A014-EN.02

resDetect™ Anti-CD28 Antibody ELISA Kit(Enzyme-Linked Immunosorbent Assay)

Catalog Number：CRS-A014
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
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A014-EN.02

INTENDED USE
The kit is developed for the detection of anti-CD28 antibody in Bioprocess manufacturing
applications. It is intended for research use only (RUO).

BACKGROUND
CD28 antibodies can be used to stimulate the proliferation and activation of T cells in CIK cell therapy.
Under the cooperation of other cytokines, such as IL2 and IL1a, CIK cells with rapid proliferation, hightumoricidal activity, broad tumor killing spectrum and non-MHC-restricted tumor killing characteristics
are generated, which has significant effects on the treatment of cancer, chronic leukemia, liver disease
and neurological diseases. Obviously, it is necessary to control the residues of raw materials in the final
cell therapy products.

PRINCIPLE OF THE ASSAY
This assay kit is used to measure the titer of Anti-CD28 Antibody by employing an indirect ELISA.
Immobilize Human CD28 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 thesubstrate into the wells and monitor color development in proportion with the amount of antibody
present. 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.

PRECAUTIONS
1. 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. Bring all reagents and samples 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.
1 /8
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A014-EN.02

MATERIALS PROVIDED
Table 1. Materials provided
Catalog

ComponentsSize(96 tests)

Storage

FormatUnopened

OpenedCRS014-C01

Pre-coated Human CD28 Microplate

1 plate

Solid

2-8℃

2-8℃

CRS014-C02

Anti-CD28 Antibody Standard

20 μg

Powder

2-8℃-70℃

CRS014-C03

HRP-Goat anti-Mouse IgG

10 μg

Powder

2-8℃-70℃

CRS014-C04

10xWashing Buffer

50 mL

Liquid

2-8℃

2-8℃

CRS014-C05

2xDilution Buffer

50 mL

Liquid

2-8℃

2-8℃

CRS014-C06

Substrate Solution

12 mL

Liquid2-8℃, avoid light

2-8℃, avoid light

CRS014-C07

Stop Solution

7 mL

Liquid2-8℃2-8℃

STORAGE AND EXPIRATION DATA
Unopened kit should be stored at 2°C -8°C upon receiving.
Find the expiration date on the outside packaging and do not use reagents past their expiration date.
The opened kit should be stored per components table. The shelf life is 30 days from the date ofopening.

REAGENTS/EQUIPMENT NEEDED BUT NOT SUPPLIEDSingle 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 450 nm and 630 nm filter;
Tubes: 1.5mL,10mL;
Timer;
Reagent bottle;
Deionized or distilled water.
2 /8
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A014-EN.02

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 37ºC 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 not beless than 5 μg.
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.

CRS014-C02

Anti-CD28 Antibody Standard

20 μg

100 μg/mL

200 μL water

CRS014-C03

HRP-Goat anti-Mouse IgG

10 μg

100 μg/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.

3 /8
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A014-EN.02

2. Preparation of Standard curve
Make serial dilutions of the Anti-CD28 Antibody Standard as a Standard curve with Dilution Buffer
as recommended in Figure 1.
Figure 1. Preparation of 1:1 serial dilutions of the Anti-CD28 Antibody

3. Add Samples
Add 100 μL serially diluted Anti-CD28 Antibody Standard curve and samples to each well. For blank
Control wells, please add 100 μL 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
the plate with microplate sealing film and incubate at 37℃ for 1 hour.
6. Washing
Repeat step 4.
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A014-EN.02

7. Substrate Reaction
Add 100 μL Substrate Solution to each well. Seal the plate with microplate sealing film and incubate at
37℃ 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: 0.078-2.5 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 to 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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A014-EN.02

QUICK GUILD

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A014-EN.02

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

PLATE LAYOUT

Note: Blank is a Blank Dilution Buffer hole.
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A014-EN.02

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

Very low readings acrossthe plate* Incorrect wavelengths* Check filters/reader* Insufficient development time

* Increase development time

Samples are reading too
high, but standard curve
looks fine

* Samples contain cytokine levels
above assay range* Dilute samples and run again* Interrupted assay set-up* Assay set-up should be continuous - have all* Reagents not at room temperature

standards and samples prepared appropriately
before commencement of theassay

High background

Drift* Ensure that all reagents are at room temperature

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

8 /8
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Tel: +1 800-810-0816

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Asia and Pacific:

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