community.elabeln.com/experiments.php?mode=edit&id=314

#314 Protein Analysis Complete

IL-6 sandwich ELISA, serum panel #8

Started on

Tags

ELISA IL-6 cytokine serum sandwich-assay

Custom ID

This template captures a sandwich ELISA for human IL-6 quantification in serum samples, with an 8-point standard curve and 16 unknown samples run in duplicate.

Goal: quantify serum IL-6 concentration in 16 patient or treatment-group samples, with the standard curve spanning 3.13 to 200 pg/mL and a high control to confirm assay performance.

Method overview: 96-well plate coated overnight at 4°C with capture antibody (anti-human IL-6, 2 µg/mL in PBS), washed and blocked with 1% BSA, samples loaded at 1:2 dilution in diluent buffer alongside standard curve dilutions, incubated 2 hours at room temperature, washed, detection antibody added (biotinylated anti-human IL-6, 0.5 µg/mL), incubated 1 hour, washed, Streptavidin-HRP added, washed, TMB developed for 15 minutes, stopped with 2N H₂SO₄, and read at OD₄₅₀ on a SpectraMax M5 plate reader.

Expected: Sigmoidal standard curve with R² > 0.99 via 4PL fit. Sample concentrations falling within the validated linear portion of the curve (typically 6.25-100 pg/mL). High control reads within ±15% of expected concentration.

Last saved: 34 seconds ago

11 fields loaded from IL-6 ELISA baseline (SOP v1.8)

Sample

Analyte

text

human IL-6

Matrix

select

serum, plasma, supernatant, urine

Sample dilution

text

1:2 in diluent

Number of samples

number

16×duplicate

Plate Layout

Capture antibody

text

mAb anti-hIL-6, 2 µg/mL

Detection antibody

text

bio-mAb anti-hIL-6, 0.5 µg/mL

Conjugate

select

Streptavidin-HRP, Strep-AP

Substrate

select

TMB, ABTS, pNPP

Standard Curve

Std curve high

number

200pg/mL

Std curve low

number

3.13pg/mL

Fit model

select

4PL, 5PL, linear, log-log

plate_layout.pdf

128 KB

spectramax_OD450_raw.csv

18 KB

4PL_fit_results.xlsx

76 KB

01

Coat plate: 100 µL/well capture Ab (2 µg/mL in PBS), seal, 4°C overnight

From last evening

16:00

02

Wash 3× with 300 µL wash buffer (PBST), tap dry

0:05

03

Block with 300 µL 1% BSA in PBS, 1 h RT

1:00

04

Prepare standard curve dilutions: 200 → 100 → 50 → 25 → 12.5 → 6.25 → 3.13 pg/mL

0:15

05

Wash 3×, add 100 µL samples + standards in duplicate, 2 h RT shaking

300 rpm orbital

2:00

06

Wash 4×, add 100 µL biotin-detection Ab (0.5 µg/mL), 1 h RT

1:00

07

Wash 4×, add 100 µL Streptavidin-HRP (1:200), 30 min RT in dark

0:30

08

Wash 5×, add 100 µL TMB substrate, develop 15 min RT dark

Blue color development

0:15

09

Add 100 µL 2N H₂SO₄ to stop (color → yellow), read OD₄₅₀ within 30 min

SpectraMax M5

0:10

10

4PL curve fit, interpolate unknowns, flag samples outside linear range

R² = 0.998

0:20

	A	B	C	D	E	F
1	Well	Type	Conc (pg/mL)	OD₄₅₀	Replicate	Notes
2	A1	STD-1 (high)	200	2.487	1	Top of curve
3	A2	STD-1 (high)	200	2.512	2
4	D1	STD-4	25	0.624	1
5	H1	STD-8 (low)	3.13	0.094	1	LLOQ
6	A3	Blank	0	0.046	—	Background
7	A4	Sample-01	38.2 (interp)	0.847	1	Within linear range
8	B4	Sample-01	38.7 (interp)	0.858	2	CV 1.3% ✓
9	A12	High ctrl	80 (expected)	1.612	—	Read 76.4 · within ±15%

EXP-2026-0331 Serum sample collection + aliquoting J. Roy Mar 31

EXP-2026-0402 IL-6 ELISA validation run (pre-study) J. Roy Apr 2

SOP-0418 IL-6 ELISA Standard Protocol v1.8 QA Team Apr 1

ABK-0211 hIL-6 antibody pair datasheet + validation Antibody DB Mar 28

METH-0061 4PL curve fitting method (SoftMax Pro) Methods DB Feb 10

Capture mAb anti-human IL-6

MAB206 · Lot AVB3194

In stock

Biotin detection mAb anti-hIL-6

BAF206 · Lot AYL3621

In stock

Recombinant human IL-6 standard

206-IL · Lot DK2147

In stock

Streptavidin-HRP conjugate

DY998 · Lot AZB1834

In stock

TMB substrate solution

34028 · Lot UE371821

Low

2 N H₂SO₄ stop solution

Lab-prep · Batch 03-29

In stock

Capture + detection Ab aliquots

-20 °C · Antibody Freezer · Rack B · Box ELISA-04

×12 each

Recombinant IL-6 standard (50 µL)

-80 °C · Freezer C-2 · Drawer 1 · Box STD-IL6

×8

Serum samples (panel #8)

-80 °C · Freezer C-2 · Drawer 2 · Box SR-2026-04

×16

Visibility

Roy LabJ. Roy

Can write

J. RoyDr. K. Liu

Color

Stroke width

Eraser

Use Ctrl + click to add text

{
    "experiment": {
        "id": 314,
        "custom_id": "ELISA-IL6-2026-04",
        "title": "IL-6 sandwich ELISA, serum panel #8",
        "template": "ELISA",
        "template_version": "v3.2",
        "category": "Protein Analysis",
        "status": "Complete",
        "started_on": "2026-05-29",
        "tags": [
            "ELISA",
            "IL-6",
            "cytokine",
            "serum",
            "sandwich-assay"
        ]
    },
    "main_text": "This template captures a sandwich ELISA for human IL-6 quantification in serum samples, with an 8-point standard curve and 16 unknown samples run in duplicate.\nGoal: quantify serum IL-6 concentration in 16 patient or treatment-group samples, with the standard curve spanning 3.13 to 200 pg/mL and a high control to confirm assay performance.\nMethod overview: 96-well plate coated overnight at 4°C with capture antibody (anti-human IL-6, 2 µg/mL in PBS), washed and blocked with 1% BSA, samples loaded at 1:2 dilution in diluent buffer alongside standard curve dilutions, incubated 2 hours at room temperature, washed, detection antibody added (biotinylated anti-human IL-6, 0.5 µg/mL), incubated 1 hour, washed, Streptavidin-HRP added, washed, TMB developed for 15 minutes, stopped with 2N H₂SO₄, and read at OD₄₅₀ on a SpectraMax M5 plate reader.\nExpected: Sigmoidal standard curve with R² > 0.99 via 4PL fit. Sample concentrations falling within the validated linear portion of the curve (typically 6.25-100 pg/mL). High control reads within ±15% of expected concentration.",
    "extra_fields": {
        "Sample": {
            "Analyte": {
                "type": "text",
                "value": "human IL-6"
            },
            "Matrix": {
                "type": "select",
                "value": "serum, plasma, supernatant, urine"
            },
            "Sample dilution": {
                "type": "text",
                "value": "1:2 in diluent"
            },
            "Number of samples": {
                "type": "number",
                "value": "16 ×duplicate"
            }
        },
        "Plate Layout": {
            "Capture antibody": {
                "type": "text",
                "value": "mAb anti-hIL-6, 2 µg/mL"
            },
            "Detection antibody": {
                "type": "text",
                "value": "bio-mAb anti-hIL-6, 0.5 µg/mL"
            },
            "Conjugate": {
                "type": "select",
                "value": "Streptavidin-HRP, Strep-AP"
            },
            "Substrate": {
                "type": "select",
                "value": "TMB, ABTS, pNPP"
            }
        },
        "Standard Curve": {
            "Std curve high": {
                "type": "number",
                "value": "200 pg/mL"
            },
            "Std curve low": {
                "type": "number",
                "value": "3.13 pg/mL"
            },
            "Fit model": {
                "type": "select",
                "value": "4PL, 5PL, linear, log-log"
            }
        }
    },
    "attached_files": [
        {
            "filename": "plate_layout.pdf",
            "type": "pdf",
            "size": "128 KB"
        },
        {
            "filename": "spectramax_OD450_raw.csv",
            "type": "csv",
            "size": "18 KB"
        },
        {
            "filename": "4PL_fit_results.xlsx",
            "type": "xlsx",
            "size": "76 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Coat plate: 100 µL/well capture Ab (2 µg/mL in PBS), seal, 4°C overnight",
            "duration": "16:00",
            "complete": true,
            "note": "From last evening"
        },
        {
            "n": 2,
            "description": "Wash 3× with 300 µL wash buffer (PBST), tap dry",
            "duration": "0:05",
            "complete": true
        },
        {
            "n": 3,
            "description": "Block with 300 µL 1% BSA in PBS, 1 h RT",
            "duration": "1:00",
            "complete": true
        },
        {
            "n": 4,
            "description": "Prepare standard curve dilutions: 200 → 100 → 50 → 25 → 12.5 → 6.25 → 3.13 pg/mL",
            "duration": "0:15",
            "complete": true
        },
        {
            "n": 5,
            "description": "Wash 3×, add 100 µL samples + standards in duplicate, 2 h RT shaking",
            "duration": "2:00",
            "complete": true,
            "note": "300 rpm orbital"
        },
        {
            "n": 6,
            "description": "Wash 4×, add 100 µL biotin-detection Ab (0.5 µg/mL), 1 h RT",
            "duration": "1:00",
            "complete": true
        },
        {
            "n": 7,
            "description": "Wash 4×, add 100 µL Streptavidin-HRP (1:200), 30 min RT in dark",
            "duration": "0:30",
            "complete": true
        },
        {
            "n": 8,
            "description": "Wash 5×, add 100 µL TMB substrate, develop 15 min RT dark",
            "duration": "0:15",
            "complete": true,
            "note": "Blue color development"
        },
        {
            "n": 9,
            "description": "Add 100 µL 2N H₂SO₄ to stop (color → yellow), read OD₄₅₀ within 30 min",
            "duration": "0:10",
            "complete": true,
            "note": "SpectraMax M5"
        },
        {
            "n": 10,
            "description": "4PL curve fit, interpolate unknowns, flag samples outside linear range",
            "duration": "0:20",
            "complete": true,
            "note": "R² = 0.998"
        }
    ],
    "spreadsheet": [
        [
            "Well",
            "Type",
            "Conc (pg/mL)",
            "OD₄₅₀",
            "Replicate",
            "Notes"
        ],
        [
            "A1",
            "STD-1 (high)",
            "200",
            "2.487",
            "1",
            "Top of curve"
        ],
        [
            "A2",
            "STD-1 (high)",
            "200",
            "2.512",
            "2",
            ""
        ],
        [
            "D1",
            "STD-4",
            "25",
            "0.624",
            "1",
            ""
        ],
        [
            "H1",
            "STD-8 (low)",
            "3.13",
            "0.094",
            "1",
            "LLOQ"
        ],
        [
            "A3",
            "Blank",
            "0",
            "0.046",
            "—",
            "Background"
        ],
        [
            "A4",
            "Sample-01",
            "38.2 (interp)",
            "0.847",
            "1",
            "Within linear range"
        ],
        [
            "B4",
            "Sample-01",
            "38.7 (interp)",
            "0.858",
            "2",
            "CV 1.3% ✓"
        ],
        [
            "A12",
            "High ctrl",
            "80 (expected)",
            "1.612",
            "—",
            "Read 76.4 · within ±15%"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0331",
                "title": "Serum sample collection + aliquoting",
                "owner": "J. Roy",
                "date": "Mar 31"
            },
            {
                "ref_id": "EXP-2026-0402",
                "title": "IL-6 ELISA validation run (pre-study)",
                "owner": "J. Roy",
                "date": "Apr 2"
            }
        ],
        "resources": [
            {
                "ref_id": "SOP-0418",
                "title": "IL-6 ELISA Standard Protocol v1.8",
                "owner": "QA Team",
                "date": "Apr 1"
            },
            {
                "ref_id": "ABK-0211",
                "title": "hIL-6 antibody pair datasheet + validation",
                "owner": "Antibody DB",
                "date": "Mar 28"
            },
            {
                "ref_id": "METH-0061",
                "title": "4PL curve fitting method (SoftMax Pro)",
                "owner": "Methods DB",
                "date": "Feb 10"
            }
        ]
    },
    "compounds": [
        {
            "name": "Capture mAb anti-human IL-6",
            "catalog_lot": "MAB206 · Lot AVB3194",
            "stock_status": "In stock"
        },
        {
            "name": "Biotin detection mAb anti-hIL-6",
            "catalog_lot": "BAF206 · Lot AYL3621",
            "stock_status": "In stock"
        },
        {
            "name": "Recombinant human IL-6 standard",
            "catalog_lot": "206-IL · Lot DK2147",
            "stock_status": "In stock"
        },
        {
            "name": "Streptavidin-HRP conjugate",
            "catalog_lot": "DY998 · Lot AZB1834",
            "stock_status": "In stock"
        },
        {
            "name": "TMB substrate solution",
            "catalog_lot": "34028 · Lot UE371821",
            "stock_status": "Low"
        },
        {
            "name": "2 N H₂SO₄ stop solution",
            "catalog_lot": "Lab-prep · Batch 03-29",
            "stock_status": "In stock"
        }
    ],
    "storage": [
        {
            "name": "Capture + detection Ab aliquots",
            "location": "-20 °C · Antibody Freezer · Rack B · Box ELISA-04",
            "count": "×12 each"
        },
        {
            "name": "Recombinant IL-6 standard (50 µL)",
            "location": "-80 °C · Freezer C-2 · Drawer 1 · Box STD-IL6",
            "count": "×8"
        },
        {
            "name": "Serum samples (panel #8)",
            "location": "-80 °C · Freezer C-2 · Drawer 2 · Box SR-2026-04",
            "count": "×16"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "Roy Lab"
            },
            {
                "type": "Person",
                "name": "J. Roy"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "J. Roy"
            },
            {
                "type": "Person",
                "name": "Dr. K. Liu"
            }
        ]
    }
}