal antibody, its occlusion body to conjunctivitis chlamydia and ornithosis virus does not have reactivity.

(10) as (8) described monoclonal antibody, antibody wherein is that hybridoma cell line AY6E2E8 (FERM BP-5154) produces, and is labeled subsequently.

(11) a kind of generation has the preparation method of the generation antibody cell of atopic monoclonal antibody to albumen, this method comprises to animal immune inoculates a kind of of natural and Denatured protein, and with other albumen booster doses, obtain a kind of cell that produces antibody, and merge the cell of this generation antibody with the myeloma cell.

(12) as (11) described method, comprise to animal immune and inoculate a kind of native protein also with a kind of Denatured protein booster dose, obtain a kind of cell that produces antibody, and merge the cell of this generation antibody with the myeloma cell.

(13) as (11) described method, Denatured protein is wherein handled native protein with detergent or Reducing agent and is obtained.

(14) as (11) described method, albumen wherein is chlamydial major outer membrane albumen.

(15) as the cell of the prepared generation monoclonal antibody of (11) described method.

(16) a kind of cell that produces monoclonal antibody, this cell can produce as (1) described monoclonal antibody.

(17), be hybridoma cell line CP-6 (FERM BP5155) or CP-11 (FERM BP-5156) as the cell of (16) described generation monoclonal antibody.

(18) a kind of preparation monoclonal antibody method comprises the cell of cultivating as (15) described generation monoclonal antibody.

(19) a kind of preparation monoclonal antibody method comprises the cell of cultivating as (16) described generation monoclonal antibody.

(20) with the method that detects and/or measure Chlamydia pneumoniae as (1) described monoclonal antibody.

(21) as (20) described method, monoclonal antibody wherein is that hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156) produce, and is labeled subsequently.

(22) with the method that detects and/or measure Chlamydia pneumoniae as (8) described monoclonal antibody.

(23) as (22) described method, monoclonal antibody wherein is that hybridoma cell line AY6E2E8 (FERM BP-5154) produces, and is labeled subsequently.

(24) a kind of reagent that detects and/or measure Chlamydia pneumoniae, comprising as (1) described monoclonal antibody.

(25) as (24) described reagent, monoclonal antibody wherein is that hybridoma cell line CP6 (FERM BP-5155) or CP-11 (FERM BP-5156) produce, and is labeled subsequently.

(26) a kind of reagent that detects and/or measure Chlamydia pneumoniae, comprising as (8) described monoclonal antibody.

(27) as (26) described reagent, monoclonal antibody wherein is that hybridoma cell line AY6E2E8 (FERM BP-5154) produces, and is labeled subsequently.

(28) a kind of usefulness is as the method for (1) described monoclonal antibody diagnosis infection involving chlamydia pneumoniae.

(29) as (28) described method, monoclonal antibody wherein is that hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156) produce, and is labeled subsequently.

(30) a kind of usefulness is as the method for (8) described monoclonal antibody diagnosis infection involving chlamydia pneumoniae.

(31) as (30) described method, monoclonal antibody wherein is that hybridoma cell line AY6E2E8 (FERM BP-5154) produces, and is labeled subsequently.

(32) a kind of reagent of diagnosing infection involving chlamydia pneumoniae, comprising as (1) described monoclonal antibody as active component.

(33) as (32) described diagnostic reagent, monoclonal antibody wherein is that hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156) produce, and is labeled subsequently.

(34) a kind of reagent of diagnosing infection involving chlamydia pneumoniae, comprising as (8) described monoclonal antibody as active component.

(35) as (34) described diagnostic reagent, monoclonal antibody wherein is that hybridoma cell line AY6E2E8 (FERM BP-5154) produces, and is labeled subsequently.

Preferably the MOMP of Chlamydia pneumoniae is had atopic monoclonal antibody and comprise, the MOMP of Chlamydia pneumoniae is had specific reaction and to the chlamydial MOMP of the conjunctivitis reactive monoclonal antibody of tool and the MOMP of Chlamydia pneumoniae had specific reaction and the elementary body of Chlamydia pneumoniae is had reactive monoclonal antibody not.Even more preferably the MOMP of Chlamydia pneumoniae had specific reaction and tool is reactive and to the monoclonal antibody of elementary the responding property of body of Chlamydia pneumoniae to the chlamydial MOMP of conjunctivitis.

The object lesson that the MOMP of Chlamydia pneumoniae is had atopic monoclonal antibody comprises, the monoclonal antibody that hybridoma cell line CP-6 produces, monoclonal antibody that produces among the hybridoma cell line CP-11 and analog.Hybridoma cell line CP-6 and CP-11 have pressed the clause of Bu Peisi treaty at the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Science andTechology (1-3, Higashi lchome Tsukuba-shi Ibaraki-ken 305 is Japan) with FERM BP5-5155 and FERM BP-5156 preservation.

Preferably to have reactive monoclonal antibody be those to the occlusion body of conjunctivitis chlamydia and the ornithosis virus reactive monoclonal antibody of tool not to the 53KDa antigen protein of Chlamydia pneumoniae.

The object lesson that the 53KDa antigen protein of Chlamydia pneumoniae is had reactive monoclonal antibody comprises, monoclonal antibody and analog that hybridoma cell line AY6E2E8 produces.Hybridoma cell line AY6E2E8 has pressed the clause of budapest treaty at the NationalInstitute of Bioscience and Human-Technology, and the Agencyof Industrial Science and Technology is with FERM BP-5154 preservation.

The invention still further relates to and to produce the celliferous preparation method that albumen is had atopic monoclonal antibody, comprising inoculating a kind of of native protein and Denatured protein to animal immune, with other albumen booster doses, obtain producing the cell of antibody, merge the cell of this generation antibody with the myeloma cell.

The method feature that preparation of the present invention produces the antibody cell is, to the animal immune inoculation, remove this it can also comprise to animal immune by common step and inoculating, cell fusion, the screening of the cell that merges, the screening that produces the cell of specific antibody, clone or the like finishes.

The used albumen of the inventive method comprises chlamydia, and particularly former the stopping of pneumonia clothing belongs to, antibacterial, and the MOMP of virus, animal comprises people and plant deutero-various albumen of institute and analog.

Native protein is defined as keeping the protein of luv space structure, comprises chlamydia, antibacterial, and viral organism itself, for example, chlamydial EB, and the proteinic part and the thing of purifying fully.Native protein is used as the antigen of immunity inoculation.The deutero-antigen of various bacterial strains of Chlamydia pneumoniae can be used as special proteantigen.For example, can use YK-41 bacterium target and EBS and TWAR.

The albumen of degeneration is defined as the protein under the following condition, its luv space structure in physiological solution is destroyed by certain processing, and the protein under the following condition, its cystine linkage (S-S key) also disconnects, and preferably its aminoacid sequence is identified as antigen site.Denatured protein also can be used as the antigen of immunity inoculation.Because the immunity inoculation Denatured protein has strengthened producing the cell of the antibody in anti-low antigenicity site in the Denatured protein fully, even, also can easily obtain producing the antibody cell so be difficult to obtain to produce at an immunity inoculation native protein under the situation of cell of monoclonal antibody of anti-native protein.

When making the antigen of immunity inoculation, only bring out the antibody that forms anti-high originality site in the albumen, and be difficult to bring out effectively the antibody that forms anti-low antigenicity site with native protein or Denatured protein.

As the MOMP of the antigenic Chlamydia pneumoniae of immunity inoculation and EB can by for example hereinafter the method described of embodiment from the YK-41 bacterial strain, make.

Make the method for protein denaturation include but not limited to following method, handle with Reducing agent or detergent, heat treatment is with the organic solvent processing etc.The preferred method of handling with Reducing agent or detergent.Reducing agent includes but not limited to 2 mercapto ethanol.Detergent includes but not limited to SDS (dodecylbenzene sodium sulfonate).

Mammal by immunity inoculation comprises mice, Mus, rabbit, sheep, chicken etc.Mice is particularly preferred.

Mammal does to inoculate at last with a kind of immunity inoculation of native protein and Denatured protein and with other albumen, preferably uses the native protein immunity inoculation and with the last immunity inoculation of Denatured protein.Under the situation of Chlamydia pneumoniae, suggestion is inoculated the reuse Denatured protein with the EB or the natural MOMP of Chlamydia pneumoniae to mammalian immune, and the MOMP that for example has been washed agent or Reducing agent processing degeneration carries out last immunity inoculation.

In addition, a kind of adjuvant of preferred use in immunity inoculation.The preferred example of adjuvant comprises Freund's complete adjuvant (hereinafter slightly " FCA "), incomplete Freund (hereinafter slightly " FIA ") and analog.

Immunity inoculation generally can be finished by 1-5 or injection more frequently.Antigenic injection volume generally can be every mammal 1ng-10mg, and can change with antigenic difference.

Last immunity inoculation generally can be finished by 1-5 or injection more frequently.Antigenic injection volume generally can be every mammal 1ng-10mg, and can change with antigenic difference.Usually, the antigen amount that is used for last immunity inoculation is 1/100 to 1/10 of an immunity inoculation institute consumption.

After the immunity inoculation of said method capacity, from being isolated splenocyte the mammal of immunity inoculation, bone-marrow-derived lymphocyte or analog are as the mother cell that can produce antibody.

General available myeloma cell is as other mother cells of preparation hybridoma cell line.For example, can use the P3U1 of mice, NS-1,653, SP2, X63, MPC-11 and analog, the AG1 of Mus, AG2, AG3, RCY3,210 and analog, people's SKO-007 and analog.The cell of mice preferably, preferred especially P3U1,653 and analog.

Cell fusion can be used the Polyethylene Glycol method, Sendai virus method, electro fusion method or the like.Preferably use the method for Polyethylene Glycol.

Hybridoma screening can be undertaken by cultivating in the screening culture medium that can only make interested hybridoma growth.For example, HAT (hypoxanthine aminopterin thymidine) culture medium, HAz (hypoxanthine azaserine) culture medium etc. is preferred culture medium.

Screening produces the cell of specific antibody, be by the supernatant in the recovery well, the required hybridoma of having grown in the culture medium in this well, whether reuse desirable proteins (as the EB or the MOMP of Chlamydia pneumoniae) inspection has the antibody that is formed by enzyme antibody technology or immunoblot assay is finished.

The clone is defined as obtaining with the form of a deutero-cell mass of clone the process of the cell of generation specific antibody.The clone can pass through the limiting dilution culture method, and bacterium colony is placed method on the soft agar, unicellular facture, and methods such as FACS method are carried out.The limiting dilution cultural method is simpler, so be preferred.

The cell that produces monoclonal antibody can obtain with said method.

The cell of the generation monoclonal antibody that obtains by the inventive method comprises hybridoma cell line CP-6 and CP-11.

The cell of the generation monoclonal antibody that obtains with described method can be cultivated in the suitable culture base with the generation monoclonal antibody.Perhaps, by the hybridoma cell line that obtains to injections such as mices, regather ascites and obtain monoclonal antibody.

Culture medium comprises, mend with fetus sura serum improve Eagle culture medium (MEM culture medium), Dulbecco improves Eagle culture medium (DMEM culture medium), RPM11640 culture medium or the like.

From the supernatant of culture fluid, reclaim monoclonal antibody and can pass through to use the protein A post, the affinity chromatography of Protein G post etc., ion exchange chromatography, the Polyethylene Glycol fractionating process, the ethanol fractionating process, ammonium sulfate fractionating process and similar method are carried out.Affinity chromatography preferably, the preferred protein A post that is to use.

Can measure it to required proteic idiosyncrasy to the monoclonal antibody of gained.For example, screen the monoclonal antibody that specific reaction is arranged with desirable proteins, used Organic substance in this method through appropriate solvent processing and electrophoretic process with the Western blotting.More specifically, can measure the idiosyncrasy of monoclonal antibody to the MOMP or the EB of Chlamydia pneumoniae, and/or with the reactivity of 53KDa antigen protein.Idiosyncrasy to the MOMP of Chlamydia pneumoniae can be by quantitative assay, method is to measure the existence that has atopic antibody with the MOMP of the about 39.5KDa of molecular weight with the Western blotting, uses the pneumonia clothing EB originally with suitable solution-treated and electrophoretic process in this method.Can believe by quantitative assay and discern MOMP aminoacid sequence as the Chlamydia pneumoniae of epitope for the MOMP of Chlamydia pneumoniae there being atopic monoclonal antibody.

The antigen protein of research and monoclonal antibody reactive is performed such: the cDNA storehouse for preparing Chlamydia pneumoniae with λ gtll and analog, express cDNA and obtain peptide, make monoclonal antibody and resulting reactive polypeptide, the codon of the cDNA of analysis and monoclonal antibody specific reaction, this codon inserts bacteriophage lambda.

The present invention prepares the antibody that monoclonal antibody method can prepare any kind (globulin type).Can use the fragment and the molecule that monoclonal antibody fragment is arranged of monoclonal antibody.

The monoclonal anti physical ability of the inventive method preparation is identified as epitope with low antigenicity site in the albumen, and this former being difficult to accomplishes.More particularly, they can discern protein planar structure or the aminoacid sequence as epitope.

The MOMP of Chlamydia pneumoniae there is atopic monoclonal antibody and can obtains by the following method the monoclonal antibody of the responding property of 53KDa antigen protein of Chlamydia pneumoniae, for example, cultivate the cell (hybridoma cell line) of the generation monoclonal antibody of the inventive method preparation, collect the culture supernatant, purify with said method then.Can finish with known technology in order to prepare the used immunity inoculation of cell that produces monoclonal antibody in the method.

Preferably, other kinds that the monoclonal antibody of gained of the present invention does not belong to clothing substantially originally are as conjunctivitis chlamydia and ornithosis virus reaction.Particularly, more preferably substantially not with the MOMP of other all kinds of chlamydiaceae only with the monoclonal antibody of the MOMP of Chlamydia pneumoniae reaction, with with the 53KDa antigen protein of Chlamydia pneumoniae the monoclonal antibody of specific reaction is arranged because they can be used to diagnose Chlamydia pneumoniae specific diseases kind.

If monoclonal antibody in direct or indirect fluorescent technique with the kind reaction of pneumonia clothing outside originally, but the low reactivity of a tool strictly speaking, they can be identified from be those of high response, so can be useful in the diagnosis.This monoclonal antibody also is useful.Monoclonal antibody should be not very important with frequent isolating conjunctivitis chlamydia generation cross reaction, but whether they are not so important with not frequent isolating ornithosis virus and an eye conjunctiva chlamydia generation cross reaction.If monoclonal antibody in direct or indirect fluorescent technique with the reaction of Chlamydia pneumoniae height but only with the faint reaction of other kinds (as ornithosis virus), and do not react with other kinds (as conjunctivitis chlamydia and eye conjunctiva chlamydia) basis, they just can be used for determining the disease that the chlamydia of which kind causes, so they are preferred.

Monoclonal antibody of the present invention comprises the antibody of any kind (globulin type).The fragment (Fab, fab ', the fab ' that also comprise monoclonal antibody ₂Deng) and the segmental molecule of monoclonal antibody is arranged.

The MOMP of Chlamydia pneumoniae there is atopic monoclonal antibody and can be labeled the monoclonal antibody of the responding property of 53KDa antigen protein of Chlamydia pneumoniae.

Marking to monoclonal antibody can be in conjunction with a kind of labelled reagent, as various dyestuffs, colloid, enzyme etc. on monoclonal antibody.

Dyestuff comprises fluorescent dye, as FITC (the different sulfur hydrogen salt of fluorescein 5-), rhodamine, fluorescein etc.FITC becomes preferred with its suitability and being easy to get property.FITC can be by SigmaCo., and Ltd buys.

Colloid comprises gold colloid or the like.

Enzyme comprises peroxidase, alkali phosphatase, luciferase, beta galactosidase or the like.

Can use any prior art in conjunction with last labelling for monoclonal antibody, the method described in " EXPERIMENTALIMMUNOLOGY ", people such as Ivan Lefkovits compile, NishimuraShoten, (1985).

In addition, between monoclonal antibody and labelled reagent, can insert chemical substance, as biotin, antibiont albumen, streptoavidin, digoxygenin or the like.

Because FITC is combined on the lysine residue of antibody, the result makes some antibody lose its antigen-binding activity with the FITC labelling.

Detect and/or measure Chlamydia pneumoniae with for example direct or indirect labelling monoclonal anti physical ability of the present invention afterwards.Can mark directly for monoclonal antibody of the present invention with said method.Be labeled reagent (the another kind of antibody of labelling) labelling, can discern and can give monoclonal antibody indirect labelling of the present invention with the bonded antigen of monoclonal antibody.This another kind antibody comprises anti-mouse immuning ball protein antibody of rabbit or the like.At labelled reagent is under the situation of fluorescence infection, and it also detects and/or measure the fluorescence that is taken place with ultraviolet radiation.When labelled reagent is enzyme, add color development and the color enhancing of substrate detection and/or mensuration by the catalytic reaction generation of enzyme.If desired, all can use sensitizer in both cases.

The reagent that detects and/or measure Chlamydia pneumoniae comprises monoclonal antibody of the present invention itself of unlabelled or labelling and composition thereof, comprises a kind of of following composition at least.

(1), the material (as, the serum albumin of cattle) that suppresses nonspecific immune reaction

(2), be used as the substrate of the enzyme of labelled reagent

(3), sensitizer

(4), control antigen (pneumonia clothing MOMP originally, 53KDa antigen protein)

(5), made the another kind of antibody of labelling

These compositions no matter use separately or two or more mix use, preferably make freeze-dried products so that clinical practice.

The reagent that detects and/or measure Chlamydia pneumoniae can be directly as the reagent of diagnosing infection involving chlamydia pneumoniae.

Monoclonal anti physical ability of the present invention is used to diagnose infection involving chlamydia pneumoniae.Infection involving chlamydia pneumoniae can be diagnosed with the reagent of reagent that detects and/or measure Chlamydia pneumoniae or diagnosis infection involving chlamydia pneumoniae.In the test of these reagent on probation and diagnostic reagent, when presenting the positive, sample just confirms the generation of infection involving chlamydia pneumoniae.

The preferred mixture that uses monoclonal antibody of the present invention is because antigenic amount is with the sample difference, so it can discern different antigen.

The example that can mix monoclonal antibody comprises to the monoclonal antibody of the responding property of MOMP of Chlamydia pneumoniae with to the monoclonal antibody of the responding property of 53KDa antigen protein of Chlamydia pneumoniae.

In addition, monoclonal antibody of the present invention can be mixed with other monoclonal antibodies or multiple grand antibody.

Those antibody that monoclonal antibody of the present invention and the inventive method make can be used in the method that detects desirable proteins, particularly use immunoreation, as radioimmunoassay, in the method for enzyme immunoassay etc.They also can be as the composition of diagnostic reagent in the scientific research.If desired, they can be directly or are used in the pharmaceutical preparation with the form of intercalate agent or potus or antibody.

In addition, those antibody capables of monoclonal antibody of the present invention and the inventive method preparation are used in the diagnostic tool case, and they combine by polymerase chain reaction therein.

The present invention produces the cell of antibody can also be as the source of antibody group.The antibody group can show in the various cells.

More than describing is general description of the present invention.Can understanding more completely be arranged to the present invention with reference to following specific embodiment, these examples are used for the purpose of explanation at this, rather than limit the scope of the invention.Embodiment 1 preparation has specific monoclonal antibody to the MOMP of Chlamydia pneumoniae (1)

1.1 the EB of preparation Chlamydia pneumoniae

In order to prepare pneumonia clothing EB originally, use the YK-41 bacterial strain.Use YK-41 strain infection people's pneumonocyte (hereinafter referred to as " HL cell ") according to people's such as Kishimoto method [KENSA TO GIFUTU (test and technology) 18 (7), 959-964 (1990)].The infection cell of cultivating in 24-or 6-well petri plate is scraped also and culture medium reclaims together, then with 6000rpm centrifugalize 30 minutes.After removing the supernatant, precipitate is suspended in sucrose, [contains 7.5 (w/v) sucrose, 3.8mMKH in the mixed solution of phosphate and glutamic acid ₂PO ₄, 7mM K ₂HPO ₄With 5mM glutamic acid, (PH7.4), hereinafter referred to as " SPG "].With the broken float of homogenizer then with 2500rpm centrifugalize 10 minutes.Remove the supernatant, precipitate is suspended among the SPG.Repeat these processes 5 times, reclaim the supernatant then.The Urografin (Nihon Schering K.K.) of the supernatant and 23% (v/v) of reclaiming and the sucrose of 50% (w/v) are by the supernatant: Urografin: the volume ratio of sucrose=2: 2: 1 is mixed, then with 8000rpm centrifugalize 1 hour.After removing the supernatant, recovery lower floor's [50% (w/v) sucrose layer] also is suspended among the SPG, and the washing back was with 10000rpm centrifugalize 30 minutes.Siphon away the supernatant, precipitate is suspended among the SPG, is that the Urografin of 26.6-38% (v/v) mixes with 1: 4 volume ratio and density gradient, and with 8,000rpm centrifugalize 1 hour is reclaimed the intermediate layer and also is suspended in SPG., inhale and go the supernatant, precipitate to be suspended among the SPG of Sq, after 30 minutes with the 10000rpm centrifugalize so obtain EB solution.After the distribution, this solution is stored in 4 ℃ or-70 ℃.

1.2 the MOMP of preparation Chlamydia pneumoniae

Prepare electrophoretic separation and the above EB solution of 1.1 gained of purifying with disc type, obtain the MOMP of Chlamydia pneumoniae thus.In brief, 0.5ml [0.31M Tris-HCl (Tris-HCl) (PH6.8) with 0.5ml 2X sample buffer for the EB solution of gained [albumen (EB) concentration: 727 μ g/ml], 1.6% (w/v) sodium lauryl sulphate (hereinafter referred " SDS "), the 1mM dithiothreitol, DTT, 16% (v/v) glycerol, 0.005 (w/v) bromophenol blue] mix, boiled 3 minutes so that the protein dissolving at 100 ℃.Then, whole volumes are joined disc type gel (basic unit's gel: 4%; Separating gel: 10%), [equipment: disc type preparation electrophoresis equipment NA-1800 Nippon Eido K.K. makes to carry out electrophoresis; Operating condition: 100V (DC voltage)] and in certain volume recovery (0.26ml/ part).Carry out SDS disk electrophoresis (equipment: DNA-PAGE electrophoresis equipment NB-5000, Nippon Eido K.K. manufacturing for every part; Operating condition: 100V (DC voltage)].The result proves that molecular weight is that the MOMP of 39.5K is recovered (protein concentration: 0.21mg/ml) in the 33rd to 45 part.

1.3 preparation hybridoma cell line and monoclonal antibody

The normal saline of the EB that said process obtains (0.5ml) mixes with 0.6mlFCA (Difco).Handle the gained mixture with sonicator (Branson) and obtain water-in-oil emulsification, with its antigen as immunity inoculation.With the BALB/c female mice in five 8 weeks, (amount of EB is every intraperitoneal administration 200 these antigens of μ l in the potion: 10 μ g/ mices).After the administration 6 days and 12 days, carry out immunity inoculation with FIA (Difco).Give the animal blood-letting after this immunity inoculation after 6 days.Measure the antibody titer of every part of serum serosity that obtains with an immunity bundle algoscopy (dot immuno binding assay) (hereinafter referred is " DIBA ").Begin from measuring antibody titer that the animal of high antibody titer carries out three days last immunity inoculations to presenting that day, the purification MOMP (amount of the MOMP in the potion: 1 μ g/ mice) that obtains by said method from tail vein administration 200 μ l.Put to death and take out spleen with every mouse carotid dislocation next day after last immunity inoculation was finished in three days.Then thereby unicellular dispersion is made the splenocyte that is used for cell fusion.The coupling cell of cell fusion is the P3U1 murine myeloma cell, and it is at CO ₂In the cell culture apparatus (Napco), 37 ℃, 5%CO ₂And under the saturated humidity, cultivate with Dulbecco MEM culture medium (hereinafter state and be " 10F culture medium ") the lining advance notice of 10% (v/v) fetal bovine serum in benefit.

Above-mentioned splenocyte (4.8 * 10 ⁷) and P3U1 murine myeloma cell (1.2 * 10 ⁷) mix, with 1000rpm centrifugalize 10 minutes.Siphon away the supernatant then.Impose slight vibration, 0.2ml cell fusion accelerator [50% (W/V) Polyethylene Glycol; Molecular weight: 4000; Merck makes) make cell pellets loose.After 1 minute 45 seconds, with 1 minute adding 10ml culture fluid.Add the 20ml culture fluid again so that cell suspension.Then, with the dispersion liquid centrifugalize with separate (300rpm3 minute, 500rpm3 minute, 700rpm3 minute) step by step.After siphoning away the supernatant, (Dainippon Pharmaceutical Co., Ltd.) (hereinafter referred to as " 10F+Bri culture medium ") is with preparation cell dispersion liquid to add 5%Briclone in cell pellets.This cell dispersion liquid is dispersed in the flat multilayer disc of 96-well (Corning), and to obtain cell density be 4 * 10 ⁴Myeloma cell/well/0.1ml, then at 37 ℃, 5%CO ₂With constant temperature culture under the saturated humidity.

Next day, the 10F culture medium that contains HAT (is mended with 1 * 10 ^-4The M hypoxanthine, 4 * 10 ^-7The M aminopterin, 1.6 * 10 ^-5Dulbecco ' s MEM the culture medium of thymidine and 10% (v/v) fetal bovine serum; Hereinafter referred to as " HAT culture medium ") by volume join in the well for the 0.1ml/ well.In addition, replaced the culture fluid supernatant of half in per 3 to 4 days with the HAT culture medium; So, the process that is called the HAT screening is carried out about 2 weeks.The well that can observe hybridoma cell line growth clear liquid at the middle and upper levels is collected and with the DIBA screening, prepares the antibody of anti-EB.

Carry out DIBA (Corning) with multilayer disc at the bottom of the 96-well u shape.To be fixed in (Advantec on 3mm side's film filter as the amount that antigenic EB is ordered with 0.02 μ g/ in advance; 47mm diameter lattice filter; Cat.No.A045b047A), add 10% (v/v) FCS (fetus sura serum), at room temperature reacted 15 minutes to this filter.Then, remove the supernatant, the hybrid cell line that adds 100 μ l is cultivated the supernatant, at room temperature reacts 1 hour.Remove the supernatant, add the anti-mouse immuning ball protein antibody of rabbit (Cappel Inc.) of the peroxidase labelling of 500 times of 100 μ l dilutions, reaction is 1 hour under the room temperature.At last, add 4-chloro-1-naphthols and make the reaction solution colouring as substrate.Can bore hole observe well that color occurs and predicated in Antibody Preparation and be positive being fixed with on the antigenic membrane filter.

Obtain conclusion in above-mentioned screening and be hybridoma cell line in male those wells of Antibody Preparation, by limiting dilution culture method time cloning.Briefly, the Antibody Preparation positive hybridoma cell is suspended in the HAT culture medium, measures the cell density of suspension.With the suspension dilution, be dispersed on the new shallow bid then, cell density is from 2 to 100 cells/100 μ l/ wells, constant temperature culture.Use HT+5%Briclone to cultivate and make diluent.

When in the well of observing dilution for many times the growth of hybridoma being arranged, detect the ability that assorted oncocyte produces antibody, and microexamination confirms that it is a monoclonal, repeat the clone of same operating process with the monoclonal antibody of the anti-EB of preparation generation then, it has very high ability and keeps production of antibodies.To each clone who so obtains, the cultivation scale strengthens gradually.At last, each clone cultivates in 50ml 10F culture medium, and checks that the culture supernatant produces the characteristic of antibody.

Carried out twice to this described testing regulations, obtained the clone strain that 10 shown in the table 1 produce monoclonal antibody at last.

The globulin class of the monoclonal antibody that is produced by above-mentioned 10 hybridoma cell clones adopts ISOTYPE-Ab-STAT-1 kit utility (Kit) (Sang Stat Medical) to measure.

As shown in table 1, monoclonal antibody CP-1, CP-2, CP-4, CP-5, CP-6, CP-7 and CP-8 do not present reactivity to the conjunctivitis chlamydia, but Chlamydia pneumoniae is presented reactivity.

Next step, will be by the 10F culture medium culturing each produce the hybridoma clone of monoclonal antibody as shown in table 1 and the propagated cell that obtains with 1 * 10 ⁷The amount of individual cell/mice is to (Wako Purechemical Co., the BALB/C mice of Ltd.) handling is inoculated by the abdominal cavity through 0.5ml Pristan in advance.Inoculation two to three weeks of back obtain ascites in every mice body, obtain monoclonal antibody of the present invention from wherein purifying.

With the resultant ascites of following method purification inoculation hybridoma clone No.6, wherein used the affinity post of immobilization protein A.Briefly, in mouse ascites, add the 1.5M glycine of equivalent and the mixture of 3M NaCl (PH8.9).The gained mixture is sent into protein A-Poros (NGK Insulators Ltd.) affinity post by the nylon filter (Corning) of 0.45 μ m, and washs with the mixture of 1.5M glycerol and 3M HaCl (PH8.9).Then, with containing the phosphate-citrate salts buffer (PH6.0) of 150mMNaCl with antibody elution.

After 1M Tris-HCl buffer (PH9.0) neutralization, eluate is cooled, and spends the night with the PBS dialysis.(use Model U-2000, Hitachi Ltd) by 0.2 μ m cellulose acetate filter (Corning), stores down in-80 ℃, thereby makes the solution of purification antibody at the absorbance of 280nm to measure the solution that is reclaimed.

1.4 the specificity analyses of monoclonal antibody

Carry out the specificity analyses of monoclonal antibody with immunoblotting.EB solution (the EB concentration: 727 μ g/ml that makes with 1.1 described methods above, mensuration is to the reactivity of Chlamydia pneumoniae, specifically, the EB solution and the sample buffer agent [0.063MTris that will contain 50 μ g EB, 5% (v/v) 2 mercapto ethanol, 10% (v/v) glycerol, 2.3% (w/v) SDS, 0.001% (w/v) BPB (bromophenol blue) is (PH6.8)] mix with 1: 1 volume ratio, boiled 10 minutes.Then, the gained mixed liquor carries out electrophoresis [equipment: AE-6560, the manufacturing of Atto company limited; Gel: Pagel 1020 DL; The electrophoresis buffer agent: 0.025M Tris, 0.192M glycerol, 0.1% (w/v) SDS, (PH8.3); Operating condition: DC current 20mA, 80 minutes].With NA-1510 (Nippon Eido K.K.) as transfer equipment, electrophoresis arteries and veins body, filter paper that two-layer 3mm is thick (Whatman) and nitrocellulose membrane (Bio Rad) were immersed in transferring buffered dose [0.025 M Tris, 0.192M glycerol, 20% methanol (PH8.3)] of pre-preg in ice 15 minutes.On a pad, be placed with filter paper, gel, nitrocellulose membrane and according to this order filter paper, then, another pad is placed on the top, and they is placed in the conversion equipment.This equipment is placed in the ice, opens 2 hours down in the 50V DC voltage, and protein transduction is moved on on the nitrocellulose membrane.With PBS (Nissui Pharmaceutical Co., Ltd) the washing nitrocellulose membrane is (each 2 minutes, twice totally, jolting), stop (jolting is 30 minutes under the room temperature), reuse PBS (Nissui Pharm Pur GmbH) with 10%FCS/PBS then, washing nitrocellulose membrane (each 2 minutes, totally three times, jolting), and with the culture supernatant reaction (jolting is 1 hour under the room temperature) of listed all hybridomies of each table 1.After PBS (each two minutes, totally three times, jolting) washing, nitrocellulose membrane reacted 1 hour under room temperature, jolting with the anti-mouse immuning ball protein antibody of rabbit (Cappel Inc.) of the peroxidase labelling of 500 times of dilutions.With PBS (each two minutes, totally three times, jolting) washing nitrocellulose membrane, add quality solution (the 3mg/ml 4-chloro-1-naphthols of 1ml, 5ml PBS, 2 μ l aqueous hydrogen peroxide solutions) then and react, occur up to protein band.Use distilled water wash film cessation reaction for several times then.

Under above-mentioned conditions of similarity, carry out immunoblotting, with conjunctivitis chlamydia (the bacterial strain L that purifies ₂) EB analyze the chlamydial reactivity of conjunctivitis.

The result is that two unit clonal antibodies of CP-6 of the present invention and CP-7 do not present reactivity to the chlamydial MOMP of conjunctivitis, and the MOMP of Chlamydia pneumoniae is presented reactivity.

1.5 analyze antibody (to the conjunctivitis chlamydia, ornithosis virus and the chlamydial reactivity of eye conjunctiva) with fluorescent antibody technique

The conjunctiva chlamydia, ornithosis virus and the chlamydial educt difference point sample of eye conjunctiva (1 μ l) are on microscope slide, with the culture supernatant of all hybridomies shown in each table 1 (8 μ l) placed on it.Then, microscope slide is placed in wet tank, is reflected at wherein and carried out 3 hours in 37 ℃.With PBS and distilled water wash microscope slide, air drying then.The anti-mice globulin/lowlenthal serum (KPL Inc.) that after the air drying 8 μ l is diluted 10 times FITC labelling is placed on microscope slide loam cake residence spottiness.Microscope slide is placed in wet tank, reacts on and carried out under 37 ℃ 30 minutes.Washed and air drying then.Add 100 μ l encapsulating solution (50% glycerol-, detect with fluorescence microscope PBS) to form occlusion body.As a result, two kinds of monoclonal antibodies of CP-6 of the present invention and CP-7 only have bright spot with Chlamydia pneumoniae together the time at them, illustrate that these antibody and EB react.

The hybridoma that produces said monoclonal antibody CP 1 is deposited in the National Institute of Bioscience and Human-technology, Agency of Industrial Science and Technology preserving number (Accession No.): FERM P-14436 with hybridoma and CP-6; BP-5155).

Table 1

Hybridoma clone number 123456789 10 monoclonal antibody CP-1 CP-2 CP-3 CP-4 CP-5 CP-6 CP-7 CP-8 CP-9 CP-10 globulin type IgG2b IgG2b IGM IgG2b IgM IgG1 IgG3 IgM IgM IgM of the present invention are to the reactivity of CPN^*1++++++++++ to the chlamydial reactivity of conjunctivitis ^*2--+-----++

^*1: with the reactivity of DIBA method mensuration to Chlamydia pneumoniae.

^*2: measure conjunctivitis chlamydia L with the DIBA method ₂The reactivity of bacterial strain.Have only monoclonal antibody CP-3, CP-9 and CP-10 are found and are positive.

Embodiment 2

Preparation has specific monoclonal antibody (2) to the MOMP of Chlamydia pneumoniae

With with embodiment 1 in 1.1-1.3 essentially identical methods obtain to produce the clone's (CP-11) of monoclonal antibodies a bacterial strain, the solution of preparation antibody CP-11.

Measure with ISOTYPE Ab-STAT-1 kit (Sang Stat Medical), found that the globulin type of the monoclonal antibody that hybridoma cell clone CP-11 produces is IgG1.

With with embodiment 1 in the specificity of the monoclonal antibody that produces of 1.4 essentially identical methods analyst hybridoma cell clone CP-11.This monoclonal chlamydia does not present reactivity to the chlamydial MOMP of conjunctivitis, and the MOMP of Chlamydia pneumoniae is presented very strong reactivity, and the MOMP of ornithosis virus is just presented extremely weak reactivity.

In addition, use with embodiment 1 in 1.5 essentially identical methods, analyze this monoclonal antibody with indirect fluorescent antibody technique.The result is as shown in table 2.

Table 2

Reactive ^*

Chlamydia pneumoniae +++

Ornithosis virus ±

The conjunctivitis chlamydia-

Eye conjunctiva chlamydia- ^*Extremely strong reactivity: +++strong reactivity: ++ weak reactivity :+utmost point weak reactivity: ± do not observe reactivity :-

Shown in data, according to the indirect fluorescent technology with this monoclonal antibody, strong reaction can be concluded the expression Chlamydia pneumoniae; Extremely weak reaction can be concluded the expression ornithosis virus; Do not react and to conclude expression conjunctivitis chlamydia or eye conjunctiva chlamydia.

The hybridoma that produces this monoclonal antibody is deposited in the Natioal Institute of Bioscience and Human-technology with hybridoma cell line CP-11, Agency of Industrial Science and Technolgy (Accession No.FERM P-14592; Bp-5156).

Be exposed to that part of aminoacid sequence of EB film outside from the MOMP of this monoclonal anti physical ability identification Chlamydia pneumoniae of above conclusion deducibility.

Embodiment 3

The monoclonal antibody that preparation hybridoma cell line AY6E2E8 produces.

The monoclonal antibody that produces with hybridoma cell line AY6E2E8 is as monoclonal antibody.Hybridoma AY6E2E8 is preserved in the National Institute ofBioscience and Human-technology, Agency of IndnstrialScience and Technology (Accession No.FERM P-13688; BP-5154).

Give and inject the ascites that hybridoma cell line AY6E2E8 obtains containing monoclonal antibody in the mouse peritoneal, in this ascites of 1 volume, add 3 volume PBS, mix, and in 3000rpm centrifugalize 10 minutes.With pore size is that the filter of 0.22 μ m leaches the supernatant, use in advance through the super protein A post of PBS counter-balanced chromatograph Chromatop Superprotein A Column by high pressure lipuid chromatography (HPLC) (HPLC method)) (4.6mm dia. * 100mm, NGKInsulators Ltd.) purifies.

Be introduced in this post with the sample (1ml) after the filtration of 0.22 μ m filter, keep keeping adding in 4 minutes PBS with 5ml/min in 3 minutes with 1ml/min then and wash.Speed with 2ml/min in this post adds 1 liter of pure water maintenance 5 minutes, is dissolved with 8.77gNaCl in its water, 16.7g citric acid (monohydrate) and 14.72g Na ₂HPO ₄12H ₂O, the eluting monoclonal antibody.Collection contains the part of monoclonal antibody.Aqueous solution to these parts addings 2M three (methylol) aminomethane is adjusted to 7-9 with pH value.Gained solution is stored down at 4 ℃.

Embodiment 4

The antigen protein analysis of the monoclonal antibody reactive that produces with hybridoma cell line AY6E2E8

4.1 the genomic DNA of preparation Chlamydia pneumoniae bacterial strain YK-41

The EB suspension (300 μ l) of the Chlamydia pneumoniae bacterial strain YK-41 that purifies by above-mentioned 1.3 gained (protein concentration: 1.37mg/ml) under 4 ℃ with 12000rpm centrifugalize 5 minutes.Precipitate is suspended in 500 μ l to be contained in the Tris-HCl buffer (PH8.0) of 1mM EDTA (hereinafter referred to as " TE buffer ").Carry out once similarly centrifugalize again, then precipitate is suspended in the 300 μ l TE buffer.In suspension, add the SDS aqueous solution of 30 μ l 1% and the E.C. 3.4.21.64 aqueous solution of 30 μ l 1mg/ml, in 56 ℃ of constant temperature culture 30 minutes, dissolving EB.The Tris-HCl buffer (PH8.0) of adding 350 μ 1 0.1M is full in gained solution closes phenol, fully mixes with the eddy current mixer.Then, under 4 ℃,, reclaim water layer (DNA extraction liquid) with 12000rpm centrifugalize 5 minutes.Repeat this leaching process.In reclaiming water layer, add 2 μ l 10mg/ml RNase solution then,, thereby decompose RNA in 37 ℃ of constant temperature culture 2 hours.In gained solution, add the saturated phenol of 300 μ l 0.1M Tris-HCl buffer (PH8.0), chloroform and isoamyl alcohol are by the mixed liquor (hereinafter referred to as " PCI ") of 25: 24: 1 (volume ratio), fully mix with the eddy current mixer, under 4 ℃ with 12000rpm centrifugalize 5 minutes.Reclaim the gained water layer then.This operation repeats 5 times altogether.

The 10M ammonium acetate aqueous solution and 2 volume of ethanol that in gained solution, add 1/10 volume, deposit D NA5 minute, then under 4 ℃ with 12000rpm centrifugalize 5 minutes.For precipitation process, add the ethanol water of 600 μ l 70%, twice of this process with 12000rp centrifugalize 5 minutes, repeated in the mixing back under 4 ℃.Centrifuge tube was placed 15 minutes, and uncap is with dry sediment.Precipitate is dissolved among the 200 μ l TE, in-20 ℃ of storages.

4.2 preparation genomic DNA storehouse

In the genomic DNA solution of 100 μ l gained, add M-buffer of 10 μ l restriction endonucleases and the mixture of 10 μ l restriction endonucleases (by mixing AccI, each 0.4 μ l of Hae III and dilute 1/50 diluent of AluI with 20 μ l ITE), 37 ℃ of constant temperature culture 20 minutes.20 minutes response time be genomic DNA be broken down into partial hydrolysis, size is the required time of dna fragmentation of 1kbp-7kbp.Come the assaying reaction time with a spot of genomic DNA in advance.In above-mentioned reaction solution, add 100 μ l PCI, fully mix, under 4 ℃,, reclaim water layer then with 12000rpm centrifugalize 5 minutes with the eddy current mixer.In this water layer, add 3M sodium acetate aqueous solution (10 μ l) and ethanol (220 μ l), placed 15 minutes down, make the DNA precipitation of partial hydrolysis in-80 ℃.Under 4 ℃ with after the 12000rpm centrifugalize 5 minutes, the supernatant of draining.The ethanol water that adds 500 μ l 70% in precipitate mixes the back and separated 5 minutes with the 12000rpm recentrifuge.The supernatant of draining is with precipitate dried under reduced pressure.

The DNA of the above-mentioned partial hydrolysis that obtains is dissolved in the 20 μ l pure water.In 19 these solution of μ l, add 14 μ l bridging agents described below (20pmole/ μ l), 4.5 μ l 10mMATP, 4.5 μ l contain 50mM MgCl ₂0.2M Tris-HCl buffer (pH7.6), 50mM dithiothreitol, DTT and 500 μ g/ml bovine serum albumins (hereinafter referred to as " 10X complexation buffer "), 2 μ l pure water and 1 μ l T4 ligase 16 ℃ of following constant temperature culture 4 hours, thereby are connected on the DNA bridging agent.

5′-AATTCGAACCCCTTCG-3′

3′-GCTTGGGGAAGCp-5′

The DNA that connects the partial hydrolysis of bridging agent is sent into Chroma Spin 6000 posts, and mobile phase wherein is the 10mM Tris-HCl buffer that contains 0.1M NaCl and 1mM EDTA.Collect two eluates.Analyze the position of each part with 0.8% agarose-gel electrophoresis, reclaim and contain 1kbp-7kbp dna fragmentation part.In the 144 μ l part that so obtains, add 13 μ l pure water, 20 μ l 10mM ATP, 20 μ l contain 0.1M MgCl ₂0.5M Tris-HCl buffer (pH7.6), the 50mM dithiothreitol, DTT, 1mM spermidine hydrochloride and 1mM EDTA (hereinafter referred to as " 10X phosphorylation buffer ") and 3 μ l polynucleotide kinases are in 37 ℃ of following constant temperature culture 30 minutes, with 5 ' terminal phosphateization of dna fragmentation.Add PCI (200 μ l) in reaction solution, jolting makes and mixes fully, under 4 ℃ with 12000rpm centrifugalize 5 minutes.Reclaim water layer then.Add 1 μ l 20mg/ml glycogen aqueous solution in recovery layer, 20 μ l 3M sodium acetate aqueous solutions and 400 μ l ethanol are with precipitation nucleotide.Gained solution under 4 ℃ with 12000rpm centrifugalize 10 minutes.In precipitate, add 200 μ l ethanol, centrifugalize again.The supernatant of draining with the precipitate air drying, is dissolved in the 1 μ l pure water again.

In the solution that 0.6 μ l so obtains, add 1 μ l in advance with the EcoRI hydrolysis go into gtllDNA (1 μ g/ μ l, layer gene) 0.5 μ l 10X complexation buffer, 0.5 μ l10mM ATP, 0.4 μ l T4 ligase and 2 μ l pure water spend the night 4 ℃ of following constant temperature culture.With Gigapack II Gold Packaging Kit (layer gene), gained λ gtll DNA recombinant is wrapped up.

4.3 the DNA password of clone's antigen polypeptide

The Y 1090r bacterial strain loopful of Escherichia coli (E.Coli) is cultivated is adding 3ml10mM MgSO ₄, in the LB culture medium of 0.2% maltose and 50 μ g/ml ampicillins, (containing 5g NaCl in 1 premium on currency, many peptones of 10g and 5g yeast extract), in 37 ℃, constant temperature culture is spent the night under the jolting, then with 2000rpm centrifugalize 10 minutes.The MgSO that in precipitate (E.coli), adds 9ml 10mM ₄Aqueous solution also mixes.Measure the part (0.35ml) of gained E.coli suspension.λ gtll (dna library) suspension that adds 0.1-10 μ l in this suspension 37 ℃ of following constant temperature culture 20 minutes, infects E.coli with λ gtll.In the liquid LB of 47 ℃ of placements agar culture medium, add the E.coli that above-mentioned λ gtll infects in advance to 2.5ml.From the LB agar culture medium, this culture medium is scraped immediately.After the hardening of top-layer agar culture medium, cell was at 42 ℃ of constant temperature culture 3-4 hours.When observing speckle, the nitrocellulose filters (φ 82mm) that will flood in 10mM IPTG aqueous solution places above the top-layer agar culture medium, and cell is further 37 ℃ of following constant temperature culture 12 hours.Run through with the syringe needle that black ink is housed, on nitrocellulose filters, put on three asymmetric points.Remove nitrocellulose from agar culture medium then, with the 20mM Tris-HCl buffer that contains 150mM NaCl and 0.1%Tween 20 (pH7.5, " TTBS " buffer ") washing hereinafter referred to as 3 times.Agar culture medium is placed cold closet.

Nitrocellulose filters is submerged in the 20mM Tris-HCl buffer that contains 150mM NaCl and add 0.1% bovine serum albumin that (pH7.5, hereinafter referred to as " TBS buffer " shake to finish in 1 hour and interrupt reaction under 37 ℃.Then with TTBS buffer washing nitrocellulose twice, being dipped in 5-10 μ g/ml had the TTB solution of specific monoclonal antibody (AY6E2E8) to Chlamydia pneumoniae, 37 ℃ of following joltings 1 hour.Wash nitrocellulose 3 times with the TTBS buffer, (50ng/ml) of the peroxidase labelling in the TTBS buffer resists in a mouse IgG antibody solution in 37 ℃ of following joltings 1 hour then.Wash nitrocellulose 3 times with the TTBS buffer, reuse TBS buffer washing 3 times.Then filter course is soaked in the quality solution (its preparation is to add the aqueous hydrogen peroxide solution of 60 μ l 30% and the 4-chloro-1-naphthols methanol solution of 20ml 0.3% in 100ml TBS buffer), at room temperature placed about 30 minutes.When obtaining enough colors, from solution, filtering layer is reclaimed out, wash the line space air dry of going forward side by side with pure water.

Find out and agar culture medium that recognition is corresponding with color dot on the nitrocellulose on speckle.The agar that is applied in these positions with Pasteur ' s suction pipe reclaims speckle.Containing 0.1M NaCl, splash into a chloroform in the 50mM Tris-HCl buffer agent of 8mM magnesium sulfate and 0.01% gelatin (pH7.5, hereinafter referred to as " SM buffer ") after, the speckle that reclaims is put into wherein; The mixture placement is spent the night, extract the bacteriophage lambda in the speckle.Repeating said process becomes with said monoclonal antibody and can react up to all specklees.So, cloned the DNA password of antigen polypeptide.

The result has obtained expressing the bacteriophage lambda that Chlamydia pneumoniae belongs to specific antigen polypeptide, and this antigen polypeptide reacts with the Chlamydia pneumoniae monoclonal antibody specific.

4.4 cultivate the bacteriophage lambda that obtains, purification DNA

With preparing speckle with above-mentioned 4.3 essentially identical methods.Reclaim one of them speckle, put into 100 μ l SM buffer agents, place down at 4 ℃ and spend the night, extract bacteriophage lambda.In advance 250 μ l E.coli bacterium part Y1090 r-constant temperature culture in the LB culture fluid is spent the night, add bacteriophage lambda solution (5-10 μ l), placed 20 minutes down at 37 ℃, thereby infect E.coli with bacteriophage lambda.The LB culture medium that will contain 10mM magnesium sulfate in advance is heated to 37 ℃, the cell that has infected is placed on wherein cultivates, and cell causes that up to going into phage E.coli dissolves in being shaken down in 37 ℃ of constant temperature culture 5-7 hours.Add 250 μ l chloroforms then, with 3000rpm centrifugalize 10 minutes to remove the E.coli cell residue.So obtain the bacteriophage lambda float.With Wizard λ Preps Kit (Promega) purification lambda bacteriophage dna.

4.5 amplify the DNA password of antigenic polypeptide of chlamydia pneumoniae

In 600 μ l microtubules, put into 61.5 μ l pure water, (Tris-HCl buffer, pH8.3 contain 500mM KCl to 10 μ l 10X reaction buffers, 15mM MgCl ₂With 0.01% gelatin), 1 μ l 20mM dNTP, 0.1 the lambda bacteriophage dna solution that μ l obtains, 1 μ l 20nM λ gtll forward substrate (Takara Shuzo Co., Ltd.), (Takara Shuzo Co. Ltd.) with 0.5 μ l AmpliTaq archaeal dna polymerase, drips 2-3 mineral oil and is paved into layer on its top the reverse substrate of 1 μ l 20nM λ gtll then.Constant temperature culture under the following conditions, 94 ℃, 30 seconds, 55 ℃, 30 seconds, 73 ℃ 2 minutes, repeat 30 times, thereby amplify DNA.After reaction is finished, reactant mixture is carried out cold melt agarose colloid electrophoresis.The DNA that amplifies is cut off, purify with Wizard PCR Prep Kit (Promega).

4.6 analyzing DNA basis sequence

By fluorescently-labeled terminator cyclic sequence method, with Taq archaeal dna polymerase analyzing DNA basis sequence, the DNA that amplifies with PCR makes model, and product is supplied with Model373A DNA Sequencer (Applied Biosystems).With the DNA basis sequence cut of gained, ligation is measured their aminoacid and is translated the position with DNA sequence software " DNASIS " (Hitachi SoftwareEngineering).

The result is, finds the lambda bacteriophage dna of gained, is the password of aminoacid sequence of the 53K dalton antigen protein of Chlamydia pneumoniae.

The monoclonal antibody that hence one can see that hybridoma cell line AY6E2E8 produces is identified as antigen with 53 of Chlamydia pneumoniae with your antigen protein.

Embodiment 5

Prepare fluorescently-labeled antibody

According to people's such as aforementioned Ivan Lefkovits method, give hybridoma cell line CP-11 and the AY6E2E8 labeling of monoclonal antibody of generation respectively with FITC.

(Sigma, Cat No.F1628) are dissolved among the DMSO, preparation 10mg/ml solution with FITC-Celite.

On the other hand, use the NAP-10 post, use 0.1M NaHCO ₃Buffer (pH8.2-8.6) replaces being dissolved with the buffer of monoclonal antibody.

Monoclonal anti liquid solution (the monoclonal anti body burden: 2mg), add 50 μ l FITC solution, under 4 ℃, left standstill 8 hours that obtains to 1.5ml.

The gained reaction solution is used the PBS balance by the PD-10 post.Remove unreacted FITC with PBS.So obtain the monoclonal antibody of FITC labelling.

Embodiment 6

The preparation freeze-dried preparation

Monoclonal antibody with 1.9mg FITC labelling, 7.6mg Evans blue (WakoPurechemical Co., Ltd.), 1.9g bovine serum albumin (GIBCO BRL) and 1.9ml 10%NaN3 (Wako Purechemical Co., Ltd.) be dissolved in PBS (-) (Nissui Pharmaceutical Co., Ltd.) in to cumulative volume be 100ml.

Gained solution is dispensed in the 1ml bottle, places 1 hour down at-80 ℃, then (Iwaki in freeze dryer; Co., Ltd.) lyophilized overnight.So obtain freeze-dried preparation (being CP-11 preparation and AY6E2E8 preparation).

On the other hand, with method similar to the above, use the blended freeze-dried preparation of Monoclonal Antibody of the FITC labelling of hybridoma cell line CP-11 and AY6E2E8.Embodiment 7 usefulness direct fluorescent antibody techniques are given the dyeing of Chlamydia pneumoniae occlusion body

Each freeze-dried preparation by embodiment 6 preparations is dissolved in the 2ml pure water.Have at point on the microscope slide of Chlamydia pneumoniae bacterial strain YK-41 and put 8 μ l gained solution, all some upper parts are all covered by this solution.Then, microscope slide is placed in wet tank, is reflected at wherein and under 37 ℃, carried out 30 minutes.Washed and air drying.Product detects with fluorescence microscope with 100 μ l encapsulating solution [50% (v/v) glycerol-PBS] embedding.The results are shown in Table 3.

Table 3

The preparation staining power ^*

The CP-11 preparation+

The AY6E2E8 preparation+

Mix preparation ++

^*Extremely strong reactivity: ++

Strong reactivity+-

As shown in table 3, no matter the CP-11 preparation still is the AY6E2E8 preparation all presents strong reactivity, makes the detection of Chlamydia pneumoniae become possibility.Compare with unitary agent, mix preparation presents extremely strong reactivity, can detect Chlamydia pneumoniae well with this reagent.

The monoclonal antibody that major outer membrane albumen to Chlamydia pneumoniae of the present invention has a specific reaction special detect in the Chlamydia pneumoniae very useful because this monoclonal antibody has specific reaction to the MOMP that contains in a large number in the microorganism.In addition, can finish detection with a spot of this antibody.

The feature of monoclonal antibody of the present invention is that also it does not present any reactivity to the chlamydial major outer membrane albumen of conjunctivitis, is suitable for infection involving chlamydia pneumoniae and conjunctivitis chlamydia infection are made a distinction, and the latter often separates separately clinically.So it is very useful that this antibody aligning is made a definite diagnosis the infection involving chlamydia pneumoniae that breaks.

In addition, labelling of the present invention antibody make it possible to utilize material to detect and measure Chlamydia pneumoniae with this labelling.Therefore, these antibody are very useful to a large amount of samples of fast processing in the clinical trial.

The monoclonal antibody that 53K dalton antigen protein to Chlamydia pneumoniae of the present invention has reactivity and made labelling is aimed at and to be made a definite diagnosis disconnected infection involving chlamydia pneumoniae of great use, because this antibody can detect the 53K dalton antigen protein of the specific antigen that belongs to as Chlamydia pneumoniae.

The cell line that produces said monoclonal antibody can be used for continuously, prepare in large quantities these antibody.These cell lines also can be used as the source of antibody gene.

These cell lines of usefulness of the present invention prepare monoclonal antibody method can be in order to prepare above-mentioned monoclonal antibody continuously and in large quantities.

The method of detection of the present invention and mensuration Chlamydia pneumoniae and the reagent of detection and mensuration Chlamydia pneumoniae can be used in the method that very accurately detects and measure Chlamydia pneumoniae.

The diagnostic method of infection involving chlamydia pneumoniae of the present invention and the diagnostic reagent of infection involving chlamydia pneumoniae can be used in the method for very accurately diagnosing Chlamydia pneumoniae.

The method of the cell line of generation monoclonal antibody produced according to the present invention can prepare the cell line of anti-those the proteic monoclonal antibodies of effective generation, and be difficult to finish anti-those proteic MONOCLONAL ANTIBODIES SPECIFIC FOR with conventional method.The monoclonal anti physical ability that cell line produced that the present invention produces antibody is used for detecting especially the various diagnostic methods and the diagnostic reagent of target protein, and they also can be used as medicine.

The cell line of above-mentioned generation antibody can also be used as the source of antibody gene in order to produce these monoclonal antibodies continuously and in large quantities.

Preparing monoclonal antibody method with above-mentioned cell line can be in order to prepare said monoclonal antibody continuously and in large quantities.

Claims (35)

1. the major outer membrane albumen to Chlamydia pneumoniae has atopic monoclonal antibody.

2. monoclonal antibody as claimed in claim 1, it does not have reactivity to the chlamydial major outer membrane albumen of conjunctivitis.

3. monoclonal antibody as claimed in claim 1, its elementary body to Chlamydia pneumoniae has reactivity.

4. monoclonal antibody as claimed in claim 3, it does not have reactivity to the chlamydial major outer membrane albumen of conjunctivitis.

5. monoclonal antibody as claimed in claim 1, antibody wherein is labeled.

6. monoclonal antibody as claimed in claim 1, antibody wherein are that hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156) produce.

7. monoclonal antibody as claimed in claim 6, antibody wherein are that hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156) produce, and are labeled subsequently.

8. monoclonal antibody has reactivity and is labeled the 53KDa antigen protein of Chlamydia pneumoniae.

9. monoclonal antibody as claimed in claim 8, its occlusion body to conjunctivitis chlamydia and ornithosis virus does not have reactivity.

10. monoclonal antibody as claimed in claim 8, antibody wherein are that hybridoma cell line AY6E2E8 (FERM BP-5154) produces, and are labeled subsequently.

11. a generation has the preparation method of the generation antibody cell of atopic monoclonal antibody to protein, this method comprises to animal immune inoculates a kind of of natural and Denatured protein, and with other albumen booster doses, obtain a kind of cell that produces antibody, and merge the cell of this generation antibody with the myeloma cell.

12. method as claimed in claim 11 comprises to animal immune and inoculates a kind of native protein also with a kind of Denatured protein booster dose, obtains a kind of cell that produces antibody, and merges the cell of this generation antibody with the myeloma cell.

13. method as claimed in claim 11, Denatured protein is wherein handled native protein with detergent or Reducing agent and is obtained.

14. method as claimed in claim 11, albumen wherein are chlamydial major outer membrane albumen.

15. the cell of the generation monoclonal antibody that method as claimed in claim 11 is prepared.

16. a cell that produces monoclonal antibody, this cell can produce monoclonal antibody as claimed in claim 1.

17. the cell of generation monoclonal antibody as claimed in claim 16 is that the hybridoma bag is CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156).

18. one kind prepares monoclonal antibody method, comprises the cell of cultivating generation monoclonal antibody as claimed in claim 15.

19. one kind prepares monoclonal antibody method, comprises the cell of cultivating generation monoclonal antibody as claimed in claim 16.

20. detect and/or measure the method for Chlamydia pneumoniae with monoclonal antibody as claimed in claim 1.

Produce 21. method as claimed in claim 20, monoclonal antibody wherein are hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156), and be labeled subsequently.

22. monoclonal antibody as claimed in claim 8 detects and/or measures the method for Chlamydia pneumoniae.

Produce 23. method as claimed in claim 22, monoclonal antibody wherein are hybridoma cell line AY6E2E8 (FERM BP-5154), and be labeled subsequently.

24. a reagent that detects and/or measure Chlamydia pneumoniae is comprising monoclonal antibody as claimed in claim 1.

Produce 25. reagent as claimed in claim 24, monoclonal antibody wherein are hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156), and be labeled subsequently.

26. a reagent that detects and/or measure Chlamydia pneumoniae is comprising monoclonal antibody as claimed in claim 8.

Produce 27. reagent as claimed in claim 26, monoclonal antibody wherein are hybridoma cell line AY6E2E8 (FERM BP-5154), and be labeled subsequently.

28. method with monoclonal antibody diagnosis infection involving chlamydia pneumoniae as claimed in claim 1.

Produce 29. method as claimed in claim 28, monoclonal antibody wherein are hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156), and be labeled subsequently.

30. method with monoclonal antibody diagnosis infection involving chlamydia pneumoniae as claimed in claim 8.

Produce 31. method as claimed in claim 30, monoclonal antibody wherein are hybridoma cell line AY6E2E8 (FERM BP-5154), and be labeled subsequently.

32. a reagent of diagnosing infection involving chlamydia pneumoniae, comprising monoclonal antibody as claimed in claim 1 as active component.

Produce 33. diagnostic reagent as claimed in claim 32, monoclonal antibody wherein are hybridoma cell line CP-6 (FERM BP-5155) or CP-11 (FERM BP-5156), and be labeled subsequently.

34. a reagent of diagnosing infection involving chlamydia pneumoniae, comprising monoclonal antibody as claimed in claim 8 as active component.

Produce 35. diagnostic reagent as claimed in claim 34, monoclonal antibody wherein are hybridoma cell line AY6E2E8 (FERM BP-5154), and be labeled subsequently.