MONOCLONAL ANTIBODY AND POLYCLONAL ANTIBODY AND OXYTOCIN QUANTIFICATION METHOD BASED ON SAME
20230168258 · 2023-06-01
Inventors
- José Joaquín CERÓN MADRIGAL (Murcia, ES)
- Silvia MARTÍNEZ SUBIELA (Murcia, ES)
- Marina LÓPEZ ARJONA (Murcia, ES)
Cpc classification
G01N33/74
PHYSICS
C07K16/26
CHEMISTRY; METALLURGY
International classification
G01N33/74
PHYSICS
Abstract
The present invention relates to an in vitro method for the quantification of free oxytocin, protein-bound oxytocin that can be released in a reduction/alkylation treatment, and protein-bound oxytocin, in a sample, without performing a reduction/alkylation treatment, wherein said method comprising carrying out a first assay using a monoclonal antibody that binds specifically to free oxytocin and to protein-bound oxytocin that can be released in a reduction/alkylation treatment, and carrying out a second assay using a polyclonal antibody that binds specifically to protein-bound oxytocin. The invention also relates to the monoclonal antibody and polyclonal antibody, and to a composition and kit comprising same.
Claims
1. An in vitro method for the detection and quantification of free oxytocin and protein-bound oxytocin able to be released in a reduction/alkylation treatment, in a sample, said method comprising carrying out an assay comprising: adding to said sample a monoclonal antibody that specifically binds to free oxytocin and protein-bound oxytocin able to be released in a reduction/alkylation treatment, wherein said monoclonal antibody comprises: a heavy chain CDR1 region consisting of the sequence SEQ ID NO: 6; a heavy chain CDR2 region consisting of the sequence SEQ ID NO: 8; a heavy chain CDR3 region consisting of the sequence SEQ ID NO: 10; a light chain CDR1 region consisting of the sequence SEQ ID NO: 16; a light chain CDR2 region consisting of the sequence SEQ ID NO: 18; and a light chain CDR3 region consisting of the sequence SEQ ID NO: 20; determining the amount or concentration of monoclonal antibody bound to free oxytocin and protein-bound oxytocin able to be released in a reduction/alkylation treatment; and determining the amount or concentration of free oxytocin and protein-bound oxytocin able to be released in a reduction/alkylation treatment from the amount or concentration of monoclonal antibody bound to said free oxytocin and to protein-bound oxytocin able to be released in a reduction/alkylation treatment.
2. The method according to claim 1, wherein the variable domain of the heavy chain VH has at least 95% identity to the sequence SEQ ID NO: 3 and the variable domain of the light chain VL has at least 95% identity to the sequence SEQ ID NO: 13.
3. The method according to claim 1, wherein the variable domain of the heavy chain VH consists of the sequence SEQ ID NO: 3 and the variable domain of the light chain VL consists of the sequence SEQ ID NO: 13.
4. The method according to claim 1, wherein the assay is selected from the group consisting of immunofluorescence, immunohistochemistry, immunochromatography, luminescence, homogeneous amplified luminescent proximity assay, immunoelectrotransfer, ELISA, western blot, nephelometry, immunoturbidimetry, lateral chromatography, and microarrays.
5. (canceled)
6. (canceled)
7. The method according to claim 1, wherein said monoclonal antibody is attached to a labelling probe or to a sphere attached to a labelling probe.
8. The method according to claim 1, wherein said labelling probe is selected from the group consisting of a fluorophore, a chromophore, a bioluminescent probe, a radioactive probe, an enzyme, and colloidal gold.
9. (canceled)
10. The method according to claim 1, wherein said sample is selected from the group consisting of serum, plasma, saliva, urine, and cerebrospinal fluid.
11. A monoclonal antibody that specifically binds to free oxytocin and protein-bound oxytocin able to be released in a reduction/alkylation treatment, wherein said monoclonal antibody comprises: a heavy chain CDR1 region consisting of the sequence SEQ ID NO: 6; a heavy chain CDR2 region consisting of the sequence SEQ ID NO: 8; a heavy chain CDR3 region consisting of the sequence SEQ ID NO: 10; a light chain CDR1 region consisting of the sequence SEQ ID NO: 16; a light chain CDR2 region consisting of the sequence SEQ ID NO: 18; and a light chain CDR3 region consisting of the sequence SEQ ID NO: 20.
12. The monoclonal antibody according to claim 11, wherein the variable domain of the heavy chain VH has at least 95% identity to the sequence SEQ ID NO: 3 and the variable domain of the light chain VL has at least 95% identity to the sequence SEQ ID NO: 13.
13. The monoclonal antibody according to claim 11, wherein the variable domain of the heavy chain VH consists of the sequence SEQ ID NO: 3 and the variable domain of the light chain VL consists of the sequence SEQ ID NO: 13.
14. A polynucleotide encoding the monoclonal antibody according to claim 11, wherein said polynucleotide comprises the sequence SEQ ID NO: 22, which encodes the variable domain of the heavy chain, and sequence SEQ ID NO: 23, which encodes the variable domain of the light chain.
15. A method of producing the monoclonal antibody of claim 11, said method comprising: (a) culturing an hydridoma expressing the monoclonal antibody in a culture medium; and (b) purifying the monoclonal antibody from the culture medium.
16.-20. (canceled)
21. A composition comprising the monoclonal antibody according to claim 11.
22. (canceled)
23. A kit for detecting free oxytocin and protein-bound oxytocin able to be released in a reduction/alkylation treatment, in a sample, wherein said kit comprises: (i) the monoclonal antibody according to claim 11; and (ii) buffer solutions to carry out the reactions to form a complex between the monoclonal antibody and the free oxytocin and the protein-bound oxytocin able to be released in a reduction/alkylation treatment.
24. (canceled)
25. (canceled)
26. The kit according to claim 23, further comprising reagents for carrying out the reaction to form said complexes between the monoclonal antibody and the free oxytocin and protein-bound oxytocin able to be released in a reduction/alkylation treatment.
27. (canceled)
28. A method of using the kit of claim 23 in the in vitro detection of free oxytocin and protein-bound oxytocin able to be released in a reduction/alkylation treatment and/or protein bound oxytocin, in a sample.
29. The method according to claim 28, wherein said sample is selected from the group consisting of serum, plasma, saliva, urine, and cerebrospinal fluid.
30. The monoclonal antibody according to claim 11, wherein said monoclonal antibody is attached to a labelling probe or to a sphere attached to a labelling probe.
31. The monoclonal antibody according to claim 30, wherein said labelling probe is selected from the group consisting of a fluorophore, a chromophore, a bioluminescent probe, a radioactive probe, an enzyme, and colloidal gold.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0088]
[0089]
DESCRIPTION OF EMBODIMENTS
[0090] Materials and Methods
[0091] Animals and Samples
[0092] The samples used in this study were obtained from 10 female pigs (Sus scrofa domesticus), Large White. The pigs had ad libitum access to a balanced diet and water, with a temperature between 19-23° C. and samples were taken between 8:30 and 10:00 h. A sample of each pig was taken 3 times during the study: day 58 of gestation, day 7 after farrowing (during lactation) and day 45 after farrowing. The animals were housed in the experimental farm of the University of Murcia (Murcia, Spain), in groups with a minimum space of 1.64 m.sup.2 per animal on day 58 of gestation and individually on days 7 and 45 after delivery (recommendations described in Directive 2001/88/EC, 2001 and Directive 2001/93/EC, 2001).
[0093] Saliva samples were collected using Salivette tubes (Sarstedt, Aktiengesellschaft & Co.) containing a sponge. The pigs were allowed to chew the sponge, which was attached to a thin, flexible metal rod, until it was completely wet (or at least 1 minute). The sponges were then placed into the Salivette tubes which were immediately centrifuged at 3000 g for 10 minutes at 4° C. The saliva was then transferred into 1.5 mL tubes and stored at −80° C. until analysis. In all cases, at least 500 microliters of saliva were obtained.
[0094] All procedures were approved by the Ethics Committee of the University of Murcia.
[0095] Reduction/Alkylation Procedure (R/A)
[0096] The R/A procedure was carried out following the protocol described in Brandtzaeg et al. (2016). 30 samples of pig saliva were treated. 100 μl of each sample was diluted in 200 μl 50 mM Tris-HCl buffer (pH 8.0). Then, 5 μl of 0.5 M dithiothreitol (DTT) (GE Healthcare Life Sciences) was added to 100 μl of the diluted saliva sample, mixed for 30 seconds and incubated at 37° C. for 45 minutes, followed by cooling to room temperature (22° C.). Next, 15 μL of 0.5 M iodoacetamide (IAM) (GE Healthcare Life Sciences) was added to each solution, mixed for 30 seconds and incubated at 22° C. in darkness for 20 minutes. Then, 80% acetonitrile (ACN) solution (Scharlab, SL), in ultrapure water (Millipore) (v/v), previously kept on ice, was added, mixed for 30 seconds and centrifuged for 15 minutes at 14,000 rpm in an Eppendorf Sorvall ST 8R centrifuge (Thermo Fisher Scientific). After centrifugation, the supernatant was pipetted into a new Eppendorf tube and evaporated to dryness in a Speed Vac concentrator (Eppendorf Concentrator 5301) followed by reconstitution in 100 μL saline phosphate buffer (PBS). The treated samples were diluted 1:2 with each buffer corresponding to each assay.
[0097] To assess whether the R/A procedure could affect the assays used in our study regardless of the effect it has on bound oxytocin, the assay buffer used for each assay was processed using the R/A protocol and analysed as an unknown sample in each of the three assays of our study.
Example 1. Production of the Monoclonal Antibody of the Invention “P26C” which Specifically Recognizes Free Oxytocin
[0098] Generation of Hybridoma 3 mice (BALB/c) were immunized with two intraperitoneal injections separated by an interval of 15 days with 50 μg of oxytocin-KLH (Cusabio) as antigen, where KLH is Keyhole Limpet Haemocyanin, that is, the haemocyanin of the keyhole limpet (Megathura crenulata). The antigen was emulsified with Freund's complete adjuvant (CCF, Sigma F-5881) at the first injection, and with Freund's incomplete adjuvant (CIF. Sigma F-5506) for the second intraperitoneal injection. The final immunization consisted of an intravenous or intra-splenic injection of 50 μg of antigen without the adjuvant.
[0099] Three days later, the spleen was extracted from the mice and the cells were fused to the Sp2/0-Ag14 murine myeloma cell line, developed from non-immunoglobulin-secreting BALB/c mice using 50% solution polyethylene glycol (Sigma). Hybridomas were selected by growth in RPMI 1640 medium (Biowhitaker) containing hypoxanthine, aminopterin, and thymidine (HAT), which was supplemented with 10% fetal bovine serum (Gibco), 2 mM glutamine (Gibco), 1 mM pyruvate (Sigma), and 1% penicillin/streptomycin (Gibco). In the HAT medium with aminopterin, only hybrid cells, consisting of B cells and myeloma cells, grow because B cells possess the entire panel of DNA synthesis rescue pathway enzymes and, therefore, can use the hypoxanthine and thymidine from the medium.
[0100] Finally, hybridoma supernatants were analysed by ELISA assay for the presence of anti-oxytocin immunoglobulin-secreting hybridomas. Positive hybridomas were cloned by limit dilution and expanded and frozen in liquid nitrogen.
[0101] Identification of the Monoclonal Antibody
[0102] An analysis of the supernatants of the hybridomas generated by indirect ELISA assay using Oxytocin-BSA was carried out. To this end, they were incubated in 96-well flat-bottomed Nunc microplates (Maxisorp), with 50 μL of oxytocin-BSA (250 ng antigen per well) in carbonate-bicarbonate buffer pH 9.8 overnight, at 4° C., to allow the correct adhesion of the proteins to the plate. Subsequently the plates were washed with 100 μL 0.05% PBS/Tween 20 and blocked for 30 minutes at 37° C. with 100 μL phosphate buffered saline (PBS) with 5% milk powder. The plates were then incubated 1 hour at 37° C., with 100 μL per well of each supernatant from the hybridomas. Next, three washes were carried out with 0.1% PBS/Tween 20 buffer. Then, 100 μL per well of a mouse anti-IgG antibody conjugated with horseradish peroxidase was added at a 1:2500 dilution and incubated for 1 hour at room temperature. After 5 washes with 0.1% PBS/Tween 20 buffer, to remove all labelled molecules not fixed in the form of immunocomplexes, the enzymatic substrate in solution (0.1M sodium citrate, 0.1M citric acid, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and H.sub.2O.sub.2) was added. Subsequently, absorbance (OD) was measured on a spectrophotometer (BioTek) at a wavelength of 405 nm every 5 minutes to a total of 25 minutes.
[0103] The specific anti-oxytocin antibody was identified in the P26C hybridoma supernatant in said indirect ELISA assay using oxytocin-BSA. Clone P26C was selected because the highest absorbance signal was obtained with said clone in said ELISA assay, indicating a higher affinity for oxytocin than the other clones.
[0104] A dot-blot assay was carried out with clone P26C, which determined that it had reactivity against oxytocin of a high number of species, such as pig, dog, horse and human oxytocin.
[0105] Purification of the monoclonal antibody P26C
[0106] For purification of the P26C monoclonal antibody from the supernatant obtained from the selected hybridoma, a HiTrap™ Protein G HP affinity column (GE Healthcare Life Sciences) was used using a ÄKTA pure chromatography system (GE Healthcare Life Sciences).
[0107] Structural Characterisation of the Monoclonal Antibody P26C
[0108] Total RNA was extracted from hybridoma cells and the cDNA was subsequently synthesized. The monoclonal antibody variable domains were then amplified by isotype-specific PCR, subcloned into a separate standard cloning vector, and sequenced. The isotype of the monoclonal antibody was determined to be IgGk. The variable domains of 5 clones were sequenced and the sequence was found to be identical in all 5 clones. Table 1 shows the sequence details of monoclonal antibody P26C.
TABLE-US-00001 TABLE 1 Fragment/Region/Sequence SEQ ID NO Variable domain of the heavy chain SEQ ID NO: 2 VH including signal peptide Variable domain of the heavy chain SEQ ID NO: 3 VH Signal peptide of the heavy chain VH SEQ ID NO: 4 FR1 framework region of the heavy SEQ ID NO: 5 chain VH CDR1 region of the heavy chain VH SEQ ID NO: 6 FR2 framework region of the heavy SEQ ID NO: 7 chain VH CDR2 region of the heavy chain VH SEQ ID NO: 8 FR3 framework region of the heavy SEQ ID NO: 9 chain VH CDR3 region of the heavy chain VH SEQ ID NO: 10 FR4 framework region of the heavy SEQ ID NO: 11 chain VH Variable domain of the light chain VL SEQ ID NO: 12 including signal peptide Variable domain of the light chain VL SEQ ID NO: 13 Signal peptide of the light chain VL SEQ ID NO: 14 FR1 framework region of the light SEQ ID NO: 15 chain VL CDR1 region of the light chain VL SEQ ID NO: 16 FR2 framework region of the light SEQ ID NO: 17 chain VL CDR2 region of the light chain VL SEQ ID NO: 18 FR3 framework region of the light SEQ ID NO: 19 chain VL CDR3 region of the light chain VL SEQ ID NO: 20 FR4 framework region of the light SEQ ID NO: 21 chain VL Nucleotide sequence encoding the SEQ ID NO: 22 variable fragment of the heavy chain VH including signal peptide Nucleotide sequence encoding the SEQ ID NO: 23 variable fragment of the light chain VL including signal peptide
Example 2. Determination of Free Oxytocin Concentrations in Saliva Samples
[0109] The saliva samples described in Materials and Methods were thawed at room temperature and 10 μL of 1:2 sample was diluted in assay buffer (AlphaLISA® Universal buffer, Perkin elmer, Inc.). An AlphaLISA® assay (Perkin Elmer, Inc.) was carried out, in 96-well plates, with a reaction volume of 50 μL per well, using spheres coated with the monoclonal antibody P26C and oxytocin concentrations in the samples were determined.
Example 3. Production of the Polyclonal Antibody of the Invention “P0-10” that Specifically Recognizes Protein-Bound Oxytocin
[0110] A New Zealand rabbit (female, 2.5 kg, 3 months of age) was immunized with an oxytocin-KLH (Cusabio) conjugate as antigen, using 150 μg mixed in Freund's complete adjuvant for the first injection and Freund's incomplete adjuvant for the second and subsequent injections. One week after each immunization, several blood samples were collected and an ELISA assay was carried out to evaluate antibody titre using oxytocin-BSA-coated plates and a horseradish peroxidase-labelled rabbit anti-IgG secondary antibody.
[0111] Purification of Polyclonal Antibody
[0112] An N-hydroxysuccinimide (NHS)-activated HiTrap™ HP (GE Healthcare Life Sciences) affinity column was used, to which oxytocin-BSA (Cusabio) was coupled and 1 mL batches of rabbit oxytocin antiserum were passed through the column using a ÄKTA pure chromatography system (GE Healthcare Life Sciences).
Example 4. Determination of Protein-Bound Oxytocin Concentrations in Saliva Samples
[0113] The saliva samples described in the Materials and Methods section were thawed at room temperature and 10 μL of sample was diluted in assay buffer (AlphaLISA® immunoassay buffer, Perkin Elmer Inc.). An AlphaLISA® (Perkin Elmer, Inc.) indirect competition assay was carried out on 96-well plates, with a final reaction volume of 50 μL per well, using G-protein coated beads (Perkin Elmer Inc.) and oxytocin concentrations in the samples were determined. Different concentrations of biotinylated oxytocin (0-6 nM) and the polyclonal antibody of the invention PO-10 (10-20 nM) were assayed. Different concentrations of donor beads (Perkin Elmer Inc.) and G-protein coated acceptor beads were used at concentrations between 20-40 μg/mL.
Example 5. Measurement of Oxytocin by the Method of the Invention. Comparative Assay with Cayman Chemical ELISA Kit
[0114] Imprecision was calculated as variations between assays and intra-assays and expressed as coefficients of variation (CV). Five replicates of each sample (low, medium and high saliva samples) were analysed at the same time, to determine the intra-assay precision of the method. Five aliquots of each saliva sample were stored in plastic vials at −80° C. until analysis. On the day of analysis, samples were brought to room temperature prior to oxytocin measurement. These samples were measured in duplicate five times over five different days to determine accuracy between assays using freshly prepared calibration curves.
[0115] Accuracy was assessed by evaluating linearity after dilution and recovery experiments. For the linearity evaluation, two samples with high and medium oxytocin values were diluted in series. Dilutions were 1:4 to 1:128 and 1:2 to 1:64 with assay buffer (AlphaLISA® immunoassay buffer, Perkin Elmer Inc.) for the high and medium value sample, respectively. For the recovery test, a pig saliva sample with a known oxytocin concentration with different amounts of oxytocin standard (120, 80, 40, 20 and 10 ng/L) was diluted (1:2). The percentages of oxytocin concentrations measured at the expected oxytocin concentrations were then calculated.
[0116] The limit of detection (LD) was calculated from 12 replicate oxytocin assay buffer determinations (AlphaLISA® immunoassay buffer, Perkin Elmer Inc.) as mean value plus 3 standard deviations (SD). The Lower Limit of Quantification (LLOQ) was calculated based on the lowest oxytocin concentration that could be measured with 20% accuracy. For this, a saliva sample was serially diluted with assay buffer and analysed five times at the same time for each dilution.
[0117] A commercially available ELISA kit from Cayman Chemical was used for the measurement of oxytocin for comparative purposes within the validation.
[0118] Statistical Analysis
[0119] The data obtained from different days of gestation and lactation were evaluated to determine the normality of the distribution, using the Shapiro-Wilk test, which provides a non-parametric distribution. Results below the limit of detection were set at the limit of detection of each method. The values were logarithmically transformed and the paired t-test was used for the differences between before and after the R/A procedure within each method. A one-way repeated measures ANOVA was applied to the log-transformed values, followed by uncorrected Fisher's LSD to compare oxytocin values obtained at different times on day 58 before farrowing and days 7 and 45 after farrowing. Spearman's correlation coefficients were calculated to evaluate the correlation between the three methods before and after the R/A procedure (GraphPad prism 5.00 for Windows).
[0120] In the polyclonal antibody immunoassay, intra-assay coefficients of variation (CV) were in the range 4.36-11.37% and inter-assay coefficients of variation were in the range 8.52-13.38%. Dilution of saliva samples resulted in linear regression equations with a correlation coefficient ranging from 0.98 to 0.99. The beta coefficients were significantly non-zero and the run test showed no deviation from linearity. The recovery results obtained were between 80% and 116% and the limit of detection of the assay was 58.4 pg/mL.
[0121] In the assay with the Cayman Chemical commercial kit, the intra-assay coefficients of variation were in the range 11.34-15.72% and the inter-assay coefficients of variation were in the range 18.56-21.04%. Dilution of saliva samples resulted in linear regression equations with a correlation coefficient of 0.99. The beta coefficients were significantly different from zero and the run test showed no deviation from linearity and the limit of detection of the assay was 4.66 pg/mL.
[0122] Effects of R/a Treatment
[0123] The results obtained for the samples are shown in
[0124] When the buffers were analysed, the samples gave a result below the limit of detection before and after the R/A procedure in all three assays.
[0125] Biological Response of Each Assay
[0126] The biological response of oxytocin concentrations with each assay is shown in
[0127] In the AlphaLISA® assay with the polyclonal antibody without R/A procedure, salivary oxytocin concentrations increased significantly on day 7 after farrowing, with a mean value of 25695 pg/mL, compared to gestation day 58 (mean value of 6896 pg/mL) (3.72-fold; P=0.0007) and day 45 after farrowing (mean value of 5939 pg/mL) (4.33-fold; P=0.0001). When the R/A procedure was applied to this assay, salivary oxytocin concentrations increased significantly on day 7 after farrowing, with a mean value of 1677 pg/mL, compared to gestation day 58 (mean value of 534.8 pg/mL) (3.14-fold; P=0.0003) and day 45 after farrowing (mean value of 906.5 pg/mL) (1.85-fold; P=0.0157).
[0128] Finally, when the Cayman Chemical ELISA kit was used without the R/A procedure, only 6 of the analysed samples were above the detection limit, so it was not possible to apply these conditions to evaluate possible biological changes. In the ELISA assay with the Cayman Chemical kit, with R/A procedure, salivary oxytocin concentrations increased significantly on day 7 after farrowing, with a mean value of 448.6 pg/mL, compared to day 45 after farrowing (mean value of 208.8 pg/mL) (2.15-fold; P=0.039) but there were no significant differences with gestation day 58 (mean value of 275.5 pg/mL) (1.63-fold; P=0.1999).
[0129] The summary of correlations between procedures is shown in Table 2. The asterisks indicate the statistical significance (***P≤0.001; **P≤0.01; * P≤0.05).
TABLE-US-00002 TABLE 2 AlphaLISA AlphaLISA AlphaLISA AlphaLISA Cayman kit Cayman kit monoclonal no R/A monoclonal R/A polyclonal no R/A polyclonal R/A no R/A R/A AlphaLISA 0.65*** 0.63*** 0.39* 0.51*** 0.67*** monoclonal no R/A AlphaLISA 0.68*** 0.58*** 0.47** 0.53*** monoclonal R/A AlphaLISA 0.48** 0.25 0.44** polyclonal no R/A AlphaLISA 0.28 0.34 polyclonal R/A Kit Cayman 0.36* no R/A
[0130] When the R/A procedure was not carried out on the samples, the AlphaLISA® assay with the monoclonal antibody showed a significant moderate correlation with both the AlphaLISA® assay with the polyclonal antibody (P<0.001; r=0.63) and the Cayman Chemical ELISA kit assay (P<0.001; r=0.51). No significant correlation was found between the AlphaLISA® assay with the polyclonal antibody and the Cayman Chemical ELISA kit assay. When the R/A procedure was carried out, the AlphaLISA® assay with the monoclonal antibody showed a significant moderate correlation with both the AlphaLISA® assay with the polyclonal antibody (P<0.001; r=0.58) and the Cayman Chemical ELISA kit assay (P<0.001; r=0.53). No significant correlation was found between the AlphaLISA® assay with the polyclonal antibody and the Cayman Chemical ELISA kit assay.
REFERENCES
[0131] Brandtzaeg et al. (2016). Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum. Sci Rep, 6, 31693. [0132] López-Arjona et al. (2020). Oxytocin in saliva of pigs: an assay for its measurement and changes after farrowing. Domest Anim Endocrinol, 70, 106384. [0133] MacLean et al. (2018). Validation of salivary oxytocin and vasopressin as biomarkers in domestic dogs. J Neurosci Methods, 293, 67-76.