A015-EN.01

resDetect™ Anti-CD3 Antibody ELISA Kit(Enzyme-Linked Immunosorbent Assay)

Pack Size: 96 tests
Catalog Number：CRS-A015

IMPORTANT: Please carefully read this manual before performing your experiment.
For Research Use Only. Not For Use in Diagnostic or Therapeutic Procedures
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A015-EN.01

INTENDED USE
The kit is developed for the detection of anti-CD3 antibody in Bioprocess manufacturing applications.
It is intended for research use only (RUO).

BACKGROUND
Since the 1990s, CD3 monoclonal antibody has been used in CIK cell therapy to stimulates theproliferation and activation of T cells. Under the cooperation of other cytokines, such as IL2 and IL1a,
CIK cells with rapid proliferation, high tumoricidal activity, broad tumor killing spectrum and non-MHCrestricted tumor killing characteristics are generated, which has significant effects on the treatment ofcancer, chronic leukemia, liver disease and neurological diseases. Obviously, it is necessary to control theresidues of raw materials in the final cell therapy product.

PRINCIPLE OF THE ASSAY
This assay kit is used to measure the titer of Anti-CD3 Antibody by employing an indirect ELISA.
Immobilize Human CD3E & CD3G on the microplate. Then add the samples, incubate and wash the wells.
Next add Secondary antibody HRP-Anti-Mouse IgG to the plate, incubate and wash the wells. Lastly load
the substrate into the wells and monitor color development in proportion with the amount of antibodypresent. The reaction is stopped by the addition of a stop solution and the intensity of the absorbance canbe measured at 450 nm and 630 nm. The OD Value reflects the amount of antibody bound.

PRECAUTIONS1. This kit is for research use only and is not for use in diagnostic or therapeutic procedures.
2. The kit should be used according to the instructions.
3. Do not mix reagents from different lots.
4. All reagents should be balance to room temperature (20℃-25℃) before use. If crystals have formed
in buffer solution, warm to room temperature until the crystals have completely dissolved.
5. The kit should be stored at 2°C to 8°C.
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MATERIALS PROVIDED
Table 1. Materials provided
Catalog

Components

Size(96 tests)

Storage
FormatUnopened

OpenedCRS015-C01

Pre-coated Human CD3E & CD3G Microplate

1 plate

Solid

2-8℃

2-8℃

CRS015-C02

Anti-CD3 Antibody Standard

50 μL

Liquid

2-8℃

2-8℃

CRS015-C03

HRP-Goat anti-Mouse IgG

10 μg

Powder

2-8℃-70℃

CRS015-C04

10xWashing Buffer

50 mL

Liquid

2-8℃

2-8℃

CRS015-C05

2xDilution Buffer

50 mL

Liquid2-8℃2-8℃CRS015-C06

Substrate Solution

12 mL

Liquid2-8℃, avoid light

2-8℃, avoid light

CRS015-C07

Stop Solution

7 mL

Liquid2-8℃2-8℃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.

STORAGE AND EXPIRATION DATA
1. The unopened kit is stable for 12 months from the date of manufacture if stored at 2°C to 8°C.
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.
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REAGENT PREPARATION
Bring all reagents and samples to room temperature (20℃-25℃) before use. If crystals have formed inbuffer solution, place the sample in a 37 ºC incubator until the crystals have completely dissolved andbring the solution back to room temperature before use.
Reconstitute the provided lyophilized materials to stock solutions with distilled, sterile water asrecommended in Table 2 and place the materials for 15 to 30 minutes at room temperature withoccasional gentle mixing. Avoid vigorous shaking. The reconstituted stock solutions should be stored
at -70℃. It is recommended not to freeze-thaw more than 1 times, the packing specification shall notbe less than 5μg.
Note: Considering innevitable minor quantatition variations between protein batches, it is also reasonable to generate
the standard curve with specific lot of proteins used for current production for even better accuracy.
Table 2. Reconstitution methods
IDComponents

Size

Stock Solution Con.

Reconstitution Buffer and Vol.

CRS015-C03

HRP-Goat anti-Mouse IgG

10 μg

100 ug/mL

100 μL water

RECOMMENDED SAMPLE PREPARATION
1. Working fluid 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 HRP-Goat anti-Mouse IgG working fluid:
Dilute HRP-Goat anti-Mouse IgG to 0.08 μg/mL with Dilution Buffer. The prepared working fluid should
avoid light. Please prepare it for one-time use only.
2. Preparation of Standard curve
Make serial dilutions of the Anti-CD3 Antibody Standard as a Standard curve with Dilution Buffer as
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A015-EN.01

recommended in Figure 1.
Figure 1. Preparation of 1:1 serial dilutions of the Anti-CD3 Antibody

3. Add Samples
Add 100μL serially diluted Anti-CD3 Antibody Standard curve and samples to each well. For blank
Control wells, please add 100μL 1×Dilution Buffer. Seal the plate with microplate sealing film andincubate at 37℃ for 1 hour.
4. Washing
Remove the remaining solution by aspiration, add 300 μL of 1×Washing Buffer to each well, gently tap
the plate for 1 min, remove any remaining 1×Washing Buffer: by aspirating or decanting, invert the plateand blot it against paper towels. Repeat the wash step above for three times.
5. Add HRP-Goat anti-Mouse IgG
For all wells, add 100 μL HRP-Goat anti-Mouse IgG (dilute to 0.08 μg/mL) working solution. Seal theplate with microplate sealing film and incubate at 37℃ for 1 hour.
6. Washing
Repeat step 4.
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7. Substrate Reaction
Add 100 μL Substrate Solution to each well. Seal the plate with microplate sealing film and incubate at37℃ for 20 min, avoid light.
8. Termination
Add 50 μL Stop Solution to each well, and tap the plate gently for 5 min to allow thorough mixing.
Note: the color in the wells should change from blue to yellow.
9. Data Recording
Read the absorbance at 450 nm and 630 nm using UV/Vis microplate spectrophotometer.
Note: To reduce the background noise, subtract the value read at OD450 nm with the value read at OD630
nm.

CALCULATION OF RESULTS
1. Normal range of Standard curve: R2≥0.9900, detection range: 0.05-1.56 ng/mL.
2. If the OD value of the sample to be tested is higher than the highest standard, the sample shall be
diluted with dilution buffer and assay repeated.
3. To calibrate absorbance value obtained by the standard curve, the OD value of the sample to bemeasured is subtracted from the OD value of the blank control. The standard curve is plotted with thestandard concentration as x-axis and the calibrated absorbance value as y-axis. Linear regression
equation or Four parameters logistic are used to draw the standard curve and calculate the sample
concentration.

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A015-EN.01

QUICKGUILD

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TYPICAL DATA
The following data is for reference only. The sample concentration was calculated based on the resultsof the standard curve.

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PLATE LAYOUT

Note: Blank is a Blank Dilution Buffer hole.

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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* Review the manual for proper wash.

High background

washed* Make fresh wash buffer* Contaminated washbuffer
Very low readings acrossthe plate* Incorrect wavelengths* Check filters/reader* Insufficient development* Increase development time

time
Samples are reading too* Samples containhigh, but standard curve

cytokine levels above

looks fineassay range

Drift* Dilute samples and run again* 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* Ensure that all reagents are at room temperature

before pipetting into the wells unless otherwise
instructed in the antibody inserts

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