Journal: Brain

Article Title: Augmentation of phenotype in a transgenic Parkinson mouse heterozygous for a Gaucher mutation

doi: 10.1093/brain/awu291

Figure Lengend Snippet: Local accumulations of phosphorylated SNCA in the CA1 region of the hippocampus of hSNCAA53T;Gba+/L444P and hSNCAA53T;GbaWT mice. (A.1–2) The hippocampus of a 15-month-old hSNCAA53T;GbaWT mouse stained for phosphorylated S129 SNCA. The boxed area is magnified in A.2. No accumulation of abnormal (brownish) staining can be seen. (A.3–4) The hippocampus of a 15-month-old hSNCAA53T;Gba+/L444P mouse. Increased staining for phosphorylated S129 SNCA, reminiscent of synaptic accumulations, can be seen in multiple locations in the CA1 region. (B) Quantification of the positive pixels in the hippocampus of 15-month-old mice as analysed by ImageScope software (Aperio) (n = 2 for each genotype). (C.1–2) The hippocampus of 18-month-old wild-type mouse. (C.3–4) The hippocampus of a 19-month-old hSNCAA53T;GbaWT mouse. (C.5–6) The hippocampus of a 19-month-old hSNCAA53T;Gba+/L444P mouse. Positive staining can be detected in the CA1 region of both genotypes. (D) Quantification of the positive pixels in the hippocampus of 18 to 19-month-old mice (n = 2 for wild-type control mice, and n = 3 for each of the transgene genotypes). When the two sets of data were combined, the hSNCAA53T;Gba+/L444P mice exhibited a 70% increase in pSer129 SNCA staining (from 100 ± 28% to 170 ± 15%, P < 0.05 one-tailed t-test). WT = wild-type.

Article Snippet: Similar results were also obtained by western blot analysis of 19 month hippocampal lysates with a different commercial antibody against pSer129 (Wako) (S3).

Techniques: Staining, Software, One-tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: Relation of Ser129-phosphorylated α-syn levels to total α-syn ones in intra- and extracellular spaces. CHO cells were transfected with the empty vector or the indicated amounts of wild-type α-syn cDNA. Samples were loaded along with recombinant α-syn proteins and Ser129-phosphorylated α-syn proteins for standards, followed by western blotting. Bands of Ser129-phosphorylated α-syn and total α-syn, including phosphorylated and non-phosphorylated forms, were detected by EP1536Y and Syn-1 antibody, respectively. Relative band intensities of Ser129-phosphorylated α-syn and total α-syn were corrected by plotting them on the standard curves, and then normalized to the intensities of β-actin. a Relation in intracellular α-syn. Cell lystaes (10 μg/lane) were loaded. b Relation in extracellular α-syn. After TCA-precipitated proteins were resolved by Laemmli’s sample buffer, samples corresponding to 20% of CM volume were loaded. Graphs show the positive correlation between Ser-129 phosphorylated and total α-syn levels

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Transfection, Plasmid Preparation, Recombinant, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: Effects of Ca 2+ on Ser129-phosphorylation of α-syn. SH-SY5Y cell lines stably expressing wild-type α-syn (wt-aS/SH #4) were incubated in media containing 5 μM calcium ionophore A23187 except b . As vehicle control, cells were treated with DMSO at the same final concentration as reagents used. Cell lysates (15 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or anti-β-actin (AC-15) antibody. a Effect of A23187 incubation time on Ser129-phosphorylation. Cells were treated with A23187 for the indicated time points until 8 h. b Effect of A23187 concentrations on Ser129-phosphorylation. Cells were treated with A23187 at the indicated concentrations for 8 h. c, d Effect of extracellular Ca 2+ chelator EGTA ( c) or intracellular Ca 2+ chelator BAPTA-AM (B-AM) ( d ) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 5 μM A23187 with the indicated concentrations of EGTA or BAPTA-AM for 4 h. e, f Effect of CaM inhibitor W-7 ( e ) or calmidazolium (Calm) ( f ) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing A23187 with the indicated concentrations of W-7 or calmidazolium for 4 h. Representative blots are shown. In the graphs of a to f , relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances (*, P < 0.05; **, P < 0.01)

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Incubation, Control, Concentration Assay, SDS Page, Western Blot

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: Effects of mitochondrial complex I inhibitors MPP + and rotenone on Ser129-phosphorylation of α-syn. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a Effect of incubation time of MPP + and rotenone on Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 1 mM MPP + or 5 μM rotenone for the indicated time points until 16 h. Vehicle controls were treated with DMSO at the same final concentration as each reagent. b Effect of concentrations of MPP + or rotenone on Ser129-phosphorylation. Cells were incubated in media containing the indicated amounts of MPP + or rotenone for 12 h. Graphs show relative band intensities of Ser129-phosphorylated α-syn and total α-syn. They were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances (*, P < 0.05; **, P < 0.01)

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Incubation, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: Role of Ca 2+ and CaM in mitochondria complex I inhibitor-induced Ser129-phosphorylation of α-syn. As vehicle control, cells were treated with DMSO at the same final concentration as reagents used. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a, b Effect of intracellular Ca 2+ chelator BAPTA-AM ( a ) or extracelluar Ca 2+ chelator EGTA ( b ) on MPP + -induced Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 1 mM MPP + and the indicated concentrations of BAPTA-AM or EGTA for 16 h. c, d Effect of BAPTA-AM ( c ) and EGTA ( d ) on rotenone-induced Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 5 μM rotenone and the indicated concentrations of BAPTA-AM or EGTA for 16 h. e, f Effect of CaM inhibitor W-7 on MPP + ( e )- or rotenone ( f )-induced Ser129-phosphorylation. Cells were incubated in media containing 1 mM MPP + or 5 μM rotenone with W-7 at the indicated concentrations for 16 h. Representative blots are shown. In the graphs of a to f , relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P < 0.05; **, P < 0.01)

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Phospho-proteomics, Control, Concentration Assay, SDS Page, Western Blot, Incubation

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: Ser129-mediated proteasomal targeting of soluble α-syn in mitochondrial complex I inhibition by rotenone. In each treatment, the concentration of DMSO was prepared to be equal. Cell lysates (2.5 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a CHX-chase experiments for analyzing alteration in the metabolic fates of Ser129-phosphorylated and total α-syn by MG132 treatment. Wt-aS/SH #4 cells were pre-incubated in media containing either DMSO or 10 μM rotenone for 8 h. Then, they were incubated in fresh media further containing 100 μM cycloheximide (CHX) and/or 10 μM MG132 until 120 min. b Comparison of metabolic fates of rotenone-induced Ser129-phosphorylated α-syn with physiologically phosphorylated α-syn. Cells were pre-incubated in media containing DMSO or 10 μM rotenone for 8 h before CHX-chase experiments. Representative blots are shown. In a and b , graphs show the metabolic fates of Ser129-phosphorylated α-syn ( left ) and total α-syn ( right ). Black, yellow, blue and red lines represent treatment with DMSO, rotenone, MG132 and rotenone plus MG132, respectively. Data show means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P < 0.05; **, P < 0.01)

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Inhibition, Concentration Assay, SDS Page, Western Blot, Incubation, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: Effect of Ser129-phosphorylation on α-syn solubility change by mitochondrial complex I inhibition. Wt-aS/SH #4 cells were fractionated into 1% Triton X-100 soluble and insoluble fractions by centrifugation at 100,000×g for 30 min. 1% Triton X-100 insoluble pellets were resolved by 8 M urea / 2% SDS solution. In 1% Triton X-100 soluble fractions, the extract (2.5 μg / lane) were loaded onto SDS-PAGE. In 1% Triton X-100 insoluble fractions, samples corresponding to 15 μg of soluble fractions were loaded. These samples were analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a Solubility change of α-syn by rotenone treatment. Cells were incubated by 10 or 50 nM rotenone for 5 days. The representative blots are shown. b CHX-chase experiments for analyzing alteration in the metabolic fates of insoluble Ser129-phosphorylated and total α-syn by MG132 treatment. After treatment with 50 nM rotenone for 5 days, cells were incubated in fresh media further containing 100 μM cycloheximide (CHX) and either 0.1% DMSO or 10 μM MG132 until 120 min. Graphs show the metabolic fates of Ser129-phosphorylated α-syn ( left ) and total α-syn ( right ). Data show means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P < 0.05; **, P < 0.01)

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Phospho-proteomics, Solubility, Inhibition, Centrifugation, SDS Page, Western Blot, Incubation

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: Relation of Ser129-phosphorylation-mediated α-syn clearance between the proteasome and lysosome pathways. Wt-aS/SH #4 cells were fractionated into 1% Triton X-100 soluble and insoluble fractions by centrifugation at 100,000×g for 30 min. In 1% Triton X-100 soluble fractions, the extract (2.5 μg / lane) were loaded onto SDS-PAGE. In 1% Triton X-100 insoluble fractions, samples corresponding to 15 μg of soluble fractions were loaded. Samples were analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a Effect of proteasomal and lysosomal inhibitions on the metabolism of α-syn. Cells were incubated by either 100 nM epoxomicin, 100 μM chloroquine, or 100 nM epoxomicin plus 100 μM chloroquine for 24 h. Vehicle control cells were treated with the same concentration of DMSO. Upper and lower panels show the blots of 1% Triton X-100 insoluble fractions and 1% Triton X-100 soluble fractions, respectively. b Effect of Ser129-phosphorylation on the metabolism of α-syn in proteasomal and lysosomal inhibitions. Wt-aS/SH #4 cells and S129A-aS/SH #10 cells were incubated by either 100 nM epoxomicin, 100 μM chloroquine, or epoxomicin plus chloroquine for 24 h. Upper panels show the blots of 1% Triton X-100 insoluble fractions. Graph shows relative ratios of total α-syn to vehicle control cells. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P < 0.05; **, P < 0.01)

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Phospho-proteomics, Centrifugation, SDS Page, Western Blot, Incubation, Control, Concentration Assay

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: Effect of Ser129-phosphorylation on α-syn aggregate formation in a rat AAV-mediated α-syn overexpression model. Rats were sterotaxically injected with rAAV particles into the substantia nigra, and they expressed A53T mutant α-syn or A53T plus S129A double mutant α-syn. These rats were used in our previous study . Information on the expression levels and toxicity is described in this paper . Upper panels show the photomicrographs of rat striatum immunohistochemically stained with antibody specific to Ser129-phosphorylated α-syn and human total α-syn (LB509). We counted the number of α-syn-positive aggregates larger than 5 μm in diameter. Arrow heads indicate α-syn aggregates. Bar shows 100 μm. Graph shows quantitative analysis of striatal aggregates containing Ser129-phosphorylated α-syn or total α-syn. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P < 0.05; **, P < 0.01)

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Phospho-proteomics, Over Expression, Injection, Mutagenesis, Expressing, Staining

Journal: Acta Neuropathologica Communications

Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

doi: 10.1186/s40478-017-0452-6

Figure Lengend Snippet: A model of Ser129-phosphorylation role in regulating α-syn levels and forming α-syn aggregates. Mitochondrial impairment stimulates solubility change of α-syn proteins from normally soluble forms to insoluble forms. Also, mitochondrial impairment facilitates Ser129-phosphorylation of α-syn by an increase in influx of extracellular Ca 2+ . Ser129-phosphorylated α-syn, including soluble and insoluble forms, is targeted to the proteasome pathway. Proteasomal targeting of Ser129-phosphorylated α-syn is more promoted under lysosome inhibition. It acts as a suppressor complementary to the lysosome pathway against accumulation of insoluble α-syn proteins. Also, α-syn aggregates undergo Ser129-phosphorylation. However, Ser129-phosphorylation-mediated proteasomal targeting is ineffective, once α-syn aggregates turn to be degradation-resistant. Consequently, α-syn proteins deposited in aggregates are extensively phosphorylated

Article Snippet: We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai).

Techniques: Phospho-proteomics, Solubility, Inhibition

Journal: Neurochemical Research

Article Title: Identification of Novel α-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

doi: 10.1007/s11064-011-0527-x

Figure Lengend Snippet: MALDI TOF mass spectra of the brain homogenate fractions (Tris fractions ( a , b ), Triton ® fractions ( c , d ), SDS fractions ( e , f )) immunoprecipitated using the monoclonal anti-α-syn antibody Clone 42 (BD Transduction Laboratories) ( b , d , f ) or IgG from murine serum ( negative controls ) ( a , c , e )

Article Snippet: Fig. 1 MALDI TOF mass spectra of the brain homogenate fractions (Tris fractions ( a , b ), Triton ® fractions ( c , d ), SDS fractions ( e , f )) immunoprecipitated using the monoclonal anti-α-syn antibody Clone 42 (BD Transduction Laboratories) ( b , d , f ) or IgG from murine serum ( negative controls ) ( a , c , e ) To obtain accurate mass values of the tentative α-syn species and if possible verify their identity with amino acid sequence information, we developed an online top-down proteomics approach where LC–ESI–MS/MS analysis of the immunoprecipitated brain homogenate fractions was utilized.

Techniques: Immunoprecipitation, Transduction

Journal: Neurochemical Research

Article Title: Identification of Novel α-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

doi: 10.1007/s11064-011-0527-x

Figure Lengend Snippet: Nanoflow LC-ESI-FTICR full scan mass spectra showing the α-syn forms Ac-α-syn 1–139 ( a , b ) and Ac-α-syn 1–140 ( a – c ). Note also the presence of mono- and dioxidized forms. The spectra are summed over a 1.5 min retention time interval around 46 min (50 min gradient). In the spectra the 15 + charge state of the Ac-α-syn 1–139 and Ac-α-syn 1–140 forms are indicated. One SIM scan (3 microscans) of the 15 + ion of the Ac-α-syn 1–103 form ( d , e ). Spectra from the LC–ESI–MS analysis of the Tris fraction of the brain homogenate ( a and d ). Spectra from the LC–ESI–MS analysis of the Triton ® fraction of the brain homogenate ( b and e ). Spectra from the LC–ESI–MS analysis of the SDS fraction of the brain homogenate ( c ). The detected y and b fragments are indicated in the respective sequences ( f ). Cleavage sites labeled with bold lines are confirmed by database search

Article Snippet: Fig. 1 MALDI TOF mass spectra of the brain homogenate fractions (Tris fractions ( a , b ), Triton ® fractions ( c , d ), SDS fractions ( e , f )) immunoprecipitated using the monoclonal anti-α-syn antibody Clone 42 (BD Transduction Laboratories) ( b , d , f ) or IgG from murine serum ( negative controls ) ( a , c , e ) To obtain accurate mass values of the tentative α-syn species and if possible verify their identity with amino acid sequence information, we developed an online top-down proteomics approach where LC–ESI–MS/MS analysis of the immunoprecipitated brain homogenate fractions was utilized.

Techniques: Labeling

Journal: Neurochemical Research

Article Title: Identification of Novel α-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

doi: 10.1007/s11064-011-0527-x

Figure Lengend Snippet: Western blotting of biochemically fractionated temporal cortex Brodmann area 36 (BA36) ( a – d ) and dorsolateral prefrontal cortex BA9 ( e , f ) from controls, patients with dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD) using the monoclonal anti-α-syn antibody Clone 42 ( a , c , e ) and without primary antibody ( b , d , f ). Molecular markers ( lane 1 ), Tris fraction ( lane 2 and 6 ), 0.5% Triton ® fraction ( lane 3 and 7 ), 2% Triton ® fraction ( lane 4 and 8 ) and 0.5% SDS fraction ( lane 5 and 9 ) from a control and patient with DLB, respectively ( a , b ). Western blotting of the final pellet ( c – f ) using the monoclonal anti-α-syn antibody Clone 42 ( c , e ) and without primary antibody ( d , f ). Molecular markers ( lane 1 ), from controls ( lane 2, 4, 6, 8 and 10 ), patients with DLB ( lane 3, 9, 11 ) and PDD ( lane 5, 7 ) ( c ). Molecular markers ( lane 1 ), from controls ( lane 2 and 4 ), patients with DLB ( lane 3 ) and PDD ( lane 5 – 9) ( e ). Molecular markers ( lane 1 ), from a control (lane 2) and from a patient with DLB (lane 3) (d, f). The table below each Western blot ( c , e ) shows the abundance of the α-syn bands of 32, 16, 12, and 8 kDa given. The intensities of the band (+(weak), ++(moderate), +++(strong)) presented in the table panel are based on optical density for each band of α-syn subtracting the background. Empty boxes meaning no visible bands

Article Snippet: Fig. 1 MALDI TOF mass spectra of the brain homogenate fractions (Tris fractions ( a , b ), Triton ® fractions ( c , d ), SDS fractions ( e , f )) immunoprecipitated using the monoclonal anti-α-syn antibody Clone 42 (BD Transduction Laboratories) ( b , d , f ) or IgG from murine serum ( negative controls ) ( a , c , e ) To obtain accurate mass values of the tentative α-syn species and if possible verify their identity with amino acid sequence information, we developed an online top-down proteomics approach where LC–ESI–MS/MS analysis of the immunoprecipitated brain homogenate fractions was utilized.

Techniques: Western Blot

Journal: Neurochemical Research

Article Title: Identification of Novel α-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

doi: 10.1007/s11064-011-0527-x

Figure Lengend Snippet: Individual values for the ratio (α-syn/total protein) in biochemically fractionated (Tris ( a ), 0.5% Triton ® ( b ), and SDS fraction ( c )) temporal cortex Brodmann area 36 (BA36) and dorsolateral prefrontal cortex BA9 from controls ( grey circle ), patients with dementia with Lewy bodies (DLB) ( black circle ) and Parkinson’s disease with dementia (PDD) ( white circle ) using ELISA

Article Snippet: Fig. 1 MALDI TOF mass spectra of the brain homogenate fractions (Tris fractions ( a , b ), Triton ® fractions ( c , d ), SDS fractions ( e , f )) immunoprecipitated using the monoclonal anti-α-syn antibody Clone 42 (BD Transduction Laboratories) ( b , d , f ) or IgG from murine serum ( negative controls ) ( a , c , e ) To obtain accurate mass values of the tentative α-syn species and if possible verify their identity with amino acid sequence information, we developed an online top-down proteomics approach where LC–ESI–MS/MS analysis of the immunoprecipitated brain homogenate fractions was utilized.

Techniques: Enzyme-linked Immunosorbent Assay