JPH03501161A - Methods and compositions for mimicking antigenic determinants - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Methods and compositions for mimicking antigenic determinants Background of the invention [Field of invention] The present invention relates generally to the field of immunology, and more particularly to mimicking antigenic determinants. are doing. As used herein, the term "antigenic determinant" refers to intact; a chemical structure that can bind, complex, or react with immunoglobulin molecules It means. This type of structure includes hubtens, receptors, markers, and receptors. Includes pitopes, etc.

In the 1960s, fluorescence-activated cell sorters (FlnorescsnceAejiva) As a result of the appearance of led Ce1l 5orler: FAA CS), cells A small revolution occurred in biological research.

(For details on the principles of FA and CS, see, for example, FlowCytometer ic Application in Tumor Biolo! ! , 1G? 9.

Al! aR, Li5s, Inc., Nev York) FAC 3. At the beginning of this age, mainly the cell cycle (C+, S.

Sorting cell populations according to the location of individual cells in G, M, or Go) It was initially used as a tool, but gradually developed into research on cell reaction kinetics, cell receptors, etc. - Protein identification and quantification, as well as cell surface antigens and immunology studies. It has become the basic instrument used for quantifying antibodies and antibodies. This invention relates to this This is the latter field.

FAC3 in the study of cell surface antigens and the interaction of antibodies with these types of antigens position is clearly established. FAC3 is responsible for the study of ligand/receptor interactions. It has become an important tool for immunologists conducting research. For such FACS analysis The fluorescein-labeled antibody is compared with the unlabeled antibody for antigenic determinants on the antigen of interest. Competitive competition tests are included. This type of assay allows researchers to detect both antigen and competing antibodies. can provide valuable information about An antibody is more properly an anti-antibody. Antibodies are used that are conjugated with reactive fragments or therapeutic or diagnostic components. called globulin.

However, there are some limitations regarding the antigens that can be studied. example For example, internal markers are not available for FAC5 analysis. The virus antigen is approximately 0. It is present on pillions smaller than 1μ and is a troublesome analyte for FAC5 analysis. Ru. Furthermore, certain types of cells, especially bacterial cells, that carry the antigen of interest may be difficult to use due to cost issues or health concerns. FAC5 analysis is undesirable due to factors such as health risks and difficulties in cell maintenance. It is an undesirable analyte. Therefore, it has not been possible to apply FAC3 analysis to date. l; There is a need for compositions and methods useful for mimicking antigenic determinants. Desire Preferably, this type of novel m-acid and method provides a stable, cost-effective solution to target antigens. In addition to providing a suitable supply source, we have previously analyzed using FA and CS. would increase the size of the particles with antigen, which would have been difficult to do.

[Description of related literature] U.S. Pat. No. 8,437,6110 describes immunoassay methods in general and monoclonal assays in particular. Discloses improvements in two-site or sandwich assays using antibodies.

Sogarbaker er-Lee, Flow cylometry: Gen eral princi-ples and applications to 5elected 5LudiCs in tumorbioloH (Flow Cytometrini general principles and application to tumor biology research) oFIov Cy tometric Applica Lionsin Tumor Biolo ( y, p, I25-53. New York: Alan R, Li5s.

Inc., I979°. Describes the basic principles of raw cytometry.

Loken and 5jxll, J, 1mmono, l+1eth, (198 2) 5 East: R85-Rl12 is a flow controller used as analysis and separation equipment in the field of immunology. – Discloses the use of cytometry.

Rajevsky and Tskemori, Anm, l1ev, Immu Nol. (1983): 569-07, Idiotype Genetics, Expression, and Function. Discloses basic science including.

Beaumier er sl, J. Nacl, Med, (198027, 8 4-48 contains three different human melanoma-associated antigens, p97, and groteoglycans. Immunization of radiolabeled monoclonal antibodies against gangliosides, and GD, gangliosides. Using a gamma scintillation counter developed to evaluate reactivity The certification is disclosed.

Robins el al, J, Imto++o, Metb, (1986) 9 0:165-71, tumor-reactive antibodies and conjugated antibodies using flow cytometry. The present invention discloses quantitative measurements of physical activity and competitive measurements.

Summary of the invention Novel methods useful for the study of antigen/antibody reactions, antigenic determinants and immunoglobulins law is provided. Immunoglobulin which is an anti-idiotype of the target antigen 15 compound an immortal cell line capable of expressing the antigen-binding substance, an antigenic determinant to which the antigen-binding substance binds; Provides a novel method for using epitopes in place of epitopes. Cells of the cell line are immunized Engineered to produce globulins as cell surface markers. This 1” target provides a stable and effective source of antigenic determinants, previously unavailable. This makes available epitopes available for use in substances that are not amenable to FAC5 analysis. Amplify the marker seen above.

Arrogant - Masaru Danmikasa Aim FIG. 1 is a graph showing the F/', CS competition assay using the method of the present invention.

Figure 2 is Targera! -XMMME-T01 cells and concentration gradually increased Antibody when incubated with MMME-001 monoclonal antibody/ Figure 2 is a graph showing antigen interactions, where the plateau of the binding curve represents the saturating concentration of antibody.

The Town O-Kiun - East 9th period - Eiwang - Suction The present invention provides a novel method of mimicking the antigenic determinant or shrimp. child These methods are particularly suitable for the study and analysis of antigen/antibody reactions using FA CS analysis. 1. It is used, among other things, in the ITFA CS competitive assay.

According to the invention, immortal cells are used in place of substances bearing antigenic determinants of interest. . Immortal cells IJ useful in the present invention, capable of expressing immunoglobulin molecules It is.

These cells produce immunoglobulin molecules as cell surface markers. Be manipulated.

The immunoglobulin molecules of interest are typically monoclonal or polyclonal. The antibody would be an anti-idiotype immunoglobulin. For antibodies of interest such as are epitopes present on bacterial and viral pathogens, tumor cell surface markers, and cell This includes those that bind to internal markers, hormone markers, etc.

According to the invention, immunoglobulin molecules are expressed by immortal cells. these Cells can be isolated from nature or using any of the techniques known in the art. It is made by Using the antibody of interest as an immunogen, anti-idiotype immunoglobulin Hives obtained using technology to create immortal hybrid immune cells that secrete immune molecules Ridoma cells are particularly useful. Technology for creating cell lines For more information, , GodiB, J, W, , Monoelomxl AnLib odiCs+Pr1ociples and Praetiee, ()916) Second Ed, please refer to Muctde+*1cPress (this technique (The details of the procedure are cited here by reference), these hybridoma cells are Mifuzumi, fuzumihito, mat; can be totohito. Maruji, group throw D Using NA technology, the hybridoma cells used in the present invention are genetically engineered. It is also possible to manufacture

According to the present invention, immortal cells transfer immunoglobulin molecules of interest to cell surface markers on the cell. It is operated to produce as a car. A preferred method of operation is to cool the cells to approximately 4°C. secretion of the expressed immunoglobulin essentially ceases at this temperature. circle. Chemical manipulation of cells can change the binding sites on immunoglobulins. Therefore, it is not very suitable.

Once cells are obtained, they can be used as surrogates for antigenic determinants in a number of assays. used as. Preferred assays in the present invention involve binding sites on immortal cells. FA using a fluorescein-labeled antibody that competes with the antibody or conjugated antibody of interest This is a C5v one-unit verification method. This type of assay and other assays well known in the art The method provides useful data on anti/antibody responses and provides diagnostic and therapeutic immunoconjugate It is an important means for body quality control.

Another form of competitive immunoassay using the antigen of interest (in this case a substitute for the antigen of interest) (instead of using immortal cells) is a less suitable assay. Examples of this type of assay Immortal cells can then be labeled with a detectable label (e.g., a labeled antibody or similar). directly or indirectly) to react with cell surface immunoglobulins. The assay method uses the immortal cells in a competitive assay using a conjugated antibody. Dew. In such assays, a known amount of labeled immortal cells are tested for antibody binding sites. will compete with an unknown amount of the antigen of interest in the sample.

Those skilled in the art will appreciate that a wide variety of assay methods can be used within the scope of the present invention; When substituting antigenic determinant surrogates of the present invention, each assay requires slight modification. You will understand that.

Q) The following examples are for illustrative purposes only and are not intended to limit the invention. do not have.

Production of type monoclonal antibodies (MoAb) and μ-week-511 Mouse C57, American type culture Kotokushi P (A, T) CC) XMMME-0 with deposit number HB8759 (deposited on March 26, 1985) They were immunized with a monoclonal anti-melanoma antibody called 01. -Next immunization (200 μg/mouse) is completely 7 in multiple areas including the nodobud (swell of the sole) One week later, secondary immunization (400 μg/machine) was administered. Administer by incomplete Freund's adjuvant, then insert the foot pad. Three injections (400 μg/ma) with phosphate buffered saline (PBS) at multiple sites including (Us) was conducted every other week. The final immunization was performed several days before cell fusion and was performed at multiple sites. 400 μg of XMMME-001 antibody was administered.

After extraction, the tile cells were cell line SP210-Ag14 with ATCC deposit number CRL1581. fused with. These cells were incubated with hypoxanthine/aminopterin in 96 wells. ・Spread on thymidine (HAT) selection medium, then inoculate XMMME-001 antibody F(a b’) *Using the area 1. EIAL was screened for activity. number Positive wells were cloned and XMMME-OQ1 antibody or ATCC deposited. XMMME-003 antibody (deposited on July 3, 1986) with number HB9159 (deposited on July 3, 1986) Control that binds to a different epitope than that bound by XMMME-001 The activity was examined again against the monoclonal anti-melanoma antibody). positive well also investigated their reactivity in various environments. In this example, Hyb 96 locations of nitrocellulose corresponding to the 96 wells of the ri-dat manifold X M M M E -001-F (ab') at the 12:00 position of each , was scattered about 0.33j1g. XMMME-001 antibody 0.6μg 7: Dot (contact) at the 00 position and add 0.7 meg of XMMME-003 antibody to 5:OO Scattered in different locations. After that, ATCC deposit number HB 9160 (July 1986 Anti-idiotype XMMME-r01 hybridoma culture of Add the previous upper channel (diluted 1:2 with Tris-buffered saline (TBS)) to the manifold; React with the idiotype. Next, nitrocellulose was added to the control buffer (pH 7). , 2) or I; with any of various acidic pH buffers (pH 3, 4, 5, 6). Incubated. After incubation, each well received IgM and IgG, Goat anti-mouse IgG+ preadsorbed with , was added, followed by peroxidase. A rabbit anti-goat antiserum labeled with was added.

Then, the enzyme substrate 4-chloronaphthol and hydrogen peroxide were added. each well The sound was colored at the 12:00 and 7:00 positions, but there was no sound at the 5:00 position. No color, indicating the presence of anti-idiotype antibodies.

r gG, subclass clone strain XMMME-Summer O1, XMMME-001 It was found that it reacted only with the idiotype, and the interaction was dissociated at pH 5. . Additionally, the XMMME-r01 antibody is suitable for fluorescence-activated cell sorting (FAC5) analysis. binding of XMMME-001 to melanoma cell lines (specific target) The anti-idiotype activity was confirmed.

■, FA monoclonal antibody XM using anti-idiotype hybridoma cells MME-001 was used as a labeled antibody in the FAC5 competition assay: Labeled with fluorescein according to method: 2.8 mg of XMMME-001 antibody prepared in IX phosphate buffered saline (PBS), 1) H7,0 at an initial concentration of /ml . PD-10G25M Sephadex column (Pbirmicii Fill e Chcmicxls) in bicarbonate/NaCl buffer (0.1M carbonate/NaCl buffer). Carbonate, 0.5M NaCl.

Equilibrate at pH 9,0) and add antibody 2. Added sml. Antibodies are PBS buffer (pH 7, o) 3. 10 m eluted with sml and placed in an ice water bath. Cool to about 4°C in a beaker. Fluorescein inocyanate (F ITC) solution is 12mm x 75. FITC2 in mm borosilicate tube 00 μg and 200 pN of dimethyl sulfoxide (DMSO). . The tube was then shielded from light with aluminum foil prior to use. antibody solution After stabilizing at 4°C, add FITC solution drop by drop to this antibody solution with constant stirring. I added while doing so.

(FI TC/antibody ratio: 25jLg FI TC/mg antibody).

After addition of the FITC solution, the mixture was covered, protected from light, and stirred in an ice-water bath for 3 h. . Thereafter, a 2.5 ml aliquot of this mixture was added to PBS azide buffer (1. 0mM NaPO, 0.15M NaCl, 0.02% sodium azide, p Top l2 of a PD-10G25M Sephadex column equilibrated with H7,0) added. FITC-labeled antibody (XMMME-001-FITC) was purified with PBS azide. buffer solution 3. Elute with sml, protect from light with aluminum foil, and centrifuge at 15 ml. Collect I in a tube. Fluorescein/antibody ratio (F/MoAb) was calculated using an LKB spectrometer. Absorbance is at 280 nm and 49 Two F/M o A were read at 6 nm and the ratio was calculated according to the following formula: The b ratio is calculated to be 2.2. XMMME-001-FITC solution is protected from light. and stored at 4°C.

2. FAC5 competitive assay Dulbecco's minimal essential medium (DMEM), 10% fetal bovine serum (Fe2), 1% Pi XMMME-I 01 grown in Na Rubate, 1% 200mM Glutamine For hybridoma cells, add cells and medium to a 50 ml centrifuge tube and pm, harvested from supericulum cultures by centrifugation for 5 minutes at 4°C. The upper groove is a vacuum valve. It was removed using a sterile pipette attached to the lasco. This recovery method uses antigens on cells. Do not disturb the site (including some secreted immunoglobulin). The cells are as described above; 4 = suspended in 5-10 ml of DMEM. Cell viability is determined by cell suspension Dilute 1'0OJ11 with 0.4% Trivan blue 900ml and count using a cell counter. It was measured by Viability is greater than 95%, which is the viability required for this test. It was a low 93.5%. Viability after one application of fetal bovine serum (FBS) overlayer Power has reached 96%. After determining viability, cells were diluted to a concentration of 108 M1 cells/m+. I interpreted it.

All samples for FAC3 analysis were prepared in a water bath under a laminar flow hood. All reagents Stored refrigerated. Transfer a 1 ml aliquot of the cell suspension to a 12 x 75 mm tube for assay. (Falcon #2054 or equivalent). This tube is 120O Centrifuge for 5 minutes at 4°C and decant the supernatant. Then the tube is tested Place on tube rack I; place upside down on absorbent material and absorb as much of the upper groove as possible I let it happen. The remaining superior groove was removed by blotting it with gauze.

To the cells, add 1 ml of PBS-BSA/tube and shake gently to form bubbles. Cells were suspended and washed while avoiding cell formation. After washing, centrifuge the tube and remove the upper groove. Decanted and sucked off excess supernatant as above. Add PBS-BSA to each sample. Add exactly 1 ml to suspend the cells again.

Antibodies and immune complexes for competition are XMMME-001#51102 (standard’) ,XMMME-001-R,TA#60407,XMMME-001-RTA #60505, and XMMME-001 #50513 containing azide. It was. XMMME-001-RTA immunoconjugate is disclosed in U.S. Patent No. 4,590,071 (the said patents are hereby incorporated by reference) . Each competitor initially contains 4 meg/20μ! Dilute to (0.2j1gZdoor 1), then Serial dilutions of 211g/20μI, 1μir/zop+, and 0. 5 μg/20 pl was prepared. Each dilution is sufficient to prepare duplicate samples. Met. Fluorescently labeled antibody XMMME-001-Fr produced as previously described TC is in Dulbecco PBS (SiEl*) 20111 containing 1% BSA. Diluted to saturation concentration (lItg/20μm). each except for naturally occurring fluorescent controls. Aliquots of competing and labeled antibody and/or conjugate into sample tubes. added. All samples were incubated for 1 hour on water in the dark.

After incubation, the sample is separated into a single-cell suspension to avoid cell clumping. Immediately before FAC5 analysis, pipette each sample from the tube and pipette it into a 37μ Filter by redispensing from the tube through a Nitex filter. passed. Each sample was then subjected to dual laser FACS IV (Bget++++ 650 and 750 using Dickeoson, Mt4i*v, CΔ) green FLDORESBRITE microbeads (Poly The analysis was performed after correction with the following methods: 1999-2018, 2008, 2009, 2008 and 2009.Sciences, Worrin Hon. trial Samples were analyzed at 750v and forward scatter was removed to exclude dead cells from this analysis. Then, we extracted the intermediate distribution. The results of this analysis are shown in FIG.

Figure 1 shows the calculation of the average fluorescence distribution (F) and the average of the average values of the two samples. , with reciprocal, subtract spontaneous fluorescence, and with competing antibody or complex (C). It was obtained by plotting the concentration ratio of labeled antibody (L).

Using the ratio of the equilibrium constants of L (KL) and C' (Kc), we derived the following equation: 1/Fc”(Kc/Kt)×[(1/Fo)X [L] IX [C]X(1/ F.O.) In the above formula, Fc and Fo are respectively the F value of each [C] and F in the absence of C (in It exists, is linear, and has a slope S - (KC/KL)X [(1/FO)X CL]

The relative binding of antibody or conjugate (C1) and standard antibody (STD) (C2) (s i /sx-Kc+/KC2).

The results in Figure 1 demonstrate that the XMMME-101 cell line was previously used as a target It could be a suitable substitute for cell OM i n o r c e I l showed that.

Those skilled in the art will appreciate that the present invention simulates antigenic determinants that may be of interest for research and analysis. It will be appreciated that it provides a new way to imitate. This method is as follows: Antigenic determinants for which FAC5 analysis could not be applied to fr can now be studied. It is an improvement over the prior art in that it The present invention further provides a method for immunological analysis and research. Provides a stable and effective source of antigenic determinants.

The invention has been described in some detail by way of illustrations and examples for ease of understanding. However, a person skilled in the art may make certain modifications within the scope of the appended claims. It should be clear that

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Claims (26)

[Claims] 1. Using an antigen bound to an antigen-binding substance, the presence or concentration of said antigen-binding substance In the method for measuring the immunoglobulin component, which is an anti-idiotype of the substance, Immortal cells capable of expressing antibodies are used in place of said antigen, and said cells are capable of expressing said immunoglobulins. expressing said immunoglobulin as a cell surface marker containing a binding site for The method has been improved by. 2. The method uses a fluorescence activated cell sorter (Fluorescence Activz). 2. The method of claim 1, using a tcdCellSorter. 3. 2. The method of claim 1, wherein the immortal cell is a hybridoma cell. 4. Claim: the hybridoma cell is a murine/murine hybridoma cell. The method described in Section 3. 5. The murine/murine hybridoma cells have ATCC deposit number HB9160. 5. The method according to claim 4, which is an XM-secreting ME-101 hybridoma cell. 6. 2. The method according to claim 1, wherein the antigen-binding substance is an immunoglobulin. 7. The immunoglobulin is a hybridoma with ATCC deposit number HB8759. 7. The method of claim 6, produced by cell line XMMME-001. 8. The immortal cells are maintained at a temperature of about 4° C. to stop secretion of the immunoglobulin. 2. The method of claim 1, further comprising: 9. 2. The method of claim 1, wherein the immunoglobulin is labeled with a detectable label. 10. 10. The method of claim 9, wherein the label is a fluorescent label. 11. 11. The fluorescent label is fluorescein isothiocyanate. How to put it on. 12. Using a known amount of labeled antigen, the amount of labeled antigen bound to immunoglobulin is determined. In a method of determining the presence or amount of an antigen in a sample, the method comprises detecting the amount of antigen in a sample. to express an immunoglobulin molecule with a binding site similar to that of the antigen. The labeled immortal cells obtained are used in place of the labeled antigen, and the cells bind to the binding site. improved by expressing said immunoglobulin as a cell surface marker containing The above method. 13. 13. The method of claim 12, wherein the immortal cell is a hybridoma cell. 14. The hybridoma cell is a murine/murine hybridoma cell, The method according to claim 13. 15. maintaining said immortal cells at a temperature of about 4°C to stop secretion of said immunoglobulin; 13. The method of claim 12, further comprising: 16. Binding of the immune complex to the target antigen is determined by a labeled, unconjugated primary immunoglobulin. Immunity against a target antigen consists of measuring in competition with Robulin molecules. In the method for measuring the binding activity of an immune complex, the antibody component of the immune complex is Immortal cells capable of expressing a second immunoglobulin molecule, which is a diotype, are exposed to the antigen. Alternatively, using a cell surface wherein said cell contains a binding site for said second immunoglobulin. said method improved by expressing said second immunoglobulin as a marker; Law. 17. 17. The method of claim 16, wherein the method uses a fluorescence activated cell sorter. 18. 17. The method of claim 16, wherein the immortal cell is a hybridoma cell. 19. The hybridoma cell is a murine/murine hybridoma cell, The method according to claim 18. 20. The murine/murine hybridoma cells have ATCC deposit number HB9160. 20. The method according to claim 19, wherein the XMMME-101 hybridoma cell has the following. 21. 17. The method of claim 16, wherein the immune complex is an immunotoxin. 22. 22. The method of claim 21, wherein the immunotoxin is XMMME-001-RTA. Law. 23. The immortal cells are maintained at a temperature of about 4° C. to secrete the second immunoglobulin. 17. The method of claim 16, further comprising stopping. 24. 17. The labeled first immunoglobulin is labeled with a fluorescent label. How to put it on. 25. 25. The fluorescent label is fluorescein isothiocyanate. How to put it on. 26. The first immunoglobulin is a hybrid with ATCC deposit number HB8759. 26. The method of claim 25, produced by the Doma cell line XMMME-001.