A005-EN.01

resDetect™ Human Interleukin-6 (IL-6) ELISA Kit(Residue Testing)(Enzyme-Linked Immunosorbent Assay)

Catalog Number：CRS-A005
Pack Size: 96 tests

IMPORTANT: Please carefully read this manual before performing your experiment.
For Research Use Only. Not For Use in Diagnostic or Therapeutic Procedures

A005-EN.01

INTENDED USE
The Human Interleukin-6 (IL-6) ELISA Kit (Residue Testing) was developed for the detection andquantitative determination of GMP human IL-6 in samples from CAR-T product preparation processing.
It is intended for research use only (RUO).

BACKGROUNDInterleukin-6 (IL-6) is a member of the pro-inflammatory cytokine family. It induces the expression of avariety of proteins responsible for acute inflammation and plays an important role in the proliferation anddifferentiation of cells in humans. It has been demonstrated the dysregulation of the IL-6 signaling
pathways can result in inflammatory and autoimmune disorders as well as cancer development.
To support the development of CAR-T drugs, ACROBiosystems independently developed humanInterleukin-6 (IL-6) ELISA Residue Testing kit via rigorous methodological validation, which is used for
detection of GMP human IL-6 in samples from CAR-T product preparation processing for evaluation thequality of CAR-T products in drug development and CMC quality control stages.

PRINCIPLE OF THE ASSAY
This assay kit is used to measure the levels of human Interleukin-6 (IL-6) by employing a standard
sandwich-ELISA format. The micro-plate in the kit has been pre-coated with Anti-IL-6 Antibody. Firstly,
add the standard samples provided in kit and your samples to the plate, incubate and wash the wells. Then
add the Biotin-Anti-IL-6 Antibody to the plate and form Antibody-antigen-biotinylated antibody complex,
incubate and wash the wells. Next add Streptavidin-HRP to the plate, incubate and wash the wells. At last,load the substrate into the wells and monitor solution color from blue to yellow. The reaction is stopped
by the addition of a stop solution and the intensity of the absorbance can be measured at 450nm and 630nm.
The OD Value reflects the amount of IL-6 bound.

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PRECAUTIONS
1.

This kit is for research use only and is not for use in diagnostic or therapeutic applications.

2.

The kit is suitable for cell supernatant, serum and plasma samples.

3.

Do not use reagents past their expiration date.

4.

Do not mix or substitute reagents with those from other kits or other lot number kits.

5.

If samples generate values higher than the highest standard, dilute the samples with the appropriate
calibrator diluent and repeat the assay. If cell supernatant samples need step dilution, except for thefinal dilution with diluent, other intermediate dilutions can be in cell culture medium.

6.

Differences in test results can be caused by a variety of factors, including laboratory operator, pipetteusage, plate washing technique, reaction time or temperature, and kit storage.

7.

This kit is designed to remove or reduce some endogenous interference factors in biological samples,and not all possible influencing factors have been removed.

MATERIALS PROVIDED
Table1. Materials provided
Storage

Size
CatalogComponents

Format(96 tests)

Unopened

OpenedCRS005-C01

Pre-coated Anti-IL-6 Antibody Microplate

1 plateSolid

2-8℃

2-8℃

CRS005-C02

Human IL-6 Standard

20 μg

Powder

2-8℃-70℃

CRS005-C03

Biotin-Anti-IL-6 Antibody

50 μL

Liquid

2-8℃

2-8℃

CRS005-C04

Streptavidin-HRP

50 μL

Liquid2-8℃, avoid light

2-8℃, avoid light

CRS005-C05

10xWashing Buffer

50 mL

Liquid

2-8℃

2-8℃

CRS005-C06

2xDilution Buffer

50 mL

Liquid

2-8℃

2-8℃

CRS005-C07

Substrate Solution

12 mL

Liquid2-8℃, avoid light

2-8℃, avoid light

CRS005-C08

Stop Solution

7 mL

Liquid2-8℃2-8℃

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A005-EN.01

SRORAGE
1. Unopened kit should be stored at 2°C -8°C upon receiving.
2. The opened kit should be stored per Table 1. The shelf life is 30 days from the date of opening.
Note: a. Do not use reagents past their expiration date.
b. Find the expiration date on the outside packaging.

REAGENTS/EQUIPMENT NEEDED BUT NOT SUPPLIED
Single or multi-channel micropipettes and pipette tips: need to meet 10 μL, 300 μL, 1000 μL injection
requirements;
37ºC Incubator;
Single or dual wavelength microplate reader with 450nm and 630nm filter;
Tubes: 1.5mL,10mL;
Timer;
Reagent bottle;
Deionized or distilled water.

REAGENT PREPARATION
Bring all reagents and samples to room temperature (20℃-25℃) before use. If crystals have formed inbuffer solution, place the sample in an 37℃ incubator until the crystals have completely dissolved andbring the solution back to room temperature before use.
According to Table 2, prepare the provided lyophilized product into a storage solution with ultrapure water,
dissolve at room temperature for 15 to 30 minutes, and mix by gently pipetting, avoiding vigorous shaking
or vertexing. The reconstituted storage solution should be stored at -70°C. It is recommended that thenumber of freezing and thawing should not exceed 1 time, and the size of the aliquot should not be lessthan 5 μg.
Note: Considering innevitable minor quantatition variations between protein batches, it is also reasonable to generatethe standard curve with specific lot of proteins used for current production for even better accuracy.
Table 2. Preparation method
IDComponents

Size (96 T)

Storage solution concentration.

Reconstituted water Vol.

CRS005-C02

Human IL-6 Standard

20 μg

100 μg/mL

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RECOMMENDED SAMPLE PREPARATION
1.

Working Solution Preparation

1.1 Preparation of 1×Washing Buffer:
Dilute 50 mL 10×Washing Buffer with ultrapure water/deionized water to 500 mL.
1.2 Preparation of 1×Dilution Buffer:
Dilute 50 mL 2×Dilution Buffer with 1×Washing Buffer to 100 mL.
1.3 Preparation of Biotin-Anti-IL-6 Antibody working fluid:
Dilute Biotin-Anti-IL-6 at 1:1000 with 1×Dilution Buffer. Please prepare it for one-time use only.
1.4 Preparation of Streptavidin-HRP working fluid:
Dilute Streptavidin-HRP at 1:2000 with 1×Dilution Buffer. The prepared working fluid should avoid light.
Please prepare it for one-time use only.
1.5 Sample preparation
a. If the sample to be tested is the serum or plasma, dilute test sample at 1:10 with 1×Dilution Buffer.
The volume ratio of sample to diluent is 1:9.
b. If the sample to be tested is the cell supernatant, dilute test sample at 1:2 with 1×Dilution Buffer. The

volume ratio of sample to diluent is 1:1.
2.

Preparation of Standard curve

The concentration of the reconstituted human IL-6 Calibrator (CRS005-C02) is 100 μg/mL, prepare (Std.0) by diluting 6 μL the reconstituted human IL-6 Calibrator into 294 μL Sample Dilution Buffer, mix
gently well. Then prepare Std.- 1' by diluting 6 μL Std.-0 into 794 μL Sample Dilution Buffer. At last,prepare the highest concentration of standard curve, Std.-1 (150 pg/mL), by diluting 6 μL Std.- 1' into
594 μL Sample Dilution Buffer. Prepare 1:1 serial dilution for the standard curve as follows: Pipette 300µL of Sample Dilution Buffer into each tube. Make sure to mix well every time. Sample Dilution Buffer
serves as blank.

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3.

Add Samples

Add 100 µL Calibrator and samples to each well. For blank Control wells, please add 100 µL Dilution
Buffer.
Note: It is recommended to set doable holes for samples and standard curves to be tested.

4.

Incubation

Seal the plate with microplate sealing film and incubate at room temperature for 1 hour.
5.

Washing

Remove the remaining solution by aspiration, add 300 µL of 1×Washing Buffer to each well, then soakfor 10s, remove any remaining 1×Washing Buffer: by aspirating or decanting, invert the plate and blot it
against paper towels. Repeat the wash step above for three times.
6.

Add Biotin-Anti- IL-6 Antibody

For all wells, add 100 µL Biotin- Anti- IL-6 Antibody (dilute at 1:1000) working solution. Please prepare
it for one-time use only.
7.

Incubation

Seal the plate with microplate sealing film and incubate at room temperature for 1 hour.
8.

Washing

Repeat step 5.

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9.

Add Streptavidin-HRP

For all wells, add 100 µL Streptavidin-HRP (dilute at 1:2000) working solution. Please prepare it for onetime use only, avoid light.
10. Incubation
Seal the plate with microplate sealing film and incubate at room temperature for 30 min.
11. Washing
Repeat step 5.
12. Substrate Reaction
Add 100 µL Substrate Solution to each well. Seal the plate with microplate sealing film and incubate atroom temperature for 20 min, avoid light.
13. Termination
Add 50 µL Stop Solution to each well, tap the plate gently to allow thorough mixing.
Note: The color in the wells should change from blue to yellow.

14. Data Recording
Read the absorbance at 450 nm and 630 nm using UV/Vis microplate spectrophotometer within 10 minutes.
Note: To reduce the background noise, subtract the value read at OD450nm with the value read at OD630 nm.

CALCULATION OF RESULTS
1.

Calculate the mean absorbance for each standard, control and sample and subtract average zerostandard optical density (O.D.).
2.

The standard curve is plotted with the standard concentration as x-axis and the calibrated absorbancevalue as y-axis. Four parameters logistic are used to draw the standard curve and calculate the sample
concentration.
3.

Normal range of Standard curve: R2≥0.9900.

4.

Detection range: 2.34 pg/mL-150 pg/mL. If the OD value of the sample to be tested is higher than

150 pg/mL, the sample shall be diluted with dilution buffer and assay repeated. If the OD value of thesample to be tested is lower than 2.34 pg/mL, the sample should be reported.

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A005-EN.01

QUICK GUILD

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A005-EN.01

TYPICAL DATA
For each experiment, a standard curve needs to be set for each micro-plate, and the specific OD value mayvary depending on different laboratories, testers, or equipments. The following example data is forreference only. The sample concentration was calculated based on the results of the standard curve.

SENSITIVITY
The minimum detectable concentration of human IL-6 is 0.429 pg/mL. The minimum detectableconcentration was determined by adding twice standard deviations to the mean optical density value oftwenty zero standard replicates and calculating the corresponding concentration.

PRECISION
1. Intra-assay Precision
Three samples of known concentration were tested twenty times on one plate to assess intra-assay
precision.
2. Inter-assay Precision
Three samples of known concentration were tested in three separate assays to assess inter-assay precision.
Intra-assay PrecisionInter-assay Precision

Sample123123

n20

20

20

33

3

Mean(pg/mL)

116.82046.7359.995

116.25647.303

9.923

SD

4.554

1.930

0.218

3.014

1.374

0.270

CV (%)

3.9

4.1

2.2

2.6

2.9

2.7

Note: The example data is for reference only.
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A005-EN.01

RECOVERY
Three parts of blank plasma were added with different concentrations of human IL-6, and the plasma without
human IL-6 was used as background to calculate the recovery rate. The range of the recovery rate is 90.6114.8%, and the average recovery is 101.2%.
Sample Type

Average % Recovery

Range

Plasma(n=5)

101.2%

90.6-114.8%

LINEARITY
To assess the linearity of the assay, samples spiked with high concentrations of human IL-6 were seriallydiluted with calibrator diluent to produce samples with values within the dynamic range of the assay.

1:2
1:4
1:8
1:16

Cell culture
medium(DMEM)

Cell culture
medium(1640)

Citrate plasma

Average Recovery (%)

96.5

98.9

102.8

Range (%)

92.0-100.3

98.4-102.8

101.7-103.7

Average Recovery (%)

96.9

103.1

101.9

Range (%)

93.2-100.0

97.6-111.4

99.2-104.1

Average Recovery (%)

95.0

105.9

101.4

Range (%)

93.1-98.3

101.1-112.4

93.6-106.1

Average Recovery (%)

97.8

105.8

104.0

Range (%)

93.9-102.2

102.7-109.6

97.2-112.5

Note: The example data is for reference only.

SPECIFICITYThis assay recognizes natural and recombinant human IL-6. No cross-reactivity was observed when thiskit was used to analyze the following recombinant cytokines.
HumanIL-3

IL-8

IL-15

M-CSF

FGF basic

VEGF165

IL-4

IL-10

IL-18

G-CSF

Anti-CD137

Anti-CD28

IL-5

IL-11

IFN-alpha 1

GM-CSF

Flt-3 Ligand

ThrombopoietinTPO

IL-2

IL-12B

IFN-gamma

SCF

L1R

TGF-beta 1

IL-7

IL-17A

TNF-alphaBMP-2

Anti-CD3
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INTERFERING SUBSTANCES
Verify potential matrix effects by adding different levels of DMSO and HSA to the diluted buffer.
Additive

Tolerated concentration

DMSO

20%

HSA

5%

CALIBRATIONThis immunoassay is calibrated against a highly purified E. coli-expressed recombinant human IL6(89/548). Reference Reagent is calibrated by NIBSC/WHO in June 2014.
NIBSC/WHO (89/548) approximate value (U/mL) = 0.3173 × Human IL-6 value (pg/mL)

PLATE LAYOUT

Note: Blank is a Blank Dilution Buffer hole.

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A005-EN.01

TROUBLESHOOTING GUIDE
Problem

Cause

Solution

Poor standard curve* Inaccurate pipetting* Check pipettes

Large CV* Inaccurate pipetting* Check pipettes* Air bubbles in wells* Remove bubbles in wells* Plate is insufficiently washed* Review the manual for proper wash.

* Contaminated wash buffer* Make fresh wash buffer* Incorrect wavelengths* Check filters/reader* Insufficient development* Increase development timeHigh background

Very low readings acrossthe platetime
Samples are reading too* Samples contain cytokine

high, but standard curve

levels above assay range* Dilute samples and run again

looks fine
Drift

* Interrupted assay set-up* Assay set-up should be continuous - have all* Reagents not at room

standards and samples prepared appropriately

temperature

before commencement of the assay* Ensurethat all reagents are at room

temperature before pipetting into the wells
unless otherwise instructed in the antibodyinserts11 / 11

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