Product description

This kit employs the sandwich ELISA principle coupled with a biotin-streptavidin system for the detection of E.coli host cell protein (HCP) residuals in samples. The microplate wells are pre-coated with an E.coli HCP capture antibody. Samples, biotinylated detection antibody, and horseradish peroxidase (HRP)-labeled streptavidin (SA) are added and incubated together, forming an antibody-antigen-biotinylated antibody-HRP-SA complex. After thorough washing, the substrate TMB is added for color development. TMB is converted to a blue color catalyzed by peroxidase and finally turns yellow upon acid termination. The color intensity is positively correlated with the E.coli HCP content in the sample. The optical density (OD value) is measured at 450 nm using a microplate reader, and the concentration of E.coli HCP in the samples is calculated based on the standard curve.

Product specifications

This kit is for quantitative measurement of residual E.coli HCP.

Transportation and Storage

1. For unused kit: The whole kit could be stored at 2-8°C in shelf life. The shelf life is 12 months. Strong light should be avoided for storage stability.

2. For used kit: Once the microplate is opened, please cover unused wells with plate sealer and return to the foil pouch containing the desiccant pack, zip-seal the foil pouch and return to 2-8°C as soon as possible after use. Other reagents should be returned to 2-8°C as soon as possible after use.

Component list

Materials Required But Not Supplied

1. Microplate reader with 450 ± 10 nm filter (better if can detect at 450 and 650 nm wavelength).

2. Microplate shaker.

Operation Flowchart

Before You Begin

1. Bring all kit components and samples to room temperature (18-25°C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.

2. Wash buffer: dilute 20 mL of 20× concentrated wash buffer with 380 mL of deionized or distilled water to prepare 400 mL of 1× wash buffer.

3. Biotinylated detection antibody and HRP streptavidin working solution: briefly spin or centrifuge the stock solution before use. Dilute them to the working concentration with Assay Diluent.

4. TMB substrate: aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again. TMB substrate is sensitive to light, don't expose TMB substrate to light for a long time.

5. Dilute the E.coli HCP Standard (0.15 mg/mL) with Sample Diluent to concentrations of 300, 100, 40, 12, 3, 1, and 0 ng/mL. We recommend standards can be diluted as following chart:

Using protocol

1. Determine the number of strips required for the assay. Insert the strips in the frames for use. Remaining plate strips not used in this assay should be repacked in the bag with desiccant. Close the bag tightly for refrigerated storage.

2. Add 50 µL per well of the working concentration Biotinylated Detection Antibody to the microplate wells. Then add 50 µL per well of the standards or samples. Finally, add 50 µL per well of the working concentration HRP Streptavidin. If the E.coli HCP concentration in the test sample is unknown, it is recommended to test the sample at multiple dilutions using the Sample Diluent.

3. Cover the plate with a plate sealer. Incubate for 90 minutes at room temperature with shaking (500 rpm).

4. Wash step: Aspirate the solution and wash with 300 µL wash buffer to each well and let it stand for 30 s. Discard wash buffer completely by snapping the plate onto absorbent paper. Totally wash 4 times.

5. Add 100 µL of TMB substrate solution to each well. Cover the plate with a new plate sealer and incubate for 30 minutes at room temperature protected from light.

6. Add 50 µL of Stop solution to each well. Read the plate immediately using a microplate reader set to 450 nm (dual-wavelength 450 nm/650 nm is recommended).

Calculation of Results

1. Calculate the average OD value for each standard, blank, and sample. (For dual wavelength mode, use the OD value at 450 nm minus the OD value at 650 nm). Plot the standard curve with the standard concentration on the X axis and the corresponding average OD value on the Y-axis.

2. It is recommended to use professional curve fitting software such as Curve Expert 1.3 or ELISA Calc (use the four-parameter logistic (4PL) curve-fit method) to generate the standard curve and calculate sample concentrations.

Performance

1. Limit of Detection (LOD): 0.5 ng/mL

2. Limit of Quantification (LOQ): 3 ng/mL

3. Precision: Intra-assay CV≤10%, Inter-assay CV≤15%

4. Recovery: 80%-120%

5. Linear Range: 3-300 ng/mL

Considerations

1. TMB reaction temperature and time are critical, please control them according to the instruction strictly. But if the OD450 is beyond the microplate reader detection range, please reduce TMB reaction time properly.

2. In order to achieve good assay reproducibility and sensitivity, proper washing of the plates to remove excess un-reacted reagents is essential.

3. All the reagents should be mixed thoroughly prior to use and avoid bubbles during sample or reagents addition.

4. If crystals have formed in the concentrated wash buffer (20×), warm to 37°C and mix gently until the crystals are completely dissolved.

5. Avoid introducing sodium azide (NaN₃) into samples, as it inhibits HRP activity and may cause low readings.

6. Do not mix components from different kit lots. Use separate pipette tips for different reagents to avoid cross-contamination.

7. For your safety and health, wear a lab coat and gloves during experiments.

8. This product is for research use only (RUO). Not for use in clinical diagnostic procedures or other unreasonable purposes.