CN1057072A - Monoclonal anti IgM antibody, their production and purposes, and the hybridoma that produces same material - Google Patents
Monoclonal anti IgM antibody, their production and purposes, and the hybridoma that produces same material Download PDFInfo
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Abstract
A main aspect of the present invention relates to rat monoclonal antibody, it
(a) with IgM hypotype antibody selective binding;
(b) be IgGs;
(c) do not combine with IgG1 or IgG2 hypotype.
Preferred embodiment is that of 2G10 done in name in these antibody, with and function equivalent.
Description
The invention belongs to immunology and monoclonal antibody and produce the field.More specifically, it relates to monoclonal anti IgM antibody, produces the hybridoma of these antibody, the immuno-chemical substance that makes from these antibody, and the purposes of those immuno-chemical substances.
Since Kohler and Milstein delivered the report of manufacture order clonal antibody is described after, production can produce the immortal lymphocytic technology that is predetermined specific antibody and be developed, this develops clinical and basic scientific research, and is applicable to that the diagnosis of very large-scale pathology disease and the therapeutic method of treatment have significant effects.
Antibody is the endogenous protein that immunity system is replied the antigenic stimulation deposits yields.These albumen combine with specific site (epi-position) specificity on the antigen molecule.Polyclonal antibody is from the animal derived with antigen immune, and they combine with antigen in multiple site (epi-position).On the other hand, monoclonal antibody is the antibody group of a specific qualification, and they are derived from the single clone (mono-clonal) of the cell of generation specific antibody.Opposite with polyclonal antibody, monoclonal antibody only combines with a specificity epitope on antigen molecule.
Had some times though produce the technology of monoclonal antibody, existing method is time-consuming, effort, though and usually generation has specificity to target antigen, but, therefore, in various application, has the purposes of limitation to the antibody of this antigen tool than low-affinity.
In producing difficulty useful and that related monoclonal antibody ran into clinically, have one to be the rich of the IgM hypotype antibody that obtains from hybridoma, hybridoma by in the body or external standard immunoassay method produce.IgM hypotype antibody is low-affinity normally, more difficult purifying, and usually contain the most of antibody that produce by hybridoma.In addition, in the mixed culture of the hybridoma of secreting IgM and IgG, the growth of the cell of secretion IgM usually surpasses the hybrid cell of IgG secretion.
The part of producing the effort step of hybridoma is to eliminate the hybridoma of the production IgM that produces after cytogamy.This is done in such a way that usually by the limited dilution cloning cell, makes individual cells grow into the clone, tests the clone that each clone determines to produce IgG hypotype antibody alone.Usually, further analyze the antigen-specific of the hybridoma of generation IgM then with definite antibody that is produced.
Though reported the antibody that resists epi-position on IgM antibody, antibody that all are surveyed so far and IgG hypotype antibody is responding property also.
Applicant and other people disclose cytotoxic reagent have been linked to each other with antibody to produce the method for " immunotoxin ".Focus on recently and combining (Nevelle on the immunotoxin that produces by different-double function reagent with the enzymic activity part (A chain) of the toxin of bacterium or plant-sourced by monoclonal antibody, D.M.and Youle, R.J., 62:75-91 Immunol Rev(1982); Ross, W.C.J., et al., European J Biochem(1980) 104; Vitteta, E.S., et al., Immunol Rev(1982) 62:158-183; Raso, V., et al., Cancer Res(1982) 42:457-464; Trowbridge, I.W and Domingo, D.L., Nature(Cond) (1981) 294:171-173.
A main aspect of the present invention is about rat monoclonal antibody, they
(a) with IgM hypotype antibody selective binding;
(b) be IgGs;
(c) not with IgG
1With IgG
2The hypotype combination.
The preferred embodiment of these antibody is that 2G10 and functionally equivalent thereof are done in name.Producing the rat * big murine hybridoma of above-mentioned antibody and the offspring of these hybridomas is others of the present invention.
The present invention also comprises and prepares the method for hybridoma as defined above, comprises rat tumor cell and the rat spleen cells that obtains from the rat with mouse IgM hypotype immunogen immune are merged, and selects as above institute's fixed output quota to give birth to the hybridoma of antibody then.
The present invention further comprises the method that produces the antibody be defined as above, comprises cultivating having the hybridoma that produces this antibody ability, or the hybridoma that at random prepares by aforesaid method.
Another aspect of the present invention relates to immunotoxin and their preparation method, by combination
(a) said monoclonal antibody, and
(b) cytotoxicity part or magnetic bead.
Another part of the present invention relates to the labeled derivative thing of said monoclonal antibody, and antibody is by the detectable label substance markers, and derivative can be used for deciding target, and specificity is selected or classification.
Another aspect of the present invention relates to kills the method that produces IgM hybridoma or B cell, contacts with the one or more above-mentioned immunotoxin of cell killing significant quantity by making cell.
Others of the present invention are direct or indirect immunoassays, whether produce IgM antibody to measure cell, or measure whether antibody is the IgM abnormal shape.These analyses relate to hatches cell and monoclonal antibody or its labeled derivative thing.When the applying marking derivative, can directly read out in the existence of mark binary immunocomplex on the cell.When using unmarked antibody, cell is further hatched with the traget antibody of anti-monoclonal antibody, reads out in the existence of mark ternary immunocomplex on the cell then.
Fig. 1 explanation is screening IgM specific antibody in the hybridoma supernatant liquor.
Fig. 2 illustrates the dose-dependently specificity combination of antibody 2G10.
Fig. 3 explanation increases the effect that absorbs rat immune globulin in the 2G10 combination.
Fig. 4 is illustrated in 2G10 selectivity identification mouse IgM in the indirect ELISA.
Fig. 5 is by the purity of SDS-PAGE explanation 2G10 antibody.
Fig. 6 represents rat 2G10 antibody subclass feature by the Wu Hetelangshi immunodiffusion(ID).
Fig. 7 illustrates that utilization 2G10 combines with the cell of expressing IgM subclass antibody.
The elution profile of Fig. 8 explanation immunotoxin (binding substances of 2G10 and gelonin) on gel-filtration matrix.
Fig. 9 is by the purity of SDS-PAGE explanation 2G10-gelonin immunotoxin.
Figure 10 illustrates that immunotoxin (2G10-gelonin) combines with mouse IgM specificity.
For making easier being well understood of the present invention of describing here, provide following detailed description.
As used here, term " monoclonal antibody " refers to have homologous antibody group's antibody compositions. Mode about antibody source or its manufacturing is also unrestricted.
As wherein used, resist-mouse IgM antibody about the rat monoclonal that increases, term " functionally equivalent " refers to monoclonal antibody, it: the monoclonal antibody that the blocking-up that (a) intersects is increased; (b) selectively be combined with mouse IgM antibody; (c) has the G abnormal shape; And (d) different I gG1Or IgG2Special-shaped combination.
As used here, about the hybridization knurl of product monoclonal antibody of the present invention, term " offspring " comprises all derivatives of parental generation hybridization knurl, offspring and descendants, and this kind hybridization knurl produces the anti-mouse IgM of the monoclonal antibody that the parent produces, no matter age or caryogram uniformity.
The present invention can be used to produce the antibody of being combined with any kind IgM antibody. The present invention teaches and only need be used for obtaining the stable hybridization oncocyte system that produces continuously for the anti-IgM of immune kind that reaches. Preferably, anti-IgM monoclonal antibody of the present invention is for mouse or people IgM.
Being used for producing antibody that the present invention hybridizes knurl produces and merges the partner by producing with mouse IgM antibody mediated immunity rat. With the mouse IgM antibody in Freunds adjuvant of immune commercial weight to inoculating in subcutaneous rat and the peritonaeum, and then with the immunogene in adjuvant of analog quantity to promote. Obtain spleen from immune rat in last lifting after several days, from then on prepare then cell suspension and be used for merging.
With conventional somatocyte hybriding technology (Kohler, B.and Milstein, C., Nature(1975) 256:495-497[as modified by Buck, D.W., et al, In Vitro(1982) 18:377-381] preparation hybridization knurl from splenocyte and rat tumor homologue. Available available rat bone myeloma in hybridization, such as YB2/0 and Y3-Ag1.2.3. basically, the technology that merges tumour cell and splenocyte relate to use former such as the fusion of polyethylene glycol. After the fusion, isolated cell and being grown in the selective growth culture medium from merge culture medium such as the HAT culture medium, is not hybridized parental cell to remove. If necessary, amplified hybridization Knurl is by the immune detection method of tradition (such as radioimmunoassay, EIA enzyme immunoassay, or fluoroimmunoassay), with immunizing agent IgG1，IgG
2With IgM(mouse IgM antibody) as antigen, analyze anti-mouse IgM activity in the supernatant. Identify further whether positive colony has reached the standard of antibody of the present invention to measure them.
Can be in body or the growth of external use known method produce the hybridization knurl of these antibody. Monoclonal antibody can be separated from culture medium or body fluid, described in case, and traditional immunoglobulin (Ig) purification step, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and hyperfiltration, if necessary.
The important feature of monoclonal antibody is (1) their immunoglobulin class, (2) their selective to mouse IgM antibody, and (3) they differentiate and with the purposes of producing mouse IgM hybridization oncocyte and being combined.
Selective and the scope of given antibody is produced IgG by its anti-(1) of test1，IgG
2And the hybridization oncocyte of IgM and (2) IgG1，IgG
2Measure with IgM antibody hurdle. When selecting described antibody, the super first hybridization knurl culture that has screened about 162 growths. 9 clones and mouse IgM antibody response, but do not react with IgG. Select one of them clone with further evaluation.
With N-amber acid imide acyl-3-(2-pyridine two sulfo-s) propionic ester (SPDP) or imino group mercaptan alkane (IT) makes expression can accept the selective antibody of being connected with scope as coupling agent to be connected with gelonin. Whether the coated plate (Figure 11) of the test anti-Igm of bond and IgG has still preserved the antibody specificity to be determined at chemical coupling behind toxin.
The feature of this antibody further is provided in following embodiment in detail.
In the immunochemistry derivative of monoclonal antibody of the present invention significant be immunotoxin (bond of antibody and cytotoxicity part) and mark (such as radio-labeled, enzyme labeling, magnetic mark or fluorochrome label) derivative, wherein mark provides discriminating and/or classification to comprise the method for the immune complex of labelled antibody.
The cytotoxicity part of immunotoxin may be a cytotoxic drug, or the enzyme activity toxin of bacterium or plant-sourced, or the enzymic activity fragment of this toxin (" A " chain).Be preferably enzyme activity toxin and fragment thereof, their example is: gelonin, diphtheria A chain, the non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, yearning between lovers legumin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii albumen, dianthin albumen, Phytoiacca americana albumen (PAP I, PAP II, and PAP-S), momordica charantia inhibitor, curtin, crotin, saponaria officianalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.The best is gelonin.Available various double functional protein coupling agent makes the binding substances of monoclonal antibody and this cytotoxicity part.The example of these reagent has SPDP, IT, the double functional deriv of imido-ester; as dimethyl adipimidate-HCl; active ester is as two succinimide acyl suberates, aldehyde such as glutaraldehyde, and two-azido cpd is as two (p-azidopenzoyl) hexanediamine; two-overlappingization derivative is as two (para diaminobenzene formyl radical) quadrol; two isocyanic ester are as 2, and the two isocyanic acid benzyl support of 6-ester reaches two-active fluorine cpd as 1; 5-two fluoro-2, the 4-dinitrobenzene.
When being used for killing the external hybridoma that produces mouse IgM antibody, the binding substances typical case who joins in the cell culture medium is with the concentration at least about 10nm.The preparation of external use and form of medication are undemanding.Usually use and culture or the suitable mutually aqueous compositions of diffusion substratum.Can read cytotoxicity by routine techniques, produce existing or its degree of IgG hybridoma to measure.
When the body planted agent produced the IgM cell in order to compacting, immunotoxin gave immune animal (i.e. the amount that the IgM splenocyte is produced in elimination or minimizing) with the treatment significant quantity.Usually they are non-enteron aisle ground, are preferably intravenous administration.Dosage and dosage regimen depend on the characteristic of the product IgM cell that will suppress, the feature of specific immunotoxin, and as its therapeutic index, and effect is initial.The immunotoxin amount that gives typically arrives within the scope of about 10 ml/kg body weight about 0.1.
During parenterai administration, immunotoxin will be made unitary dose injectable type (solution, suspension, milk sap) with pharmaceutically acceptable non-enteron aisle vehicle.These vehicle are atoxic from birth, and right and wrong are curative.The example of these vehicle is a water, salt solution, Ringer's solution, glucose solution, and 5% human serum albumin, also available non-water quality vehicle such as solidified oil and ethyl oleate.Liposome can be used as carrier.Vehicle can contain the material that minor amounts of additives if can increase isotonicity and chemical stability, as damping fluid and preservatives.Immunotoxin typically is made into the concentration of about 1 mg/ml to 10 mg/ml in this figuration body.
Being used to eliminate the radioactive cytotoxic drugs that produces the IgM hybridoma can (as Y, Pt) prepare with antibodies by making high linear energy transfer (LET) emitting isotope.Wherein used term " cytotoxicity part " meaning comprises this isotropic substance.
The mark that is used to prepare the antibody labeling translation comprises the part that can directly detect, as fluorescence dye and radio-labeling, and must react or derive just can be detected part, as enzyme.The example of these marks is
32P,
125I,
3H,
14C, fluorescein and derivative thereof, rhodamine and derivative thereof, dansyl, umbelliferone, fluorescein, 2,3-dihydro naphthyridine diketone, horseradish peroxidase, alkaline phosphatase, N,O-Diacetylmuramidase and G-6-P salt desaturase.Antibody can be connected with these marks by currently known methods.For example, coupling agent such as aldehyde, carbodiimide, dimaleimide, imidate, succinimide, two-diazotization benzadine and analogue thereof can be used to make antibody with the fluorescence of above-mentioned description, and chemiluminescent and enzyme labelling is connected.The also available marked by magnetic bead of antibody is to be used for the magnetic sorting technique.
Antibody and traget antibody can use in various cell divide methods, separate with product IgG hybridoma so that produce the IgM hybridoma, perhaps eliminate from the culture that contains these cells and produce the IgM hybridoma.
Analytical technology commonly used comprises directly and indirect analysis.Direct analysis comprises hatches hybridoma or unknown haterotypic antibody and traget antibody of the present invention.Produce the IgM cell if contain in the sample, traget antibody will combine with these cells.With after removing non-binding not traget antibody, read sample existing at washed cell with the certification mark immunocomplex.In indirect analysis, cell sample is hatched with labeled monoclonal antibody not.Sample uses the traget antibody of anti-monoclonal antibody (as mark Chinese People's Anti-Japanese Military and Political College murine antibody) to handle then, and washing and reading exist with mark note ternary complex.
For diagnostic use or analyze to measure existing of IgM abnormal shape, antibody typically is dispensed in the diagnosis box form.These test kits will typically comprise: the antibody of mark or unmarked type is hatched and washing reagent in suitable containers, if mark Chinese People's Anti-Japanese Military and Political College murine antibody diagnosis box is to be used for indirect analysis, and the substrate or the derivatization reagent that depend on the mark characteristic.Also can comprise IgM antigen control and guidance.
Following examples provide the preparation of monoclonal antibodies representative of the present invention, the qualitative and detailed description of using.These embodiment do not limit the present invention in any way.
The source of rat anti mouse IgM monoclonal antibody and qualitative
From Dr.Joanne Trial, Department of Immunology, M.D.Anderson Cancer Center obtains name and makes 58.6 big murine hybridoma.Originally, 58.6 clones secretion rat antibody.Clone unstable and also go down to posterity for four times cultivate after, 58.6 cells stop to produce any antibody.An original race of cell clones by limiting dilution then, to obtain to stablize and produce continuously the clone of IgM antibody.
A) clone by limiting dilution
58.6 cell at Iscove ' s, in 37 ℃ of cultivations 3 days, is containing 5%CO in the substratum
2In the moist atmosphere environment of air, when the amplifying cells culture reached 50% fusion, centrifugal cell harvesting was counted with hematimeter.Cell is in 50% fresh culture and 50% conditioned medium (wherein 7 days substratum of 58.6 cells growth) dilution, and then, the cell in about 1 hole is put on 96 orifice plates.When the individual cells in the hole grows up to microcoenosis (about 12 days), remove substratum, as the anti-IgM antibody of analysis as described in the embodiment 2.Increase positive cell with scale operation antibody, frozen cell group, further specialization antibody.When further giving the clone type and producing the specific characteristics of antibody, suitable clone is heavily cloned and is increased with the frozen cell original seed, is injected into pristane and handles nude mice to produce ascites.
B. freezing hybrid cell
When the hybrid cell that is fixed in the T75 bottle reaches 70% when merging, centrifugal collecting cell also is resuspended to spherical centrifugal thing in 0.9 milliliter the foetal calf serum.Moment in cryovial, joins cell transfer in each bottle with 0.1 milliliter of methyl-sulphoxide before freezing.Bottle is stored in the liquid nitrogen.
C) generation of ascites liquid
About 10
7Hybridoma washs in the serum-free medium, and intraperitoneal injects nude mice, and these nude mices were injected into 0.5 milliliter of pristane before 7 to 14 days.Usually in 1 to 3 week, form ascites, collect ascites from the film intraperitoneal with gauge needle.Liquid collecting goes into to contain in the test tube of 5 milliliters of PBS and 20mM EDTA.After 2000 * g is centrifugal 10 minutes, collect supernatant liquor, make it in sodiumazide, to become 0.1%, be divided into little branch sample 4 ℃ of storages or freezing at-20 ℃.This liquid provides the abundant source (about 5-10 mg/ml) of monoclonal antibody.
Produce the selection and the analysis of the hybridoma of rat anti mouse IgM antibody
The hybridoma group that is chosen in the about 500-1000 cell of stand density in 2 weeks further analyzes.Produce antibody with mouse IgM antibodies for measuring which hybridoma, carry out the existence that Enzyme Linked Immunoadsorbent Assay (ELISA) detects rat anti mouse IgM in the hybridoma substratum of these groups according to people's such as Voller (ref) method.(Sigma chemical Company, St.Louis MO.) are cushioned liquid (50mM NaHCO at bag for 100 milligram of 1 mg/ml purifying mouse IgM or IgG protein
3, pH9.8) in the dilution, by in moist chamber 4 ℃ hatch, on 96 hole titer plate, absorb and spend the night.With the phosphoric acid buffer salt solution (PBS-Tween) that contains 0.2%Tween-20 the hole is washed three times.Remove in the hole behind all liquid traces washing back and patting on paper handkerchief gently, and 100 microgram hybridoma supernatant liquors are added bag by in the hole of IgM, then incubated at room 2 hours.Plate washs once more with PBS-Tween, and hatches at room temperature combines goat-anti-rat IgG with peroxidase in PBS-Tween 100 microlitre 1:1000 diluents.As above-mentioned the hole is washed once more after, they and 100 microlitre 1mM ABTS(2,2-azino-two (3-ethyl-benzthiazoline sulfonic acid) at 0.1M sodium citrate buffer solution pH4.2 in 20-60 minute (damping fluid contains 0.03% hydrogen peroxide) of 37 ℃ of reactions.On MicroElisa Reader in 405nm measuring light density.
For estimation is on the heavy chain or light chain of antibody by the epi-position of anti--IgM antibody recognition, tested 9 portions of hybridoma supernatant liquors with IgM-lambda and IgM-Kappa protein bag combining by plate.It is all similarly good that two of hybridoma IC2 and 2G10 and IgM-Kappa and IgM-lambda bag are combined by the hole, and the binding specificity that shows anti-IgM antibody is on weight (mu) chain of IgM antibody.Other 7 antibody of test are also with mouse IgM combination, and this is represented by the result on Fig. 1.As Figure 1A and 1B finding, 9 different hybridomas of test and coat a kind of combination in IgM-Kappa or the IgM-lambda plate.Carry out the standard ELISA analysis to measure and each plate bonded rat immunity.Find that all antibody are all combined by the hole with IgM-K and IgM-1 bag.In the topnotch binding antibody, select 1C2 and 2G10 with further research.Because its growth and the feature of producing antibody have been selected antibody 2G10 at last.This provides 0.3 ± 0.03(standard error) optical density(OD).Background with the substratum that does not have rat monoclonal antibody is 0.06 ODU (O.D.).Preserve the hole that surpasses or equal 0.3 O.D. down with anti-mouse IgM antibody response.
Test the specificity of rat mono-clonal 2G10 antibody under various conditions by ELISA.For measuring the selectivity identification of 2G10, make 100 nanogram(ng) mouse IgM-lambda albumen or IgG to IgM vs IgG
3-lambda protein adsorption is on titer plate (100 microlitre), with the 2G10 antibody incubation that increases concentration.As shown in Figure 2, when antibody dosage in 2-1000 nanogram(ng) scope, 2G10 and IgM bag is closed greater than being hardened with the IgG bag by the titer plate combination degree.Under 50 nanogram(ng)s and following 2G10 antibody dosage, surveys to go out combination at the IgG bag on by plate, and IgM-wraps and is discerned by 2G10 by the combination on the hole.
Also having checked increases the immunoglobulin (Ig) bag by the effect of concentration on titer plate, and the identification of antibody 2G10.Represented as Fig. 3,2G10 antibody (dosage with 100 nanogram(ng)s in 10 microlitres adds) is incubated in IgG
3Wrap by in the hole with IgM., expressed with IgM and wrap by under the concentration at all bags by the plate selective binding.Compare with IgM-Kappa at 10000 nanogram(ng) IgM or IgG(IgG3-Kappa) bag by under the concentration, by ELISA, measure IgM bag by plate than IgG bag reactive big 10 times by on the plate, 2G10 antibody has shown the selective binding with IgM once more.
Also tested the ability of antibody 2G10 selectivity identification IgM in indirect ELISA is analyzed., on titer plate, and contain the hybridoma developing medium of discerning these antigenic antibody and hatch antigen coated.In analyzing, this has used IgM and IgG
1Antibody subclass.Behind antigen coated microplate and their interreaction antibody incubations, in the hole, add the rat 2G10 antibody that increases concentration, assess the combination of 2G10 by ELISA.As shown in Figure 4,2G10 discerns the closed circle of corresponding antigen bonded IgM(with it sensitively and optionally), under similarity condition, can not survey the IgG(open circle), though IgG
1Existence can easily use with the antibody of all responding property of rat immune globulin hypotype and survey.These results show the selectivity identification of 2G10 rat monoclonal antibody to mouse IgM subclass immunoglobulin (Ig).Rat monoclonal antibody also can be identified in the IgM antibody of pico-gram dosage, shows its high affinity to mouse IgM antibody.
The evaluation of rat anti mouse IgM antibody
Various hypotypes (IgM-Kappa and lambda, IgG
1, IgG
2And IgG
3) rat immune globulin be coated on the titer plate as described in the embodiment 2, further identify the combination (in embodiment 2, being male) of the rat anti mouse IgM antibody that produces by hybridoma.With Bio-Tek elisa plate reader, all plates are read at the place at the 405nm wavelength.Absorption value (compared with the control) is used as the sign that anti-mouse IgM antibody exists.
The purifying of 2G10 rat monoclonal antibody and qualitative
Selection is named representative rat anti mouse IgM monoclonal antibody into 2G10 with further qualitative.As shown among the embodiment 3,2G10 antibody is male to IgM kappa and lambda.By centrifugal and ammonium sulfate grading purification antibody.
The representative hybridoma of called after 2G10 lies in and is deposited in American type culture collection (ATCC) April 23 nineteen ninety, and (Rockville, the Md. U.S.), preserving number is HB 10436.According to other the national patent law and the rules of the U.S. and this patent application, the preservation thing is available.The available of preservation thing does not give license licensed licenser licence and implements the present invention, to destroy the present invention who has applied for, reaches any division or the continuation application of this application case.
By slow adding equal-volume 90% saturated ammonium sulphate solution, make the 2G10 culture supernatant (or from nude mice ascites) of 45% saturated (saltouing) in ammonium sulfate liquid.Sample is stirred 30 minutes at 4 ℃, at 20,000 * g centrifugal 30 minutes then.Globe is resuspension in 40% saturated ammonium sulphate solution, stirs the centrifugal as mentioned above then thing of glomeration again 30 minutes.Globe is resuspension in water, and to 100 volume PBS dialysis.The aliquot of solution is used to measure protein content (by the optical density(OD) of 280nm), purity (passing through SDS-PAGE), and binding specificity (passing through ELISA).Last antibody-solutions is freezing during until needs at-20 ℃.
IgM antibody is further purified by ammonium sulfate precipitation and gel-filtration, filters to contain agarose, carry out on 2.6 * 40 centimetres of chromatographic resins of dextran and/or allylamine, with the PBS/0.01% sodiumazide in room temperature with 1 ml/min flow velocity wash-out.
The subclass of 2G10 rat monoclonal antibody is measured (Ouchterlony and Nilsson(1958) in Handbook of Exp.Immun.Weir by Wu Hetelang Nissl method, ed., Blackwell Scientific, London, pp 19.1-19.44), with passing through ICN Immunobiologicals(Lisle, IL.) commercial available immunodiffusion(ID) diagnosis box.
Antibody 2G10 subclass is made a difference on this molecule of purifying how assessing.Analyze utilization Wu Hetelang Nissl immunodiffusion technique diagnosis box for carrying out subclass.In brief, in each satellite hole, add the antiserum(antisera) of anti-various rat immunoglobulin hypotypes.In medium pore, add known standard sample or unknown sample, and allow it to spread to semi-solid medium.Precipitation line in specific antiserum(antisera) site has been represented hypotype.As shown in Figure 6, the positive control sample that contains all rat hypotype antibody is expressed the reactivity in all hypotype holes.On the other hand, 2G10 antibody only with IgG
2aThe reaction of hypotype antiserum(antisera), this shows that rat antibody 2G10 is IgG
2aAntibody.
For assessing combining of commercial available rat antibody and mouse IgM, assessed rat monoclonal antibody LO-MM-9(from Serotec, cat#MCA 199) the ELISA reactivity of anti-mouse IgM.In each hole of 96 orifice plates, add 100ngmu-k, gamma-k, or gamma-l.The LO-MM-9 rat antibody that adds various amounts then, the elisa assay that carries out rat antibody as discussed previously.As shown in table 1, this rat antibody and mouse IgM bag is not had by the hole and combines.
Table 1
The assessment of the commercial anti-mouse IgM of rat antibody that gets
The ELISA reactivity of mouse Ig
Concentration Mu-k gamma-k gamma-l
1000ng 0.058 0.008 0.037
500ng 0.034 0.004 0.031
100ng 0.032 0.001 0.021
50ng 0.030 0.001 0.020
10ng 0.021 0.001 0.002
5ng 0.018 0.001 0.024
1ng 0.009 0.001 0.020
0.1ng 0.020 0.001 0.023
Therefore, think that this antibody is useless to further research.In addition, shown in Fig. 6 hurdle 3, measure as SDS-PAGE, this antibody preparation contains at least three major protein bands, and at least 5 a few eggs leukorrhea.
Rat anti mouse IgM combines with generation IgM cell
For proof 2G10 rat anti mouse IgM antibody not only with bag by the purifying IgM antibodies of 96 orifice plates, and with the cell combination that produces IgM antibody, in that 10C1 cell and the mouse 238-57ADR hybridoma of IgG secretion and IgM carry out facs analysis successively.In brief, 1 * 10
6Cell centrifugal 3 minutes at 500 * g is with PBS washing three times, resuspending in 3 milliliters of PBS then.
Fluorescein is in conjunction with parentization purifying F(ab)
2The anti-rat immune globulin IgM(Cappel of fragment goat) at PBS(1x) in dilution in 1: 100, in 20 microlitre cell suspending liquids, add the 20-40 microlitre.After incubated at room 15-20 minute, cell washs secondary with PBS in the dark, at 500rpm centrifugal 3 minutes.
The aliquot that adds 300 microlitre Paraformaldehyde 96s (1% in PBS) is with fixed cell.Cell is hatched until being classified by the wandering cells reader at 4 ℃.
When dyeing indirectly, hybridoma is at first hatched with rat anti mouse IgM antibody 2G10, and fluorescein F(ab is used in washing then)
2The anti-rat immune globulin IgM dyeing of fragment goat is classified by the wandering cells counter.
As table 2 and shown in Figure 7, antibody 2G10 combines with the IgM specificity on mouse hybridoma surface at secretion IgM, and does not combine with the IgG secretory cell.
Table 2
IgM and IgG express the facs analysis of mouse hybridoma
The percentage ratio of fluorescein-labelled cell
IgG IgM
Handle the heterozygote heterozygote
0 0.87 0.37
Uncorrelated rat IgG
+ FITC goat Chinese People's Anti-Japanese Military and Political College mouse 0.23 0.96
2G10 rat anti mouse IgM
AbY
+ FITC goat Chinese People's Anti-Japanese Military and Political College mouse 1.58 89.99
As shown in table 2, the background fluorescence element of no untreated cell.Uncorrelated rat IgG does not combine with IgG heterozygote or IgG heterozygote yet.2G10 antibody and generation IgG hybridoma debond (1.58% cell positive, Fig. 7 A and table 2).But as shown in Fig. 7 B and table 2,90% cell that produces mouse IgM antibody shows with 2G10 antibody is strong and combines.Therefore, owing to be identified in the mouse IgM of cell surface, produced combining of antibody 2G10 and cell.
What appreciated is that the character of the antibody checked is unique correlated characteristic of corresponding hybridoma effectively, and promptly for purposes of the present invention, hybridoma comes qualitative by the ability that their produce the specific antibodies with above-mentioned character.
The cytotoxicity assessment
Described antibody with as by Carlsson, J. waits the described SPDP(Biochem J(1978 of people) 173:723-737) ricin toxin A chain (RTA) combination handled, or with combining with imino-mercaptan alkane (IT) processing.
2G10 and Gelonin coupling
The stoste of SPDP reagent (the N-succinimide acyl 3-(2-pyridyl two sulphur) propionic ester (6 mg/ml) of preparation in dry DMF.In 12 * 75mm glass test tube that the 1 milliliter of PBS solution that contains 1 milligram of 2G10 antibody is housed, slowly add the about 10 microlitre stostes of SPDP(of excessive five times of molar weights).Mixture made it into swirl shape in per 5 minutes in room temperature in 30 minutes.
(1 * 24cm) goes up column chromatography and removes from sample excessive unreacted SPDP, and this post is with the 100mM sodium phosphate buffer pH7.0 pre-equilibration that contains 0.5mM EDTA at Sephadex G-25 post by gel-filtration.Collect each fraction (0.5 milliliter), come analyzing proteins composition (Bradford, (1976) Anal.Biochem.72:248-254) with Bradford dyeing binding analysis.Monitor the absorption value (600nm) of 96 orifice plates with Bio-TEK Microplate automatic reading device.Antibody is collected these components and is remained on 4 ℃ at void volume place wash-out (fraction 14-20).
From the seed of gelonium multiflorum, extract the gelonin toxin, method with people such as Stirpe is purified to evenly (Stirpe et al., J.Biol Chem.255:6947-6953(1980)), in triethylamine chlorine hydride (TEA/HCl), add 1 milligram of sublimed gelonin(2 mg/ml in PBS), to ultimate density be 60mM TEA/HCl, regulate pH to 8.0.Make solution become 1mM EDTA.Add 2-imino-mercaptan alkane stoste (0.5M in 0.5M TEA/HCl, pH8.0) to ultimate density be 1mM, sample was hatched under nitrogen 90 minutes in 4 ℃.
Excessive 2-imino-sulfane alcohol reagent is by (1 * 24cm) goes up gel-filtration removes this post 5mM two-triacetate damping fluid (pH5.8) pre-equilibration that contains 50mM HCl and 1mM EDTA at Sephadex G-25 post.Collect each fraction (0.5 milliliter), on 96 hole titer plate, dye in conjunction with the test analysis protein ingredient with Bradford.Gelonin collects these fractions at fraction 14-20 wash-out, 4 ℃ of preservations.SPDP modified antibodies 2G10 mixes with the gelonin that the 2-imino-mercaptan alkane of many 5 times of molar weights is modified.Regulate the PH to 7 of mixture by adding 0.05M TEA/HCl damping fluid (pH8.0), mixture was hatched under nitrogen 20 hours in 4 ℃.Add iodo-acid amide (0.1M is in PBS) to ultimate density and be 2mM to block any remaining free sulfhydryl group, be incubated in 25 ℃ and proceed 1 hour.
Purifying 2G10 Gelonin mixture
For removing low-molecular-weight product and non-binding gelonin, with sample on the reaction mixture on sephadex S-300 post with the PBS pre-equilibration.Collect each fraction (1.0 milliliters), dye in conjunction with the protein ingredient in the test analysis 50 mul aliquots samples with Bio-Rad.For removing not in conjunction with 2G10, go up sample on the affinity column of Blue Sepharose CL-6B(1 * 24cm) from the high molecular weight peak (fraction 17-23) of S-300 post, this post is with 10mM phosphate buffered saline buffer (pH7.2) pre-equilibration that contains 0.1MNaCl.Behind last sample, with 30 milliliters of damping fluid washing columns, until the non-binding antibody of complete wash-out.Post is with the 10mM phosphoric acid buffer pH7.2 wash-out that has from 0.1 to 2MNaCl linear salt gradient.By the protein component in the previous described dyestuff binding analysis mensuration wash-out fraction.
Contain free 2G10 antibody, the coupling mixture of 2G10 gelonin and free gelonin filters purifying by glue earlier on the S-300 post.As shown in Figure 8, detect a high molecular weight peak (cut 25-42), also detect a lower molecular weight peak (fraction 55-67).Collect fraction 26-42 to analyze binding substances purity and reactivity.
2G10 gelonin binding substances to purifying carries out the PAGE analysis.As seen, 2G10 gelonin coupling mixture (hurdle 3) contains free 2G10, free gelonin(arrow in Fig. 9), 2G10 and 1 gelonin molecule coupling (monomer arrow), and 2G10 and 2 gelonin molecules coupling (disome arrow).As in the hurdle 1 as seen, the 2G10 gelonin binding substances major part of final purifying is 2G10 and the coupling of 1 gelonin molecule, is 2G10 and the coupling of 2 gelonin molecules on a small quantity.Preparation is not polluted by free gelonin or free antibody.
Whether improved the identification of 2G10 antibody to mouse IgM for measuring this 2G10 antibody and gelonin chemical reaction and coupling, 96 orifice plates wrap as shown in Figure 2 by last mouse IgG or IgM antibody.Replace 2G10 antibody, 2G10 gelonin binding substances is added in the hand-hole with various concentration.Carry out the standard ELISA analysis then to measure rat antibody.As shown in Figure 10, the 2G10gelonin binding substances is hardened with the IgM bag soon to be closed, and has only small part and IgG bag to be hardened and closes.Only at the maximum concentration (1000 nanogram(ng)s/hole) of test, 2G10 gelonin binding substances is just combined by the hole with the IgG bag largely.On the contrary, in similar concentration, 2G10 gelonin binding substances wraps by the hole combination with IgM in large quantities.Therefore, 2G10 gelonin binding substances has been saved to the immunoreactivity of IgM.In addition, 2G10 gelonin binding substances not with mouse IgG cross reaction, so the selectivity of 2G10 gelonin binding substances is not changed yet.These data show that 2G10 gelonin binding substances should be combined by the mouse cell with the IgM bag with the same manner of antibody 2G10.
Produce the ODC monoclonal antibody
The production of A. external immunity and monoclonal antibody
The scheme of the production mouse monoclonal antibody of usefulness causes producing the hybridoma of secretion IgM subclass now.Be the typical consequence of explanation mouse monoclonal antibody production decision, used the monoclonal antibody of two technology with production Chinese People's Anti-Japanese Military and Political College mouse protein ornithine decarboxylase (ODC).First technology relates to the outer immune mouse spleen cell of ODC proteoplast, promptly in culturing bottle.
The rats'liver ODC that this technology is used 25 microgram purifying by the Luben method carry out (Luben and Mohler, (1980) Molec Immunol, 17:635-639).
In brief, ODC protein and mouse cell were hatched 72 hours in the presence of thymocyte conditioned medium (a kind of source of specificity somatomedin of IgSC type) in 37 ℃.As fusogen splenocyte and MPC myeloma cell are merged with polyoxyethylene glycol.The secretion of the somatic antibody with responding property of ODC protein of test gained heterozygosis.The result lists in table 3.
Table 3
External immunity and hybridoma produce guide look
Produce the guide look words
Porose 48 2 24 orifice plates, 1 * 10 fixed
6
Cells/well
porosely have heterozygote/institute 48/48 alive to be represented as 1: 10
6Minimum melt
Porose fixedly sum of fundamental frequencies rate
First ELISA shows all 48/48 in ELISA, in them
The hole positive 14 than above-mentioned control media
Big more than 4 times
12/14 of male of the active precipitation of ODC has been tested in ELISA
Positive maximum 14 of hole count/test hole count
Amplification porosely be confined to 12 best grams to clone 12/48
Grand
Receive 70 minimumly gathers in the crops from the polyclone hole from 12 original polyclone holes
1 of the single ELISA sun of all that obtain, maximum 18
The property mono-clonal
All are active to the ODC precipitation that the active precipitation sun 50/70 of ODC adds
Maximum 48%, minimum 4% of mono-clonal/all tests of property
Mono-clonal/70/70 of all secretion IgM
All clones
The mono-clonal of all IgG secretion/0/70
All mono-clonals
After originally screening with ODC albumen, each clone of hybridoma and test once more.Obtain 70 and see hybridoma with the ODC proteins react from several standards.But, as shown in table 3, all clone's secretion IgM haterotypic antibodies, none IgG secretion antibody.Find that these antibody have limited purposes in various monoclonal antibodies are used.
B. the production of immunity and monoclonal antibody in the body
Second technology of used manufacture order clonal antibody is the ODC protein immunized mice by the injection purifying.Mice spleen cell is separated in the immunity back, makes they and P3 * 63Ag8.653 myeloma cell merge (using PEG as mentioned above).Separating clone is tested itself and the proteic reactivity of ODC, and is further qualitative with as specificity ODC identification agent.Test result is displayed in Table 4.
Table 4
Immunity and intraspecific cross knurl are produced guide look in the body
Produce the guide look words
In porose many holes of fixing on 1,200 96 orifice plates
Fix 2.5 * 10
5Cells/well
Heterozygote alive 148 about fixed 12%(about 1 are arranged in porose:
10
7Fusion frequency)
For the first time among porose positive 30/148 ELISA of ELISA institute, in conjunction with ODC
The property on wherein 6 comparisons according to significantly
High
And clone
27 3 polyclone holes do not produce from 30 original polyclone holes
All individual ELISA positive monoclonals that obtain
Positive monoclonal
The not comment of mono-clonal/27/27 of all secretion IgM
The test number
The mono-clonal of all IgG secretion/0/27
The test number
The active list 1/5 of all precipitation ODC has only a comparison heavy according to medium
The form sediment ODC of suitable volume of clone/test number lives
The property
27 monoclonal cell systems have been developed, the secretion of 100% in them IgM subclass antibody.Find again that later on these antibody are unsuitable for as ODC specificity reagent.
FACS(flow activation cell divide) method
The centrifugal secretion of 500 * g IgM hybridoma 238-57 ADR3 minute, with PBS washing three times, and in 3 milliliters of PBS resuspending.
Fluorescein is in conjunction with the F(ab of affinity purification)
2The anti-rat immune globulin of fragment goat (Cap-pel) is at PBS(1X) in 1: 100 the dilution, the 20-40 microlitre is joined in the 20 microlitre cell suspending liquids.
Room temperature is hatched 15-20 minute in dark after, with PBS washed cell secondary, at 500rpm centrifugal 3 minutes.
The five equilibrium reagent that adds 300 microlitre polyoxymethylene (1% in PBS) is with fixed cell.Cell is hatched until being sorted out by the wandering cells counter at 40 ℃.
When dyeing indirectly, hybridoma is hatched with rat anti mouse IgM antibody 2G10 earlier, and fluorescein F(ab is used in washing then)
2The anti-rat immune globulin IgM of fragment goat dyes, and is sorted out by the wandering cells counter.
Embodiment 9
The magnetic cellular segregation
Mouse hybridoma 238-57ADR washs three times in containing the Iscove ' s substratum of 1% foetal calf serum and 0.1% gentamicin.Cell was counted in room temperature at 500 * g in centrifugal 3 minutes then.Globe is resuspending in 0.5 milliliter of substratum, (uses 0.1 milliliter per 10 approximately on ice with about 0.425 mg/ml of the concentration of purifying
6Cells/ml) rat anti-mouse IgM antibody (2G10) was hatched 60 minutes.Behind the washing secondary, cell is resuspending and counting in 0.2 milliliter of substratum in cold Iscove ' s substratum.Magnetic bead washs three times with magnetic sheet in serum free medium.The cell spheroid thing mixes with the ratio of pearl globe with 20 pearls/cell.Cumulative volume should be above 0.4 milliliter.Cell/pearl mixture was hatched on ice 0.5 hour, jolted once in per 10 minutes.
Cell/pearl mixture resuspending at least 2 milliliters of substratum is magnetically perpendicular to gravity separation.In case separate and to finish, do not stir the magnetic globe and remove supernatant liquor.Pearl is resuspending in 1-2 milliliter substratum, and examines under a microscope.
Fully described the present invention now, concerning the those of ordinary skill of present technique field, clearly can not leave the spirit and scope of the present invention and do many modifications and modification.
Claims (12)
1, a kind of monoclonal antibody is characterized in that it
(a) selectivity is with the IgM antibodies;
(b) not with IgG
1Or IgG
2Antibodies; And
(c) has the G abnormal shape.
2, a kind of rat monoclonal antibody is characterized in that it
(a) optionally with mouse IgM antibodies;
(b) not with IgG
1Or IgG
2Antibodies; With
(c) tool G abnormal shape.
3, monoclonal antibody according to claim 1 and 2 is characterized in that it combines with the hybridoma that produces anti-IgM antibody.
4, monoclonal antibody according to claim 1 and 2 is characterized in that it combines with the hybridoma that produces anti--mouse IgM antibody.
5, according to any the described monoclonal antibody in the claim 1 to 3, it is characterized in that it by one in the following hybridoma produce:
（a）2G10
(b) 1C2, or
In a kind of monoclonal antibody, it and above-mentioned antibody any is functionally equivalent.
6, produce the big murine hybridoma of rat X according to any described monoclonal antibody among the claim 1-5, and the offspring of described hybridoma.
7, a kind of hybridoma, it is
(a) HB10436; Or
Its offspring.
8, a kind of immunotoxin is characterized in that it is according to the monoclonal antibody of any and the binding substances of cytotoxicity part in the claim 1 to 3.
9, a kind of method that produces mouse IgM antibody cell of killing comprises described cell is contacted with the immunotoxin of cell killing significant quantity, as defined in claim 6.
10, according to any the described monoclonal antibody in the claim 1 to 3, use the detectable marker mark.
11, a kind of method of collecting the hybridoma that produces the special-shaped monoclonal antibody of IgG, comprise described any antibody treatment hybrid cell group with claim 1-3, described immune compound cells is classified in cell sorter, collect not and described antibody compound cell.
12, a kind of method for preparing immunotoxin comprises making as the described antibody in the claim 1 combining with the cytotoxicity part.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US51597490A | 1990-04-27 | 1990-04-27 | |
| US515,974 | 1990-04-27 |
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|---|---|
| CN1057072A true CN1057072A (en) | 1991-12-18 |
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ID=24053580
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|---|---|---|---|
| CN91102913A Pending CN1057072A (en) | 1990-04-27 | 1991-04-27 | Monoclonal anti IgM antibody, their production and purposes, and the hybridoma that produces same material |
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| Country | Link |
|---|---|
| EP (1) | EP0538267A4 (en) |
| JP (1) | JPH06504424A (en) |
| CN (1) | CN1057072A (en) |
| CA (1) | CA2079901A1 (en) |
| IE (1) | IE911172A1 (en) |
| IL (1) | IL97827A0 (en) |
| NZ (1) | NZ237784A (en) |
| PT (1) | PT97482B (en) |
| RU (1) | RU2105062C1 (en) |
| TW (1) | TW349995B (en) |
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| ZA (1) | ZA912695B (en) |
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| CN108384761B (en) * | 2018-03-30 | 2020-08-11 | 四川迈克生物新材料技术有限公司 | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof |
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| KR101729056B1 (en) | 2008-09-05 | 2017-04-24 | 세키스이 메디칼 가부시키가이샤 | Monoclonal antibody, and immunoassay using same |
| WO2023088443A1 (en) * | 2021-11-20 | 2023-05-25 | 东莞市朋志生物科技有限公司 | Anti-human igm antibody and preparation method therefor and use thereof |
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| US4792447A (en) * | 1981-07-23 | 1988-12-20 | Board Of Regents, The University Of Texas System | Anti-immunoglobulin toxin conjugates useful in the treatment of B cell tumors |
| AU9016591A (en) * | 1990-10-25 | 1992-05-26 | Tanox Biosystems, Inc. | Glycoproteins associated with membrane-bound immunoglobulins as antibody targets on B cells |
-
1991
- 1991-04-08 IE IE117291A patent/IE911172A1/en unknown
- 1991-04-10 NZ NZ237784A patent/NZ237784A/en unknown
- 1991-04-11 ZA ZA912695A patent/ZA912695B/en unknown
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- 1991-04-17 TW TW080103007A patent/TW349995B/en active
- 1991-04-24 WO PCT/US1991/002822 patent/WO1991017257A1/en not_active Application Discontinuation
- 1991-04-24 CA CA002079901A patent/CA2079901A1/en not_active Abandoned
- 1991-04-24 JP JP3509144A patent/JPH06504424A/en active Pending
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| CN108384761B (en) * | 2018-03-30 | 2020-08-11 | 四川迈克生物新材料技术有限公司 | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof |
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| Publication number | Publication date |
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| RU2105062C1 (en) | 1998-02-20 |
| EP0538267A1 (en) | 1993-04-28 |
| WO1991017257A1 (en) | 1991-11-14 |
| AU7866491A (en) | 1991-11-27 |
| AU650659B2 (en) | 1994-06-30 |
| EP0538267A4 (en) | 1994-06-01 |
| JPH06504424A (en) | 1994-05-26 |
| IL97827A0 (en) | 1992-06-21 |
| PT97482A (en) | 1992-01-31 |
| CA2079901A1 (en) | 1991-10-28 |
| TW349995B (en) | 1999-01-11 |
| IE911172A1 (en) | 1991-11-06 |
| NZ237784A (en) | 1992-09-25 |
| ZA912695B (en) | 1993-02-24 |
| PT97482B (en) | 1998-08-31 |
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