Anti-human mutant erythropoietin mouse monoclonal antibody, applications and human recombinant erythropoietin stimulant detection method
By combining anti-human mutant erythropoietin mouse monoclonal antibody and bidirectional immunopurification with protein immunoblotting, the problem of the inability to distinguish between rEPO and VAR-EPO in existing technologies has been solved, enabling accurate detection of human urine samples, reducing the risk of false positives, and making it suitable for doping detection in various populations.
Patent Information
- Application Number
- CN202211320393.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-10-26
- Publication Date
- 2025-11-07
- Estimated Expiration
- 2042-10-26
AI Technical Summary
Existing Western blotting methods cannot effectively distinguish between recombinant human erythropoietin (rEPO) and mutant erythropoietin (VAR-EPO), leading to a risk of false positives in doping tests, especially among individuals carrying the EPO gene polymorphism c.577del, which affects the fairness of testing.
A detection method was designed using a mouse monoclonal antibody against human mutant erythropoietin, combined with bidirectional immunopurification and Western blotting. The method includes reverse immunopurification, forward immunopurification and Western blotting steps, and can specifically recognize VAR-EPO and distinguish between WT-EPO, VAR-EPO and rEPO.
It achieves accurate differentiation of WT-EPO, VAR-EPO and rEPO in human urine samples, reduces the risk of false positives, optimizes the doping detection process, and is suitable for accurate detection in various populations, especially carriers of the EPO gene polymorphism c.577del.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of antigen-antibody proteins, in particular to anti-human mutant erythropoietin mouse monoclonal antibodies, applications and human recombinant erythropoietin stimulant detection methods. BACKGROUND
[0002] Human recombinant erythropoietin (rEPO) has been widely abused by endurance athletes because it can stimulate erythropoiesis and improve the oxygen-carrying capacity of the human body.
[0003] Recent studies have found that the incidence of human EPO gene polymorphism c.577del in East Asian populations is 0.5-1%, and this polymorphism can produce mutant erythropoietin p.Arg193AspfsTer28 (VAR-EPO). VAR-EPO contains all the amino acid structures of wild-type erythropoietin (WT-EPO) and is extended by 26 amino acids at the C-terminus, so the currently commercially produced EPO monoclonal antibodies can recognize both WT-EPO and rEPO, as well as VAR-EPO.
[0004] The molecular weight of VAR-EPO is 3.1 kDa higher than that of WT-EPO, which is close to that of rEPO. The existing protein immunoblotting stimulant detection method cannot distinguish rEPO and VAR-EPO, and there is a risk of false positive reports of rEPO in stimulant detection. For the above reasons, the World Anti-Doping Agency has revised the rEPO detection process and judgment standard, but has not yet established an effective stimulant detection method for low-dose use of rEPO in people carrying EPO gene polymorphism c.577del, thereby affecting the fairness of stimulant detection. SUMMARY
[0005] The technical problem to be solved by the present application is to provide anti-human mutant erythropoietin mouse monoclonal antibodies, applications and human recombinant erythropoietin stimulant detection methods.
[0006] The technical solution of the present application to solve the above technical problem is as follows: the present application provides a hybridoma cell strain for secreting anti-human mutant erythropoietin mouse monoclonal antibodies, the Latin name of the hybridoma cell strain is Mus musculus, the classification name is mouse hybridoma cell, the accession number is CGMCC No.45302, the preservation unit is China General Microbiological Culture Collection Center, the address is No.1, Beichen West Road, Chaoyang District, Beijing, the preservation date is August 26, 2022.
[0007] The present application also provides an anti-human mutant erythropoietin mouse monoclonal antibody, which is produced by the hybridoma cell strain described above.
[0008] Further, the human mutant erythropoietin is human mutant erythropoietin p.Arg193AspfsTer28, and the amino acid sequence is shown as SEQ ID NO: 1.
[0009] The application further provides a polypeptide antigen for preparing the mouse monoclonal antibody, and the amino acid sequence of the polypeptide antigen is shown as SEQ ID NO: 3.
[0010] The application further provides application of the mouse monoclonal antibody in detection of a human recombinant erythropoietin stimulant.
[0011] The application further provides a method for detecting a human recombinant erythropoietin stimulant, and the mouse monoclonal antibody is used to detect the human recombinant erythropoietin stimulant in a sample to be detected.
[0012] Further, when the sample to be detected contains only the human mutant erythropoietin and / or the wild-type erythropoietin, the detection result is negative; and when the sample to be detected contains the human recombinant erythropoietin stimulant, the detection result is positive.
[0013] Further, the detection method is a bidirectional immunopurification combined with a protein immunoblotting method, and comprises at least one round of reverse immunopurification, forward immunopurification and protein immunoblotting.
[0014] Further, the step of the reverse immunopurification in each round is that the sample to be detected or supernatant solution obtained in the last round is mixed with the mouse monoclonal antibody, and then the steps of incubation, addition of immunomagnetic beads, incubation and magnetic bead enrichment are sequentially performed to obtain the supernatant solution of the reverse immunopurification in the present round.
[0015] The application further provides application of the mouse monoclonal antibody in preparation of a reagent for detecting human EPO gene polymorphism c.577del.
[0016] The application has the following beneficial effects:
[0017] (1) The anti-VAR-EPO mouse monoclonal antibody can specifically recognize VAR-EPO, and can specifically and efficiently bind to trace VAR-EPO in a urine sample, and does not recognize WT-EPO and rEPO;
[0018] (2) The rEPO stimulant detection method can distinguish WT-EPO, VAR-EPO and rEPO in a human urine sample by using the bidirectional immunopurification combined with the protein immunoblotting technology established by using the antibody, and can directly and accurately detect rEPO in urine samples from various populations, and can eliminate the risk of false positive;
[0019] (3) The rEPO detection method of the present application optimizes the doping detection process and makes up for the defects in the current world anti-doping field about rEPO detection.
[0020] (4) The anti-VAR-EPO mouse monoclonal antibody of the present application can be used for preparing a detection kit for human EPO gene polymorphism c.577del, thereby being used for screening of the gene polymorphism. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 The non-denaturing SDS-PAGE electrophoretogram of the antibody of the present application;
[0022] Figure 2 The denaturing SDS-PAGE electrophoretogram of the antibody of the present application;
[0023] Figure 3 The immunoblotting figures obtained by detecting each sample by using the double-directional immunopurification combined with protein immunoblotting and the forward immunopurification combined with protein immunoblotting in the present application, respectively. DETAILED DESCRIPTION
[0024] The principles and characteristics of the present application will be described below in combination with specific examples and drawings, and the examples are only used for explaining the present application and are not used for limiting the scope of the present application.
[0025] The present application designs and synthesizes a peptide segment as an antigen based on the C-terminal difference amino acid chain of VAR-EPO and WT-EPO, obtains a hybridoma cell strain by using hybridoma cell technology, and obtains an anti-VAR-EPO mouse monoclonal antibody by expression and purification. The antibody is the first mouse monoclonal antibody in the world which specifically recognizes VAR-EPO, can specifically and efficiently bind to trace VAR-EPO in a urine sample, and does not recognize WT-EPO and rEPO. The double-directional immunopurification combined with protein immunoblotting method established by using the antibody can distinguish WT-EPO, VAR-EPO and rEPO in a human urine sample, directly and accurately detects rEPO in urine samples derived from various populations, excludes the risk of false positive, optimizes the doping detection process, and makes up for the defects in the current world anti-doping field about rEPO detection.
[0026] Specifically, the double-directional immunopurification combined with protein immunoblotting method used in the rEPO doping detection method of the present application includes the steps of reverse immunopurification at least one round, forward immunopurification and protein immunoblotting. The steps of each round of reverse immunopurification are as follows: mixing the sample to be detected or the supernatant solution obtained in the last round with a mouse monoclonal antibody, then sequentially performing the steps of incubation, adding immunomagnetic beads, incubation and magnetic bead enrichment, to obtain the supernatant solution of the present round of reverse immunopurification.
[0027] The determination mode of the detection result is that when the sample to be detected contains only VAR-EPO and / or WT-EPO, the detection result is negative; and when the sample to be detected contains rEPO, the detection result is positive.
[0028] The ELISA plate eluate of the sample to be detected is used to detect rEPO.
[0029] For the negative detection result, the specific genotype carried by the sample can be further judged in combination with the electrophoresis band of the magnetic bead eluate.
[0030] The human recombinant erythropoietin stimulant detection method provided by the application adopts a method of bidirectional immune purification combined with protein immunoblotting to detect rEPO in urine, and can detect trace VAR-EPO in urine.
[0031] The test methods used in the following examples are conventional methods unless otherwise specified; and the materials, reagents and the like used are reagents and materials that can be obtained through commercial channels unless otherwise specified.
[0032] Example 1 Preparation of antigen
[0033] Since VAR-EPO only increases 26 amino acids at the C terminal of WT-EPO on the basis of containing all amino acids and glycosyl structures of WT-EPO, in order to prepare a monoclonal antibody that only recognizes VAR-EPO but does not recognize WT-EPO and rEPO, a polypeptide antigen is designed and synthesized based on the difference between VAR-EPO and WT-EPO in the C terminal amino acid chain on the basis of fully considering immunospecificity.
[0034] The amino acid sequence of the VAR-EPO amino acid chain is shown in SEQ ID NO: 1; wherein the amino acid sequence of the C terminal difference amino acid chain between VAR-EPO and WT-EPO is shown in SEQ ID NO: 2, and SEQ ID NO: 2 is GDDDQVCPPGHIHHLPHQHCLCHTLPRHS.
[0035] The antigen polypeptide is designed and synthesized by Beijing Yiqiao God State, and the design process fully considers immunogenicity, specificity, hydrophilicity and hydrophobicity, surface accessibility and spatial conformation and other factors. The amino acid sequence of the antigen is shown in SEQ ID NO: 3, SEQ ID NO: 3: CHTLPRHSGGGSHTLPRHS.
[0036] The antigen polypeptide obtained after synthesis is coupled with virus-like particles (VLP) to prepare an antigen that can be used for animal immunization.
[0037] Example 2 Preparation of anti-VAR-EPO mouse monoclonal antibody hybridoma cell strain and mouse monoclonal antibody
[0038] The antigen prepared in Example 1 is used to prepare mouse monoclonal antibody, and the specific experimental steps are as follows:
[0039] (1) Animal immunization
[0040] Balb / c mice were immunized by injecting antigens. The immunization method was to mix the immunogen with an equal amount of adjuvant and inject it subcutaneously in multiple points on the abdomen of the mice, wherein the injection dose of the immunogen for each mouse was 50 ug. After the first immunization, the second immunization was performed after 2 weeks, and the third immunization was performed after 3 weeks.
[0041] (2) Blood sampling and titer detection
[0042] After the third immunization, blood was taken to determine the serum titer. The serum titer was determined by using rEPO and recombinant VAR-EPO, respectively, and the specific steps were as follows:
[0043] One week after the third immunization, 50-60 ul of blood was taken from the orbital venous plexus of the mice, and after standing overnight at 4°C, the upper serum was separated by centrifugation and used for titer detection; an appropriate amount of protein to be detected was diluted with coating buffer to 5 ug / mL, then 100 μL was added to each well of the 96-well plate using a single-channel pipette, the plate was gently shaken to mix the sample, and the plate was sealed with plastic wrap, coated overnight at 4°C; wash the plate once with 200 μL / well of washing solution, and dry the enzyme-labeled plate; then block the enzyme-labeled plate with 300 μL / well of blocking solution for 1 hour at room temperature; after washing the plate twice with 300 μL / well of washing solution, add the sample (dilute the sample and sample diluent by gradient dilution, and add 100 μL / well), and add the detection antibody to 100 μL / well in the 96-well plate, and react for 2 hours at room temperature; wash the plate 3 times with 200 μL / well of washing solution, add 200 μL / well of color developing solution, and incubate at room temperature for 12 min; add 50 μL / well of stop solution to terminate the reaction; and detect with an enzyme-labeled instrument: the detection wavelength is 450 nm.
[0044] After the determination, select the mouse with high serum titer reacting with recombinant VAR-EPO and no reaction with rEPO, and immunize once with the immunogen, and take the spleen for fusion after 3 days.
[0045] (3) Fusion and screening
[0046] Take the spleen cells of the immunized mouse, mix with SP2 / 0 myeloma cells at a ratio of 1:1, and use the electric fusion method for fusion to obtain hybridoma cells. rEPO (1 μg / mL) and recombinant VAR-EPO (1 μg / mL & 0.1 μg / mL) are used as coating antigens, and the cell supernatant is determined by ELISA method. Select the VAR-EPO high and low concentration positive and rEPO negative wells by limiting dilution continuous cloning 2-3 times to obtain a hybridoma cell strain stably secreting mouse monoclonal antibody, numbered 22TJY2-MM07.
[0047] (4) Hybridoma cell culture and antibody purification
[0048] Rinse the Protein A affinity chromatography column with ultrapure water, then equilibrate with the equilibration buffer; load the hybridoma cell supernatant obtained by culture into the affinity chromatography column, and after loading, rinse with the equilibration buffer. Elute with elution buffer, collect the elution peak, neutralize with Tris buffer, and desalt to PBS 7.4 to obtain the purified antibody.
[0049] After purification, the antibody is verified, and the non-denaturing SDS-PAGE electrophoretogram is as shown in Figure 1 , and the denaturing SDS-PAGE electrophoretogram is as shown in Figure 2 .
[0050] Example 3: Detection of rEPO in urine samples
[0051] This example uses the method of bidirectional immune purification combined with protein immunoblotting for detection, and the specific detection steps are as follows:
[0052] (1) Sample ultrafiltration and concentration
[0053] Take 15 mL of the urine sample to be detected, add 1.5 mL of Tris-HCl buffer (3.75 M, pH 7.4), mix well to adjust the pH value of the sample, centrifuge for 20 minutes at a centrifugal force of 4000 g. After centrifugation, pour the supernatant into a 15 mL 10 kD ultrafiltration centrifuge tube (Millipore) and concentrate to 200 μL-500 μL, and replace it with a PBS buffer system. Transfer the concentrated sample into a 1.5 mL low adsorption centrifuge tube.
[0054] (2) Reverse immune purification
[0055] Add 1 μg of the mouse monoclonal antibody prepared in Example 2 to the concentrated sample, mix well, and incubate at room temperature for 1 hour. After the incubation, add 50 μL of Anti-mouse IgG magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG, Invitrogen) to the sample, mix well, and incubate at 4°C for 90 minutes. After the incubation, place the sample on a magnetic stand to enrich the magnetic beads.
[0056] Recover the supernatant, add 1 μg of the mouse monoclonal antibody prepared in Example 2 to the supernatant, mix well, and incubate at room temperature for 1 hour. After the incubation, add 50 μL of Anti-mouse IgG magnetic beads to the sample, mix well, and incubate at 4°C overnight.
[0057] After the incubation, place the sample on a magnetic stand to enrich the magnetic beads, and collect the magnetic beads and the supernatant, respectively.
[0058] After washing the enriched magnetic beads three times, add 25 μL of SDS loading buffer, and incubate at 95°C for 5 minutes to elute the EPO protein. After the incubation, cool the EP tube, centrifuge briefly, and place the tube on a magnetic stand for loading.
[0059] It should be noted that in actual detection, the reverse immunopurification can be performed in multiple rounds, and each round of reverse immunopurification continues to repeat the above experimental steps on the supernatant obtained from the previous round of reverse immunopurification.
[0060] (3) Forward immunopurification
[0061] Transfer the supernatant recovered in step (2) to a 0.5 mL 30 kD ultrafiltration centrifuge tube (Millipore), and concentrate to about 20 μL. Transfer the concentrated sample to an ELISA plate coated with EPO antibody (Stemcell), and incubate at 37°C for 2 hours. After the incubation, wash the ELISA plate, add 20 μL of SDS loading buffer, and incubate at 95°C for 5 minutes to elute the EPO protein. After the incubation, cool the ELISA plate for loading.
[0062] (4) Western blotting
[0063] Perform double Western blotting on the magnetic bead eluate in step (2) and the ELISA plate eluate in step (3), respectively.
[0064] SDS-PAGE gel electrophoresis was performed using 10% Bis-Tris protein gel (Invitrogen) with SDS MOPS buffer system, constant voltage 200V, 75 minutes. The protein was transferred to 0.45 μm PVDF membrane (Millipore) using Towbin transfer buffer with semi-dry transfer method, 1.0 mA / cm2transfer for 60 minutes. After the transfer, the PVDF membrane was blocked in 5% LFM / PBS for 60 minutes. After blocking, the PVDF membrane was washed with PBS and then 20 mL of the primary antibody solution (1 μg / mL, MAB2871, R&D) diluted with 1% LFM / PBS was added for overnight incubation at 4°C.
[0065] After the incubation of the primary antibody, the PVDF membrane was washed with PBS and cleaned for 3 times, and then semi-dry transfer method was used for the second protein immunoblotting to transfer the EPO antibody combined with the first PVDF membrane to the second membrane, the transfer buffer system was 0.7% acetic acid solution, and the transfer condition was 0.72 mA / cm2for 10 minutes. After the transfer, the PVDF membrane was blocked in 5% LFM / PBS for 60 minutes. After blocking, the PVDF membrane was washed with PBS and then 40 mL of the biotin-labeled secondary antibody solution (0.65 μg / mL, Goat anti-Mouse IgG (H+L) Secondary Antibody Biotin, Invitrogen) diluted with 1% LFM / PBS was added for incubation at room temperature for 60 minutes. After the incubation, the PVDF membrane was washed with PBS and cleaned for 3 times, and then 40 mL of the streptavidin-labeled HRP solution (0.25 μg / mL, Streptavidin Protein HRP, Thermo) diluted with 1% LFM / PBS was added for incubation at room temperature for 60 minutes. After the incubation, the PVDF membrane was washed with PBS and cleaned for 5 times, and then HRP chemiluminescence substrate coloration reagent (SuperSignal West Femto) was used for coloration.
[0066] (5) Analysis of the detection results
[0067] Whether the sample contains VAR-EPO encoded by EPO gene polymorphism c.577del can be detected by the band of the magnetic bead eluent, and whether the sample contains rEPO can be further detected by the band of the ELISA plate eluent. The results of the detection are shown in Table 1 by comprehensive analysis of the ELISA plate eluent and the magnetic bead eluent of the same sample:
[0068] Table 1
[0069]
[0070] In Table 1, a-g represent the following results, respectively:
[0071] a: not carrying EPO gene polymorphism c.577del, rEPO negative;
[0072] b: not carrying EPO gene polymorphism c.577del, rEPO positive;
[0073] c: not carrying EPO gene polymorphism c.577del, rEPO positive;
[0074] d: EPO gene polymorphism c.577del heterozygote, rEPO negative;
[0075] e: EPO gene polymorphism c.577del homozygote, rEPO negative;
[0076] f: EPO gene polymorphism c.577del heterozygote, rEPO positive;
[0077] g: EPO gene polymorphism c.577del homozygote, rEPO positive.
[0078] Comparative Example 1
[0079] Each sample was detected by forward immunopurification combined with protein immunoblotting to obtain the immunoblotting results.
[0080] Each sample was detected by the experimental methods of Example 3 and Comparative Example 1, respectively, to verify the detection effect of the rEPO stimulant detection method.
[0081] Among them, the samples to be detected are known, and the electrophoresis bands of each sample are as shown in Table 2. Figure 3 Among them, the electrophoresis band numbers and detection methods are as shown in Table 2:
[0082] Table 2
[0083]
[0084] By Figure 3As can be seen, for the blank urine samples 1-4 without the addition of rEPO, when detecting the population carrying the non-EPO genetic polymorphism c.577del (1 and 2), whether the forward immunopurification or the bidirectional immunopurification of the present application is used for detecting the eluent of the ELISA plate, only the band of WT-EPO is detected, and no band of rEPO is detected. When detecting the population carrying the EPO genetic polymorphism c.577del heterozygously (3 and 4), only the forward immunopurification is used, and in the eluent of the ELISA plate, not only the band of WT-EPO is detected, but also a band above it is shown. When the bidirectional immunopurification of the present application is used for detecting the eluent of the ELISA plate, only the band of WT-EPO is detected, and no band above it is detected. As can be seen, when only the forward immunopurification is used, even if the urine sample does not add rEPO, because it is the population carrying the EPO genetic polymorphism c.577del heterozygously, the VAR-EPO contained in the urine sample will cause the detection of rEPO to be positive, so that the detection result is inaccurate. The detection result of the bidirectional immunopurification of the present application shows that rEPO is negative, which shows that the method of the present application can effectively distinguish the VAR-EPO generated due to the carrying of the EPO genetic polymorphism c.577del from rEPO, so that the detection result is accurate.
[0085] For the urine samples 5-8 with the addition of rEPO, whether the forward immunopurification or the bidirectional immunopurification method of the present application is used, whether the urine sample is from the population carrying the non-EPO genetic polymorphism c.577del or the population carrying the EPO genetic polymorphism c.577del heterozygously, a band above the band of WT-EPO can be detected, which shows that the detection method of the present application can effectively detect rEPO.
[0086] For samples 2' and 4', the bidirectional immunopurification method of the present application is used to detect the eluent of the magnetic beads, as can be seen, for the population carrying the non-EPO genetic polymorphism c.577del, the method of the present application does not detect a band, and for the population carrying the EPO genetic polymorphism c.577del heterozygously, the method of the present application can detect a band, which shows that the detection method of the present application can effectively detect VAR-EPO.
[0087] As can be seen from the above experimental results, the anti-VAR-EPO mouse monoclonal antibody of the present application has high specificity and good affinity for VAR-EPO, and the bidirectional immunopurification combined with protein immunoblotting can effectively distinguish WT-EPO, VAR-EPO and rEPO, so that the sample from the population carrying the EPO genetic polymorphism c.577del can be avoided to be misjudged as containing rEPO. At the same time, for the population carrying the EPO genetic polymorphism c.577del, if rEPO is contained in the urine sample, it can also be detected, which effectively prevents missed detection.
[0088] Therefore, for a population of people with completely unknown genotypes, no matter what genotype they carry, the mouse monoclonal antibody and the bidirectional immunopurification method of the present application can effectively determine whether the urine sample contains rEPO at one time, without subsequent follow-up investigation, effectively improving the detection efficiency and accuracy.
[0089] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A hybridoma cell line for secreting a murine monoclonal antibody against human mutant erythropoietin, characterized in that, The Latin name of the hybridoma cell strain is Mus musculus, and the preservation number is CGMCC No. 45302, and the preservation unit is China General Microbiological Culture Collection Center.
2. An anti-human mutant erythropoietin mouse monoclonal antibody, characterized in that, The hybridoma cell strain of claim 1 is produced.
3. The anti-human mutant erythropoietin murine monoclonal antibody according to claim 2, characterized by The human mutant erythropoietin is human mutant erythropoietin p.Arg193AspfsTer28, and the amino acid sequence is shown as SEQ ID NO:
1.
4. Use of the murine monoclonal antibody according to claim 2 or 3 for the detection of human recombinant erythropoietin stimulants, characterized in that, The detection method is a bidirectional immunopurification combined with a Western blot method, which comprises the steps of reverse immunopurification at least one round, forward immunopurification, and Western blot.
5. A method for detecting human recombinant erythropoietin stimulant, characterized by, The mouse monoclonal antibody of claim 2 or 3 is used to detect human recombinant erythropoietin stimulants in a sample to be tested; the detection method is a bidirectional immunopurification combined with a Western blot method, which comprises the steps of reverse immunopurification at least one round, forward immunopurification, and Western blot.
6. The method for detecting recombinant human erythropoietin stimulants according to claim 5, characterized in that, When the sample to be tested contains only human mutant erythropoietin and / or wild-type erythropoietin, the detection result is negative; when the sample to be tested contains human recombinant erythropoietin stimulants, the detection result is positive.
7. The method for detecting recombinant human erythropoietin stimulants according to claim 5, characterized in that, The steps of each round of reverse immunopurification are as follows: the sample to be tested or the supernatant solution obtained in the last round is mixed with the mouse monoclonal antibody, and then the steps of incubation, addition of immunomagnetic beads, incubation, and magnetic bead enrichment are sequentially performed to obtain the supernatant solution of the reverse immunopurification in the present round.
8. Use of the mouse monoclonal antibody of claim 2 or 3 in the preparation of a reagent for detecting human EPO gene polymorphism c.577del.