A 17a-hydroxyprogesterone monoclonal antibody, its preparation method and application

Abstract

The application provides a 17alpha-hydroxyprogesterone monoclonal antibody, a heavy chain variable region (VH) of the antibody comprises CDR regions with sequences as follows: a heavy chain CDR1 is shown as SEQ ID NO:1; a heavy chain CDR2 is shown as SEQ ID NO:2; a heavy chain CDR3 is shown as SEQ ID NO:3; a light chain variable region (VL) of the antibody comprises CDR regions with sequences as follows: a light chain CDR1 is shown as SEQ ID NO:4; a light chain CDR2 is shown as SEQ ID NO:5; a light chain CDR3 is shown as SEQ ID NO:6. When the antibody is applied to progesterone detection (such as sandwich method immunofluorescence detection) as a blocking agent, the interference of high-concentration 17alpha-hydroxyprogesterone can be effectively blocked, and the detection accuracy is significantly improved.

Description

Technical Field

[0001] This invention relates to a 17α-hydroxyprogesterone monoclonal antibody, its preparation method, and its application, belonging to the field of antibody technology. Background Technology

[0002] Currently, progesterone (PROG), also known as luteal hormone, is an important steroid hormone mainly secreted by the corpus luteum of the ovary, placenta, and adrenal cortex, playing a central role in female reproductive health and pregnancy maintenance. Clinical testing of progesterone has wide applications, playing a crucial role in assessing ovarian function and ovulation, diagnosing luteal insufficiency, and assisting in the diagnosis of ectopic pregnancy. The main methods for progesterone detection currently available on the market are immunoassays, including biochemical immunoassays, enzyme-linked immunosorbent assays (ELISA), immunochromatography, and chemiluminescent immunoassays.

[0003] 17α-Hydroxyprogesterone is a key intermediate in the synthesis of cortisol, aldosterone, and sex hormones in the adrenal cortex and gonads, and its synthesis is mainly regulated by adrenocorticotropic hormone (ACTH). Clinically, the diagnosis and screening of congenital adrenal hyperplasia (CAH) is the most important clinical application of 17α-hydroxyprogesterone testing, especially since 21-hydroxylase deficiency leads to elevated 17α-hydroxyprogesterone levels. The molecular structure of 17α-hydroxyprogesterone differs from progesterone only by having one more hydroxyl group at position 17. Most commercially available progesterone antibodies cannot distinguish between 17α-hydroxyprogesterone and progesterone, leading to false positives in samples containing 17α-hydroxyprogesterone, where the measured progesterone level is higher than the actual value. Therefore, developing an antibody that can only recognize 17α-hydroxyprogesterone and not progesterone would solve this 17α-hydroxyprogesterone interference problem and reduce false positives in progesterone testing. Summary of the Invention

[0004] This invention provides a 17α-hydroxyprogesterone monoclonal antibody, its preparation method, and its application, to solve the problem of false positives caused by the influence of 17α-hydroxyprogesterone in the detection of small molecule compound PROG.

[0005] This invention provides a 17α-hydroxyprogesterone monoclonal antibody, wherein the heavy chain variable region (VH) of the antibody comprises a CDR region with the following sequence: Heavy chain CDR1 is shown in SEQ ID NO: 1; Heavy chain CDR2 is shown in SEQ ID NO: 2; Heavy chain CDR3 is shown in SEQ ID NO: 3; The antibody's light chain variable region (VL) contains a CDR region with the following sequence: The light chain CDR1 is shown in SEQ ID NO: 4; The light chain CDR2 is shown in SEQ ID NO: 5; The light chain CDR3 is shown in SEQ ID NO: 6. In some embodiments, the amino acid sequence of the heavy chain variable region of the 17α-hydroxyprogesterone monoclonal antibody is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8. In some embodiments, the heavy chain amino acid sequence of the 17α-hydroxyprogesterone monoclonal antibody is as shown in SEQ ID NO: 9; and the light chain amino acid sequence of the antibody is as shown in SEQ ID NO: 10. This invention provides a method for preparing the 17α-hydroxyprogesterone monoclonal antibody, comprising the following steps: 1) Balb / c mice were immunized with 17α-hydroxyprogesterone-BSA immunogen; 2) Fuse spleen cells from immunized mice with myeloma cells to obtain hybridoma cells; 3) Screening for hybridoma cell lines that specifically recognize 17α-hydroxyprogesterone but not progesterone to obtain the 12C3 cell line; 4) Antibodies were obtained by expressing and purifying the cells described above. The present invention provides a hybridoma cell line that secretes the 17α-hydroxyprogesterone monoclonal antibody. This invention provides the application of the 17α-hydroxyprogesterone monoclonal antibody or the cell line described herein in the preparation of 17α-hydroxyprogesterone detection products or as a progesterone detection interference blocker. The present invention provides a test kit for detecting progesterone, wherein the kit contains the 17α-hydroxyprogesterone monoclonal antibody as an inhibitor.

[0006] The beneficial effects of this invention are: When the antibody of the present invention is used as a blocking agent in progesterone detection (such as sandwich immunofluorescence detection), it can effectively block the interference of high concentrations of 17α-hydroxyprogesterone and significantly improve the detection accuracy. Attached Figure Description

[0007] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0008] Figure 1 This is a comparison diagram of the molecular structures of 17α-hydroxyprogesterone and progesterone. Figure 2 This is an SDS-PAGE electrophoresis image of the purified 12C3 monoclonal antibody. Figure 3 The activity curves of 12C3 antibody against different antigens were detected by ELISA. Figure 4 Clinical linearity of progesterone detection without the addition of blocking agents (R) 2 = 0.9332). Figure 5 Clinical linearity of progesterone detection after the addition of a 12C3 inhibitor (R) 2 = 0.9932). Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0010] Example 1: Preparation of 17α-hydroxyprogesterone antibody The reagents or kits involved in this application and their sources are as follows: Freund's Adjuvant Complete (Catalog No. 77140, Thermo Fisher); Freund's Incomplete Adjuvant (Catalog No. 77145, Thermo Fisher); HAT Media Supplement (50×) (Catalog No. 21060017, Thermo Fisher); HT Media Supplement (50×) (Catalog No. H0111067030, Thermo Fisher); PEG (Catalog No. P7181, Sigma); RPMI 1640 (Catalog No. L210KJ, Shanghai Yuanpei Biotechnology); Fetal Bovine Serum (FBS) (C04001-500, Shanghai Xiaopeng Biotechnology); DMEM (Catalog No. L310KJ, Shanghai Yuanpei Biotechnology); Penicillin-Streptomycin (Catalog No. 15140122, Gibco); HRP-labeled Goat Anti-Mouse Antibody (Catalog No. D110087, Shanghai Sangon Biotech); Protein A Resin (Catalog No. SA023010, Changzhou Tiandi Renhe Biotechnology Co., Ltd.)

[0011] 1. Mouse immunization Dissolve the above immunogen 17α-hydroxyprogesterone (purchased from Bio-Tech, catalog number BSA-0218A) and emulsify it with an equal volume of Freund's Adjuvant, Complete (Thermo Fisher, catalog number: 77140) until homogeneous. Take 6 Eight-week-old SPF-grade Balb / c mice (Fuzhou Wu's Animal Experiment Center) were injected subcutaneously at multiple sites with 200 μg / mouse. Two weeks later, an equal volume of Freund's Adjuvant, Incomplete (Thermo Fisher, catalog number: 77145) was injected subcutaneously at multiple sites to emulsify the antigen. Two booster immunizations were given. Three days before fusion, 100 μg / mouse was injected intraperitoneally for shock treatment.

[0012] 2. Preparation of feeder cells Balb / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and then, under aseptic conditions in a laminar flow hood, the abdominal skin was cut open to expose the peritoneum. 5 mL of RPMI 1640 basal culture medium containing 1% penicillin-streptomycin was injected into the peritoneal cavity using a syringe. The cells were repeatedly rinsed, and the rinse fluid was collected. The cells were centrifuged at 1000 rpm for 5 minutes, and the pellet was resuspended in RPMI 1640 complete culture medium containing 1% HAT (RPMI 1640 basal medium containing 10% fetal bovine serum and 1% penicillin-streptomycin). The cell concentration was adjusted to 1×10⁶ cells / mL. 5 Add 150 μL / well to a 96-well plate and incubate overnight at 37°C with 5% CO2.

[0013] Penicillin-Streptomycin: Gibco, Product No.: 15140122.

[0014] RPMI1640 basic culture medium: Shanghai Yuanpei Biotechnology, catalog number: L210KJ.

[0015] HAT culture medium: HAT Media Supplement (50×), Thermo Fisher, catalog number: 21060017.

[0016] 3. Preparation of immune spleen cells Three days after the last immunization of the mice, the spleens were removed under sterile conditions, placed in a petri dish, rinsed once with RPMI 1640 basal culture medium, and then ground and filtered on a nylon mesh (the nylon mesh was placed in a small beaker) to prepare a cell suspension. The cells were centrifuged, the supernatant was discarded, and the suspension was resuspended in RPMI 1640 basal culture medium. This process was repeated three times, and the cells were counted.

[0017] 4. Cell fusion (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium (Shanghai Yuanpei Biotechnology, catalog number: L310KJ) and 1 mL of 50% PEG (M12000, Sigma, catalog number: P7181) and place them in a 37°C water bath for preheating; (2) Take mouse myeloma cells Sp2 / 0 (2×10⁷ cells) and the above-mentioned immune spleen cells (1×10⁷ cells) respectively. 8 Add the suspension to a 50mL centrifuge tube, mix well, and add DMEM serum-free culture medium to a final volume of 40mL. Centrifuge for 10 minutes, discard the supernatant, and mix well. (3) Place the centrifuge tube in water preheated to 37°C, take 0.7 mL of 50% PEG solution preheated to 37°C, and let it stand for 90 seconds. Immediately add 15 mL of serum-free culture medium preheated to 37°C; (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and discard the supernatant. Add 40 mL of HAT culture medium containing 15%-20% fetal bovine serum (FBS). Mix well with a pipette and add 2 drops to each well of the 96-well cell culture plate containing feeder cells. Incubate at 37°C and 7% CO2.

[0018] Fetal bovine serum: Shanghai Xiaopeng Biotechnology, product number: C04001-500.

[0019] 5. Selective culture of hybridoma cells On days 1, 3, 5, and 7 after cell fusion, the culture medium was changed with HAT medium containing 15%-20% fetal bovine serum. The cells that survived were hybridoma cells, while the non-hybridoma cells died, and the true hybridoma cells were selected.

[0020] 6. Detection of specific antibodies and cloning of hybridoma cells Supernatant was collected from each culture well, and an indirect ELISA was used to detect the culture medium containing culture wells that specifically recognized 17α-hydroxyprogesterone-BSA but not progesterone-BSA. The OD of the supernatant from these wells that recognized 17α-hydroxyprogesterone-BSA was then measured. 490 A value greater than 2 indicates the OD value of progesterone-BSA on an ELISA test. 490 Cells with a value less than 0.2 are considered positive hybridoma cell lines. The day before cloning, prepare feeder cells according to step 3 and plate them; use a pipette to mix the selected positive hybridoma cells to be cloned, and dilute the cells in each well to 1 cell per well using HT medium; incubate at 37°C and 5% CO2 for 7-10 days; antibody detection is possible when visible clones appear; observe under an inverted microscope and mark the wells with only a single clone growing, thus initially screening for the 12C3 monoclonal antibody hybridoma cell line.

[0021] 8. Antibody sequencing Cloned 12C3 antibody cells were sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The amino acid sequence of the monoclonal antibody 12C3 was obtained as follows: Heavy chain CDR1 sequence: CETSQI (SEQ ID NO:1) Heavy chain CDR2 sequence: KEFSGGHVKL (SEQ ID NO:2) Heavy chain CDR3 sequence: YRGDWQVFM (SEQ ID NO:3) Light chain CDR1 sequence: LATPHEVCS (SEQ ID NO:4) Light chain CDR2 sequence: KFWYGD (SEQ ID NO:5) Light chain CDR3 sequence: SPHCAYNL (SEQ ID NO:6) The heavy chain variable region sequence is: QVQLQQPAAELVRPGASVKMSCKASGYNFCETSQIWVKQRPGRGLEWIGKEFSGGHVKLKATLTVDKSSSTTYIQLSGLASEDSAVYYCTRYRGDWQVFMWGQGTTLTVSS(SEQ ID NO:7) The light chain variable region sequence is: DIVMTQSTSSLPVSLGDRVSITCLATPHEVCSNTYLEWYQQKPDGTVKLLIYKFWYGDVPSRFSASGSGTDYSLTISNLEQEDFATYFCSPHCAYNLYTFGGGTKLEIK(SEQ ID NO:8) The antibody heavy chain sequence is: QVQLQQPAAELVRPGASVKMSCKASGYNFCETSQIWVKQRPGRGLEWIGKEFSGGHVKLKATLTVDKSSSTTYIQLSGLASEDSAVYYCTRYRGDWQVFMWGQGTTLTVSSSTPPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ IDNO:9) The antibody light chain sequence is: DIVMTQSTSSLPVSLGDRVSITCLATPHEVCSNTYLEWYQQKPDGTVKLLIYKFWYGDVPSRFSASGSGTDYSLTISNLEQEDFATYFCSPHCAYNLYTFGGGTKLEI KRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:10) 9. Antibody expression and purification Balb / c mice were injected intraperitoneally with 0.5 ml of liquid paraffin. Ten days later, the selected hybridoma cell lines were inoculated with 1×10⁻⁶ cells. 6 The fluid was injected into the peritoneal cavity of Balb / c mice. After about 10 days, the mice's abdomens began to swell. The mice were then euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and the ascites was extracted in a single procedure.

[0022] Ascites supernatant was purified by protein A affinity chromatography, following the procedures outlined in Cytiva's handbook: Affinity Chromatography, Vol. 1: Antibodies. 17α-hydroxyprogesterone monoclonal antibody 12C3 was prepared from the purified sample. The purified SDS-PAGE image is shown below. Figure 2 The purified antibody has clear and single heavy and light chain bands, indicating high purity.

[0023] The above operations are known to those skilled in the art, and other plasmid construction, cell transfection, culture methods, separation and purification techniques in the known field can also be used to obtain the above monoclonal antibodies.

[0024] Example 2: ELISA detection of 17α-hydroxyprogesterone antibody 12C3 activity 1. Take an ELISA plate and add 2 μg / mL of 17α-hydroxyprogesterone-BSA (purchased from Bio-Tech, catalog number BSA-0217A) or progesterone-BSA (purchased from Bio-Tech, catalog number BHYD-903) to each well of the coating solution (10 mM phosphate buffer, pH 7.4). Coat the wells with the coating solution, cover the plate, and incubate overnight at 2–8°C.

[0025] 2. Aspirate the liquid from each well and wash each well once with 200 µL of washing buffer (containing 0.05% Tween 20 mM phosphate-buffered saline). After washing, invert the plate and gently tap it on absorbent paper to remove any remaining liquid.

[0026] 3. At room temperature, add 200 µL of blocking buffer (10% skim milk) to each well and block for 1 hour.

[0027] 4. Suction, invert the perforated plate and gently pat it on absorbent paper to remove residual liquid.

[0028] 5. Add 100 μL to the blocking buffer to dilute different concentrations of 12C3 solution. Incubate at room temperature for 1 hour.

[0029] 6. Aspirate the liquid from each well and wash 6 times with 200 µL of washing buffer to each well.

[0030] 7. Add 10,000 times diluted HRP-labeled goat anti-mouse antibody (catalog number D110087, Shanghai Sangon Biotech), 50-100 μL per well, incubate at 37°C for 30 min, and wash 6 times.

[0031] 8. Add 100 μL of freshly prepared substrate development solution (purchased from Sangon Biotech, catalog number D110098) to each well, incubate at 37°C for 15 min.

[0032] 9. Terminate the reaction with 2 mol / L H2SO4 and read the OD on an ELISA reader. 450 The values ​​and results are shown in Table 1 below. Figure 2 .

[0033] Table 1. ELISA data of 112C3 with 17α-hydroxyprogesterone-BSA and progesterone-BSA

[0034] From Table 1 and Figure 2 Looking at the top: the 12C3 antibody shows almost no binding to PROG-BSA; however, as the antibody concentration increases, the OD... 450 The results remained largely unchanged, while the 12C3 antibody bound to 17α-hydroxyprogesterone-BSA with a good gradient, indicating that the 12C3 antibody is a 17α-hydroxyprogesterone-specific antibody and has no cross-reactivity with PROG, and can be used for the development of PROG reagent blocking agents.

[0035] Example 3: Evaluation of blocking effect 1. Blocking effect of 12C3 antibody on PROG immunofluorescence reagent 17α-hydroxyprogesterone small molecules were diluted to 20, 100, and 500 ng / mL. PROG immunofluorescence kits (purchased from Baotai Biotechnology, catalog number 3.5.02.05.0002) were used. Sample dilutions were prepared with 1 μg / person, 5 μg / person, 10 μg / person, and no 12C3 antibody. The diluted 17α-hydroxyprogesterone samples were tested, and the results are shown in Table 2 below. 1 μg / person of 12C3 antibody completely blocked the effect of 20 ng / mL 17α-hydroxyprogesterone. Increasing the 12C3 dosage to 10 μg / person essentially blocked the effect of 500 ng / mL 17α-hydroxyprogesterone, indicating a good blocking effect of 12C3.

[0036] Table 2.1.1.1.2 ...

[0037] 2. The effect of 12C3 antibody on clinical samples tested with PROG immunofluorescence reagent PROG's immunofluorescence kit was used. Sample diluents were prepared with and without 5 μg / person of 12C3 antibody. Fifteen clinical samples were tested to investigate the effect of 12C3 antibody on the linearity and fluorescence value of the clinical samples. The results are shown in Table 3 below. Figure 4 and Figure 5 The addition of 12C3 antibody had no significant effect on the overall fluorescence value of the reagent; the linearity of the addition of 12C3 antibody was improved, and the R2 value was better, especially for the two abnormal samples No. 8 and No. 12.

[0038] Table 3. Clinical test results using the PROG immunofluorescence sandwich method.

[0039] In summary, the 17α-hydroxyprogesterone monoclonal antibody 12C3 provided by this invention has shown unexpected technical effects.

[0040] First, this invention successfully overcomes the "selectivity paradox" of interference blocking in immunoassay. In the immunoassay system for progesterone (PROG), because 17α-hydroxyprogesterone (17α-OHP) differs from progesterone by only one hydroxyl group at position C17 in their molecular structure, conventional 17α-OHP antibodies in the art often exhibit varying degrees of cross-reactivity with progesterone. If added as a blocking agent to the detection system, while it can bind some interfering substances, it will inevitably bind the analyte progesterone in the sample, leading to false negatives. However, the 12C3 antibody of this invention forms a highly specific binding site through the synergistic action of specific heavy and light chain CDR region sequences. Experimental data shows that it has extremely high affinity for 17α-OHP while its cross-reactivity with progesterone is close to zero (as shown in the ELISA results in Example 2, there is essentially no binding signal for progesterone-BSA).

[0041] Secondly, this invention achieves interference shielding over an extremely wide range. The concentration of 17α-OHP in clinical samples fluctuates greatly (e.g., it may be abnormally elevated in CAH patients). Experimental results show that only a very low dose of 12C3 antibody (10 μg / person) is needed to completely neutralize 17α-OHP interference at concentrations as high as 500 ng / mL. This highly efficient blocking capability allows the progesterone test kit to maintain excellent stability even in extreme pathological samples.

[0042] Finally, the significant improvement in clinical relevance demonstrates its application value. By comparing clinical test data before and after the addition of the blocking agent, the correlation coefficient R... 2 The accuracy improved dramatically from 0.9332 to 0.9932. This leap forward is not a simple linear additive effect, but rather demonstrates that the 12C3 antibody can precisely locate and eliminate "structural analog noise" that causes detection deviations, bringing the accuracy of the immunoassay system to the gold standard level of reference methods (such as LC-MS / MS). This near-absolute recognition and blocking effect between highly homologous small molecules fully demonstrates the non-obviousness of the antibody sequence design and the outstanding technological advancements of this invention.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A 17α-hydroxyprogesterone monoclonal antibody, characterized in that, The heavy chain variable region (VH) of the antibody contains a CDR region with the following sequence: Heavy chain CDR1 is shown in SEQ ID NO: 1; Heavy chain CDR2 is shown in SEQ ID NO: 2; Heavy chain CDR3 is shown in SEQ ID NO: 3; The antibody's light chain variable region (VL) contains a CDR region with the following sequence: The light chain CDR1 is shown in SEQ ID NO: 4; The light chain CDR2 is shown in SEQ ID NO: 5; The light chain CDR3 is shown in SEQ ID NO:

6.

2. The 17α-hydroxyprogesterone monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

8.

3. The 17α-hydroxyprogesterone monoclonal antibody according to claim 2, characterized in that: The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 9; The light chain amino acid sequence of the antibody is shown in SEQ ID NO:

10.

4. A method for preparing the 17α-hydroxyprogesterone monoclonal antibody according to any one of claims 1-3, characterized in that, Includes the following steps: 1) Balb / c mice were immunized with 17α-hydroxyprogesterone-BSA immunogen; 2) Fuse spleen cells from immunized mice with myeloma cells to obtain hybridoma cells; 3) Screening for hybridoma cell lines that specifically recognize 17α-hydroxyprogesterone but not progesterone to obtain the 12C3 cell line; 4) Antibodies were obtained by expressing and purifying the cells described above.

5. The use of the 17α-hydroxyprogesterone monoclonal antibody according to any one of claims 1-3 in the preparation of 17α-hydroxyprogesterone detection products or as a progesterone detection interference blocker.

6. A test kit for detecting progesterone, characterized in that, The kit contains the 17α-hydroxyprogesterone monoclonal antibody as described in any one of claims 1-3 as an inhibitor.

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