Journal: Current Issues in Molecular Biology

Article Title: Association of OXTR , AVPR1a , LNPEP , and CD38 Genes’ Expression with the Clinical Presentation of Autism Spectrum Disorder

doi: 10.3390/cimb45100527

Figure Lengend Snippet: Correlation matrix for all analyzed parameters; Spearman’s rho value on the left side; p -value on the right side. CS = cumulative scale; SA = social affect; RRB = restrictive and repetitive behaviors; DA = domain A; DB = domain B.

Article Snippet: An expression analysis of the CD38 (Hs01120071_m1), OXTR (Hs00168573_m1), AVPR1A (Hs00176122_m1), and LNPEP (sonda Hs00893646_m1) genes was performed using TaqMan Gene Expression Assays, along with the reference gene GAPDH (Hs03929097_g1), and TaqMan Gene Expression Master Mix (Applied Biosystems, USA).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Association of OXTR , AVPR1a , LNPEP , and CD38 Genes’ Expression with the Clinical Presentation of Autism Spectrum Disorder

doi: 10.3390/cimb45100527

Figure Lengend Snippet: R-values of Pearson’s correlation between LNPEP/CD38 expression ratio, ADOS-2 subscales, and BINET IQ; DOM A—language and communication disorders; DOM B—disorder of social reciprocity; RRB—restrictive and repetitive behaviors. * designates statistically significant rho value.

Article Snippet: An expression analysis of the CD38 (Hs01120071_m1), OXTR (Hs00168573_m1), AVPR1A (Hs00176122_m1), and LNPEP (sonda Hs00893646_m1) genes was performed using TaqMan Gene Expression Assays, along with the reference gene GAPDH (Hs03929097_g1), and TaqMan Gene Expression Master Mix (Applied Biosystems, USA).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: The NLRP3 inflammasome is involved in resident intruder paradigm-induced aggressive behaviors in mice

doi: 10.3389/fphar.2023.974905

Figure Lengend Snippet: Behavioral phenotype of mice in each group. (A) Schematic representation of the experimental design. (B) Open field test representative trajectory graph. (C) Elevated plus-maze test representative trajectory graph. (D) Open field test results. (E) Elevated plus-maze test results. (F) Attack behavior test results. Data are expressed as means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Control, producers of non-aggressive behavior after stress; Model, aggressive behavior after stress; A804598, P2X7R antagonist; IL-1Ra, IL-1β blocker; NLRP3 −/− , NLRP3 knockout mice. n = 8/group. (TIFF format, 1200 dpi, 2-column fitting image).

Article Snippet: Chemicals are listed as follows: A804598 (HY-100483, MedChemExpress Co., Ltd. United States); IL-1Ra (HY-P7029A, MCE, United States); DMSO (D8370, Solarbio, China); Mouse NLRP3 ELISA kit (JYM0765Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); Mouse IL-1β ELISA kit (JYM0531Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); BCA Protein Assay Kit (PC0020, Solarbio, China); SDS-PAGE Gel Rapid Preparation Kit (G2037-50T, Servicebio, China); Recombinant Anti-beta Actin antibody (GB15003, Servicebio, China); NLRP3 Antibody (DF7438, Affinity Biologicals, Canada); Recombinant Anti-P2X7 antibody (ab259942, abcam, United Kingdom); HRP-conjugated goat anti-rabbit IgG (GB23204, Servicebio, China); Anti-Iba1 Rabbit pAb (GB11105, Servicebio, China); Cy3-labeled goat anti-rabbit IgG (GB21303, Servicebio, China); FITC-labeled goat anti-rabbit IgG (GB22303, Servicebio, China); Anti-BrdU Mouse mAb (GB12051, Servicebio, China); Anti-NeuN Rabbit pAb (GB11138, Servicebio, China); HE staining solution (G1005-100ML, Servicebio, China).

Techniques: Control, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: The NLRP3 inflammasome is involved in resident intruder paradigm-induced aggressive behaviors in mice

doi: 10.3389/fphar.2023.974905

Figure Lengend Snippet: The P2X7R was activated in the resident intruder paradigm. (A) Representative images of P2X7R (green) and DAPI (blue) labeling in the hippocampus CA1, CA3, and DG after drug interventions; scale bar = 200 and 50 µm. (B) Positive staining area of P2X7R and DAPI double-labeled (P2X7R + DAPI) proteins in the hippocampus CA1 (** p < 0.01 Control vs. Model ; n = 3/group), CA3 (** p < 0.01 Control vs. Model ; * p < 0.05 A804598 vs. Model ; n = 3/group), and DG (** p < 0.01 Control vs. Model ; * p < 0.05 A804598 vs. Model ; n = 3/group). (C) Representative immunoreactive bands showing the protein levels of hippocampal P2X7R in the Control, Model, A804598, IL-1Ra, and NLRP3 −/− mice. (D) Statistical results show that A804598, IL-1Ra, and NLRP3 −/− decreased the protein expression of P2X7R ( n = 3, **** p < 0.0001 vs. Model).

Article Snippet: Chemicals are listed as follows: A804598 (HY-100483, MedChemExpress Co., Ltd. United States); IL-1Ra (HY-P7029A, MCE, United States); DMSO (D8370, Solarbio, China); Mouse NLRP3 ELISA kit (JYM0765Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); Mouse IL-1β ELISA kit (JYM0531Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); BCA Protein Assay Kit (PC0020, Solarbio, China); SDS-PAGE Gel Rapid Preparation Kit (G2037-50T, Servicebio, China); Recombinant Anti-beta Actin antibody (GB15003, Servicebio, China); NLRP3 Antibody (DF7438, Affinity Biologicals, Canada); Recombinant Anti-P2X7 antibody (ab259942, abcam, United Kingdom); HRP-conjugated goat anti-rabbit IgG (GB23204, Servicebio, China); Anti-Iba1 Rabbit pAb (GB11105, Servicebio, China); Cy3-labeled goat anti-rabbit IgG (GB21303, Servicebio, China); FITC-labeled goat anti-rabbit IgG (GB22303, Servicebio, China); Anti-BrdU Mouse mAb (GB12051, Servicebio, China); Anti-NeuN Rabbit pAb (GB11138, Servicebio, China); HE staining solution (G1005-100ML, Servicebio, China).

Techniques: Labeling, Staining, Control, Expressing

Journal: Frontiers in Pharmacology

Article Title: The NLRP3 inflammasome is involved in resident intruder paradigm-induced aggressive behaviors in mice

doi: 10.3389/fphar.2023.974905

Figure Lengend Snippet: Correlations between the expression levels of NLRP3 in the hippocampus or serum and aggressive behaviors. (A) Representative immunoreactive bands showing the protein levels of hippocampal NLRP3 in the Control, Model, A804598, IL-1Ra, and NLRP3 −/− mice. (B) Statistical results show that A804598, IL-1Ra, and NLRP3 −/− decreased the protein expression of NLRP3 ( n = 3, **** p < 0.0001 vs. Model). (C) NLRP3 and IL-1βhippocampus and serum levels. (D) Pearson correlation analyses were performed by comparing the expression levels of NLRP3 in the hippocampus or serum and aggressive behavior latency or aggressive behavior score. Correlations were performed considering some animals ( N = 3). The coefficient r and p -value for each correlation are presented in a box. Statistically significant correlations are highlighted in red ( p < 0.05). Data are expressed as means ± SEMs. Control, producers of non-aggressive behavior after stress; Model, aggressive behavior after stress; A804598, P2X7R antagonist; IL-1Ra, IL-1β blocker; NLRP3 −/− , NLRP3 knockout mice. (TIFF format, 1200 dpi, 2-column fitting image).

Article Snippet: Chemicals are listed as follows: A804598 (HY-100483, MedChemExpress Co., Ltd. United States); IL-1Ra (HY-P7029A, MCE, United States); DMSO (D8370, Solarbio, China); Mouse NLRP3 ELISA kit (JYM0765Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); Mouse IL-1β ELISA kit (JYM0531Mo, Wuhan ColorfulGene biological technology Co., Ltd. China); BCA Protein Assay Kit (PC0020, Solarbio, China); SDS-PAGE Gel Rapid Preparation Kit (G2037-50T, Servicebio, China); Recombinant Anti-beta Actin antibody (GB15003, Servicebio, China); NLRP3 Antibody (DF7438, Affinity Biologicals, Canada); Recombinant Anti-P2X7 antibody (ab259942, abcam, United Kingdom); HRP-conjugated goat anti-rabbit IgG (GB23204, Servicebio, China); Anti-Iba1 Rabbit pAb (GB11105, Servicebio, China); Cy3-labeled goat anti-rabbit IgG (GB21303, Servicebio, China); FITC-labeled goat anti-rabbit IgG (GB22303, Servicebio, China); Anti-BrdU Mouse mAb (GB12051, Servicebio, China); Anti-NeuN Rabbit pAb (GB11138, Servicebio, China); HE staining solution (G1005-100ML, Servicebio, China).

Techniques: Expressing, Control, Knock-Out

Journal: Scientific Reports

Article Title: Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

doi: 10.1038/s41598-023-44038-1

Figure Lengend Snippet: The C-terminal domain of IRAP can be secreted into the circulation/extracellular milieu. IRAP contains two functional domains: a cytosolic N-terminal domain that contains trafficking motifs and an extracellular/intra-luminal C-terminal domain that contains the catalytic site. Following cleavage at a site near the transmembrane region, the C-terminal domain can be secreted. Current anti-IRAP antibodies that are commercially available only target the N-terminal domain.

Article Snippet: Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1% Tween-20 in 1 × TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk/TBS-T) for 1 h. The rabbit anti-IRAP antibody (1:2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 μg/ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4 °C.

Techniques: Functional Assay

Journal: Scientific Reports

Article Title: Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

doi: 10.1038/s41598-023-44038-1

Figure Lengend Snippet: The novel anti-IRAP antibodies can specifically detect human IRAP. ( a ) Representative Western blots (uncropped 12 lanes in same gel) showing binding of the novel mouse anti-IRAP antibodies (RB9, RF7, RH3, RG4; 0.5 µg/µl) to (1) full length IRAP derived from membrane preparations of HEK293T cells transiently overexpressing human IRAP (0.01 µg protein/µl) or (2) purified soluble human IRAP (0.001 µg/µl). The lanes for each antibody were imaged at either different exposure times or at the same exposure time across all antibodies (n = 3). Original, uncropped blots are provided in Supplementary Fig. . ( b ) The mouse anti-IRAP antibodies (1 µg/well) and the commercially available rabbit anti-IRAP antibody (Rb; 1:500 concentration) were tested for binding to mIRAP (10 µg protein/well) or purified sIRAP (0.001 µg/well) using an indirect ELISA. Data presented is the mean from two separate experiments performed in duplicate (n = 2). ( c ) The mouse RB9 clone (1 µg/well; red) and the rabbit antibody (1:500; purple) were tested against decreasing concentrations of purified sIRAP (0.001–0.01 µg/well) and mIRAP (10 µg protein/well). Simple linear regression was conducted (R 2 = 0.94). Data presented is the mean of absorbance readings from one experiment performed in duplicate (n = 1).

Article Snippet: Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1% Tween-20 in 1 × TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk/TBS-T) for 1 h. The rabbit anti-IRAP antibody (1:2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 μg/ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4 °C.

Techniques: Western Blot, Binding Assay, Derivative Assay, Membrane, Purification, Concentration Assay, Indirect ELISA

Journal: Scientific Reports

Article Title: Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

doi: 10.1038/s41598-023-44038-1

Figure Lengend Snippet: Specificity of the subcloned antibodies for IRAP. Western blots of the untagged and biotinylated newly subcloned monoclonal anti-IRAP antibodies RF7, RB9, RH3 and RG4 (0.0005 µg/µl) against either ( a ) purified soluble human IRAP (S; 0.001 µg/well) and full length human IRAP enriched membrane preparations of HEK293T cells transiently transfected with human IRAP cDNA (M; 0.1 µg proteins/well) or ( b ) protein lysate from cardiac tissue of 10-week old male IRAP KO or WT mice (20 µg protein/well), n = 1. All blots are imaged for the same exposure time. Original, uncropped blots are provided in Supplementary Fig. .

Article Snippet: Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1% Tween-20 in 1 × TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk/TBS-T) for 1 h. The rabbit anti-IRAP antibody (1:2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 μg/ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4 °C.

Techniques: Western Blot, Purification, Membrane, Transfection

Journal: Scientific Reports

Article Title: Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

doi: 10.1038/s41598-023-44038-1

Figure Lengend Snippet: Detection of increases in sIRAP expression in human plasma throughout the later stages of pregnancy. ( a ) Representative Western blot and quantification of the optical density (OD) of sIRAP (140–150 kDa) in plasma from healthy controls and women at later stages of pregnancy (28- & 36-weeks and full term; diluted 1:10), detected using the subcloned RF7 anti-IRAP antibody (0.0005 µg/µl). Control plasma is from three different participants and pregnant plasma is three different batches of pooled samples (n = 3). Data is expressed as mean ± SEM, was corrected for total protein concentration and analysed using a one-way ANOVA with Tukey’s post-hoc test, * p < 0.05, ** p < 0.01 compared to control. ( b ) Sandwich ELISAs using RF7-RB9-B (red) and RF7-RG4-B (green) antibody combinations was conducted to measure concentrations of IRAP in plasma from control and pregnant women (28- & 36-weeks & term) using a purified sIRAP standard curve (µg/ml). All samples were diluted 1:5. Data was analysed using simple linear regression analysis to interpolate the concentrations of each sample and a one-way ANOVA with Tukeys’s post-hoc test to compare groups to the control, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs control, n = 5–6. All data was corrected for the negative control and is presented as mean ± SEM.

Article Snippet: Following transfer, membranes were washed briefly in Tris-buffered saline-tween (TBS-T; 0.1% Tween-20 in 1 × TBS) and then placed on a shaker (70 rpm) in blocking buffer (5% skim milk/TBS-T) for 1 h. The rabbit anti-IRAP antibody (1:2000; Cell Signalling #6918) or the mouse anti-IRAP antibodies (0.0005 μg/ml) were diluted in the same blocking buffer to the appropriate concentration and incubated on membranes overnight on a shaker (70 rpm) at 4 °C.

Techniques: Expressing, Clinical Proteomics, Western Blot, Control, Protein Concentration, Purification, Negative Control

Journal: Translational oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response.

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: Fig. 2. Low expression of IL1RA in OSCC and validation of IL1RA overexpression efficiency. A. Expression levels of IL1RA in HNSCC and normal specimens available from the GEPIA database. B. H&E and IHC staining of OSCC and paracancerous specimens in vitro (n=50, scale bar=100 μm). The percentage of low and high IL1RA expressions in OSCC and paracancerous specimens is visualized. C-D. Efficiency of IL1RA overexpression by lentivirus transfection is validated in CAL27 and HN6 cells by RT-qPCR (C) and Western blot (D) in vitro. *P<0.05, and **P<0.01 vs. NC group.

Article Snippet: Transferring on the Polyvinylidene fluoride (PVDF) membrane, it was immersed in 5 % skim milk, and incubated overnight at 4◦C with primary antibodies against β-Actin (#3700, CST, 1:1000), IL1RA (NBP1-32568, Novusbio, 1:500), IFNA (18013-AP, Proteintech, 1:500) and IFNB (27506-AP, Proteintech, 1:2000).

Techniques: Expressing, Biomarker Discovery, Over Expression, Immunohistochemistry, In Vitro, Transfection, Quantitative RT-PCR, Western Blot

Journal: Translational oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response.

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: Fig. 3. IL1RA inhibits the proliferation and migration of OSCC cells in vitro. CAL27 and HN6 cells were transfected with NC and oeIL1RA. A. Cell viability at 0, 24, 48, and 72 h was detected by CCK-8 assay. B. Clone number detected by colony formation assay. C. Cell migration was detected by the cell scratch assay at 0 and 24 h (scale bar=100 μm). *P<0.05, and **P<0.01 vs. NC group.

Article Snippet: Transferring on the Polyvinylidene fluoride (PVDF) membrane, it was immersed in 5 % skim milk, and incubated overnight at 4◦C with primary antibodies against β-Actin (#3700, CST, 1:1000), IL1RA (NBP1-32568, Novusbio, 1:500), IFNA (18013-AP, Proteintech, 1:500) and IFNB (27506-AP, Proteintech, 1:2000).

Techniques: Migration, In Vitro, Transfection, CCK-8 Assay, Colony Assay, Wound Healing Assay

Journal: Translational oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response.

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: Fig. 4. IL1RA inhibits tumor growth in OSCC in vivo. Nude mice were subcutaneously implanted with 1 × 106 CAL27 cells transfected with NC and oeIL1RA. A. Gross view of OSCC xenografts at 21 days, and the mean tumor weight and volume. B. The mRNA level of IL1RA in OSCC xenografts of NC group and oeIL1RA group. C. H&E and IHC staining of OSCC xenografts in the NC group and oeIL1RA group (scale bar=100 μm). n=6. *P<0.05, and **P<0.01 vs. NC group.

Article Snippet: Transferring on the Polyvinylidene fluoride (PVDF) membrane, it was immersed in 5 % skim milk, and incubated overnight at 4◦C with primary antibodies against β-Actin (#3700, CST, 1:1000), IL1RA (NBP1-32568, Novusbio, 1:500), IFNA (18013-AP, Proteintech, 1:500) and IFNB (27506-AP, Proteintech, 1:2000).

Techniques: In Vivo, Transfection, Immunohistochemistry

Journal: Translational oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response.

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: Fig. 5. IL1RA is associated with oncogenic signaling pathways and type I interferon response in OSCC in vitro. A. Volcano plots visualizing 132 DEGs between CAL27 cells transfected with NC and oeIL1RA. Down-regulated, up-regulated and non-regulated genes were labeled in blue, red, and grey colors, respectively. B. GSEA showed less enriched cancer-related features or processes in CAL27 cells overexpressing IL1RA. C. GSEA showed that IL1RA overexpression was significantly associated with the type I interferon response. D. A Venn diagram visualizing 45 (61.6 %) OSCC patients simultaneously carrying mutations in the IL1RA, IFNA, and IFNB genes in the TCGA dataset.

Article Snippet: Transferring on the Polyvinylidene fluoride (PVDF) membrane, it was immersed in 5 % skim milk, and incubated overnight at 4◦C with primary antibodies against β-Actin (#3700, CST, 1:1000), IL1RA (NBP1-32568, Novusbio, 1:500), IFNA (18013-AP, Proteintech, 1:500) and IFNB (27506-AP, Proteintech, 1:2000).

Techniques: Protein-Protein interactions, In Vitro, Transfection, Labeling, Over Expression

Journal: Translational oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response.

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: Fig. 6. DEGs in OSCC cells overexpressing IL1RA are involved in multiple interferon response processes. A. GO enrichment analysis showed that over expression of IL1RA was associated with the type I interferon response in OSCC, with a positive correlation. B. The analysis in the Reactome Pathway Database showed that overexpression of IL1RA was associated with interferon signaling pathways, including the interferon α/βand interferon γ signaling pathways.

Article Snippet: Transferring on the Polyvinylidene fluoride (PVDF) membrane, it was immersed in 5 % skim milk, and incubated overnight at 4◦C with primary antibodies against β-Actin (#3700, CST, 1:1000), IL1RA (NBP1-32568, Novusbio, 1:500), IFNA (18013-AP, Proteintech, 1:500) and IFNB (27506-AP, Proteintech, 1:2000).

Techniques: Over Expression, Protein-Protein interactions

Journal: Translational oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response.

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: Fig. 7. Overexpression of IL1RA up-regulates type I interferon proteins in OSCC in vitro. A. H&E (the first lane) and IHC staining (the latter three lanes) of IFNA and IFNB in OSCC specimens of the high and low IL1RA expression groups (n=30, scale bar=100 μm). B-C. The mRNA (B) and protein expressions (C) of IFNA and IFNB in OSCC specimens of the high and low IL1RA expression groups. *P<0.05, and **P<0.01 vs. NC group.

Article Snippet: Transferring on the Polyvinylidene fluoride (PVDF) membrane, it was immersed in 5 % skim milk, and incubated overnight at 4◦C with primary antibodies against β-Actin (#3700, CST, 1:1000), IL1RA (NBP1-32568, Novusbio, 1:500), IFNA (18013-AP, Proteintech, 1:500) and IFNB (27506-AP, Proteintech, 1:2000).

Techniques: Over Expression, In Vitro, Immunohistochemistry, Expressing

Journal: Translational oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response.

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: Fig. 8. IL1RA promotes the expressions of IFNA and IFNB in the OSCC xenografts and their release in OSCC cells. A. IHC staining of positive expressions of IFNA and IFNB in OSCC xenografts of oeIL1RA group and NC group in vivo (scale bar=100 μm). B. The mRNA levels of IFNA and IFNB in OSCC xenografts of the oeIL1RA group and NC group in vivo. C. ELISA showed contents of IFNA and IFNB in the cell supernatant of the oeIL1RA group and NC group in vitro. *P<0.05, and **P<0.01 vs. NC group.

Article Snippet: Transferring on the Polyvinylidene fluoride (PVDF) membrane, it was immersed in 5 % skim milk, and incubated overnight at 4◦C with primary antibodies against β-Actin (#3700, CST, 1:1000), IL1RA (NBP1-32568, Novusbio, 1:500), IFNA (18013-AP, Proteintech, 1:500) and IFNB (27506-AP, Proteintech, 1:2000).

Techniques: Immunohistochemistry, In Vivo, Enzyme-linked Immunosorbent Assay, In Vitro