Human Trypsin ELISA Kit

Cat. No.: EJ1514725

GRADE & PURITY BioReagent, for Enzyme immunoassay(ELISA)

Descripción general

Trypsin is a serine protease belonging to the PA superfamily, which exists in the digestive system of numerous vertebrates and functions to hydrolyze proteins. It is generated in the small intestine when trypsinogen secreted by the pancreas is activated. This enzyme primarily cleaves peptide chains at the carboxyl side of lysine and arginine residues. Trypsin is widely applied in diverse biotechnological procedures. The degradation process mediated by trypsin is commonly termed tryptic digestion, and proteins processed with trypsin are defined as trypsinized proteins.

Experimental Principle:

This kit adopts double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotinylated detection antibodies and HRP conjugates are sequentially added into microplates pre-coated with human trypsin capture antibodies. Following incubation and washing steps, chromogenic substrate TMB is applied. Catalyzed by horseradish peroxidase (HRP), TMB turns blue and subsequently shifts to yellow under acidic conditions. The color intensity is positively correlated with the concentration of human trypsin in samples. The optical density (OD value) is measured at 450 nm via a microplate reader to calculate sample concentration.

Precautions:

1. Incubate strictly at the specified time and temperature for accurate results. All reagents must reach room temperature (20–25°C) before use. Refrigerate reagents immediately after use.

2. Improper washing may cause inaccurate results. Ensure wells are completely aspirated before adding substrate. Do not let microwells dry out for extended periods.

3. Wipe residual liquid and fingerprints from the plate bottom, as they affect OD readings.

4. TMB substrate should be colorless; do not use if it turns blue.

5. Avoid cross-contamination of reagents and samples to prevent false results.

6. Protect from direct strong light during storage and incubation.

7. Do not expose reagents to bleach or its fumes, as bleach inactivates kit bioactivity.

8. Do not use expired products. Do not mix components from different catalog numbers/lots.

9. Recombinant proteins from external sources may not be recognized by kit antibodies due to mismatch.

10. Handle all samples as potentially infectious. Dispose of samples and test devices per regulations.

Kit Components

Item No.	Appearance	Components	48T	96T	Storage
EJ1514725A	—	Pre-coated Assay Plate	48 wells	96 wells	2-8℃.
EJ1514725B	Solid	Standard	1 vial	2 vials	2-8℃.
EJ1514725C	Liquid	Universal Diluent	1×20 mL	2×20 mL	2-8℃.
EJ1514725D	Liquid	Biotin-antibody (100×)	60 μL	120 μL	2-8℃.
EJ1514725E	Liquid	Streptavidin-HRP (100×)	60 μL	120 μL	2-8℃.
EJ1514725F	Liquid	Wash Buffer (20×)	1×10 mL	2×10 mL	2-8℃.
EJ1514725G	Liquid	TMB Substrate	5 mL	10 mL	2-8℃.
EJ1514725H	Liquid	Stop Solution	3 mL	6 mL	2-8℃.
EJ1514725I	—	Plate Sealer	4 pieces	4 pieces	2-8℃.

Note: EJ1514725B、EJ1514725D、EJ1514725E and EJ1514725F shall be diluted in accordance with the instructions.

Sample Handling and Requirements

1. The detection range of the kit is not equivalent to the concentration range of the analyte in samples. It is recommended to estimate the analyte concentration in samples by referring to relevant literature before the experiment, and confirm the actual concentration through pre-experiments. Appropriately dilute or concentrate samples if the analyte concentration is too high or too low.

2. If the test sample type is not listed in the instructions, a pre-experiment is recommended to verify the detection applicability.

3. Serum: Place whole blood collected in serum separation tubes at room temperature for 2 hours or at 2–8 °C overnight. Centrifuge at 1000× g for 20 minutes and collect the supernatant. The supernatant can be stored at -20 °C or -80 °C; repeated freeze-thaw cycles must be avoided.

4. Plasma: Collect samples using EDTA or heparin as anticoagulants. Within 30 minutes after collection, centrifuge the sample at 1000× g and 2–8 °C for 15 minutes, then collect the supernatant for detection. The supernatant can be stored at -20 °C or -80 °C with avoidance of repeated freeze-thawing.

5. Tissue homogenate: Rinse tissues with pre-cooled PBS (0.01 M, pH=7.4) to remove residual blood (lysed red blood cells in homogenate will interfere with test results). Weigh and mince the tissues. Transfer minced tissues into a glass homogenizer with an appropriate volume of PBS (generally at a weight-to-volume ratio of 1:9, e.g., 1 g tissue with 9 mL PBS; the volume may be adjusted properly with experimental records. Addition of protease inhibitor to PBS is recommended). Fully grind on ice or use a homogenizer. For thorough cell lysis, perform ultrasonication or repeated freeze-thaw treatment on the homogenate. Finally, centrifuge the homogenate at 5000× g for 5–10 minutes and collect the supernatant for testing.

6. Cell culture supernatant: Centrifuge at 1000× g for 20 minutes and collect the supernatant for detection. Store the supernatant at -20 °C or -80 °C and avoid repeated freeze-thaw cycles.

7. Cell lysate: Gently wash adherent cells with pre-cooled PBS, digest with trypsin, and collect cells after centrifugation at 1000× g for 5 minutes. Suspension cells can be collected by direct centrifugation. Wash harvested cells three times with pre-cooled PBS. Resuspend every 1×10⁶ cells in 150–200 μL PBS (protease inhibitor addition is recommended; reduce PBS volume appropriately if the target content is low). Lyse cells by repeated freeze-thawing or ultrasonication. Centrifuge the extract at 1500× g and 2–8 °C for 10 minutes, and collect the supernatant for detection.

8. Other biological samples: Centrifuge at 1000× g for 20 minutes and collect the supernatant for testing.

9. Sample appearance: Samples shall be clear and transparent; suspended substances must be removed by centrifugation.

10. Sample storage: Samples to be tested within 1 week can be stored at 4 °C. For delayed detection, aliquot samples into single-use portions and store frozen at -20 °C (within 1 month) or -80 °C (within 6 months). Repeated freeze-thawing is prohibited. Hemolysis will affect test results, so hemolyzed samples are not suitable for this assay.

Sample Dilution Protocol:

Estimate the sample concentration range in advance. Refer to the following scheme if dilution is required.

100-fold dilution: Single-step dilution. Add 5 μL sample into 495 μL universal diluent.

1000-fold dilution: Two-step dilution. First, add 5 μL sample into 95 μL universal diluent for 20-fold dilution. Then take 5 μL of the diluted solution and add into 245 μL diluent for 50-fold dilution, reaching a total 1000-fold dilution.

100000-fold dilution: Three-step dilution. First, add 5 μL sample into 195 μL diluent for 40-fold dilution. Second, take 5 μL of the above solution into 245 μL diluent for 50-fold dilution. Finally, take 5 μL of the 2000-fold diluted solution into 245 μL diluent for another 50-fold dilution, achieving total 100000-fold dilution.

The volume taken for each dilution shall not be less than 3 μL, and single dilution fold shall not exceed 100 times. Mix thoroughly without foaming after each dilution step.

Self-supplied laboratory equipment required for the experiment:

1. Microplate reader (450 nm)

2. High-precision pipettes & tips: 0.5–10 μL, 5–50 μL, 20–200 μL, 200–1000 μL

3. 37°C incubator

4. Distilled or deionized water

Pre-test Preparation:

1. Take the kit out of the refrigerator 10 minutes in advance and equilibrate it to room temperature.

2. Preparation of serial standard working solutions

Add 1 mL universal diluent into the lyophilized standard. Let it stand for 15 minutes until fully dissolved, then mix gently to obtain the stock solution at 20 ng/mL. Prepare serial dilutions at concentrations of 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.312 ng/mL and 0 ng/mL.

Double dilution method: Prepare 7 EP tubes and add 500 μL universal diluent into each tube. Pipette 500 μL of 20 ng/mL standard solution into the first tube and mix well to make 10 ng/mL solution. Repeat the operation sequentially for subsequent concentrations. The last tube serves as blank control without liquid transfer from the penultimate tube. Refer to the figure below for details.

3. Preparation of biotinylated detection antibody working solution

Centrifuge 100× concentrated biotinylated detection antibody at 1000×g for 1 minute 15 minutes prior to use. Dilute the concentrate to 1× working concentration with universal diluent (e.g. 10 μL concentrate mixed with 990 μL universal diluent). Prepare freshly before use.

4. Preparation of enzyme conjugate working solution

Centrifuge 100× concentrated enzyme conjugate at 1000×g for 1 minute 15 minutes prior to use. Dilute the concentrate to 1× working concentration with universal diluent (e.g. 10 μL concentrate mixed with 990 μL universal diluent). Prepare freshly before use.

5. Preparation of 1× washing solution

Add 10 mL 20× washing solution into 190 mL distilled water. Crystallization may occur in refrigerated concentrated washing solution, which is normal. Place it at room temperature until crystals dissolve completely before preparation.

Assay Procedure

1. Remove the required plate strips from the aluminum foil pouch (equilibrated at room temperature for 10 minutes). Seal the remaining strips in a zip-lock bag and return to 4°C.

2. Sample Loading: Add 100 μL of sample/positive control/negative control to the respective wells. Add 100 μL of Universal Diluent to blank wells. Cover with a plate sealer and incubate at 37°C for 60 minutes.

Recommendation: Dilute the test sample at least 1-fold with Universal Diluent before adding to the ELISA plate to reduce matrix effects on test results. Multiply the final sample concentration by the corresponding dilution factor. It is recommended to set up duplicate wells for all test samples and positive/negative controls during the test.

3. Add Biotin-Antigen: Remove the ELISA plate, discard the liquid (do not wash). Add 100 μL of Biotin-Antigen Working Solution to each well directly. Cover with a plate sealer and incubate at 37°C for 60 minutes.

4. Wash Plate: Discard the liquid. Add 300 μL of 1× Wash Solution to each well, let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat the washing process 3 times (a plate washer can also be used).

5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a plate sealer and incubate at 37°C for 30 minutes.

6. Wash Plate: Discard the liquid and wash 5 times according to the washing method in Step 4.

7. Add Substrate: Add 90 μL of Substrate (TMB) to each well. Cover with a plate sealer and incubate at 37°C in the dark for 15 minutes.

8. Add Stop Solution: Remove the ELISA plate, add 50 μL of Stop Solution to each well directly, and immediately measure the OD value of each well at a wavelength of 450 nm.

Calculation of Experimental Results:

Result Evaluation:

1. Calculate the average optical density (OD) value of duplicate wells for standards and samples, then subtract the OD value of blank wells to obtain corrected values. Plot the four-parameter logistic standard curve on log-log graph paper with concentration as the horizontal axis and OD value as the vertical axis.

2. Retest the sample after proper dilution if its OD value exceeds the upper limit of the standard curve. Multiply the corresponding dilution factor when calculating sample concentration.

Typical Data and Reference Curve:

The following data and curve are for reference only. Establish the standard curve based on actual experimental results.

Concentration (ng/mL)	20	10	5	2.5	1.25	0.625	0.312	0
OD	2.22	1.56	0.92	0.63	0.42	0.25	0.18	0.08
Corrected OD	2.14	1.48	0.84	0.55	0.34	0.17	0.1	—

Note: This figure is for reference only. Sample content shall be calculated according to the standard curve plotted from experimental data of each test.

Kit Performance:

1. Reproducibility: Intra-assay coefficient of variation is less than 10%, inter-assay coefficient of variation is less than 10%.

2. Recovery rate: Three concentrations of human trypsin were spiked into healthy human serum, plasma and cell culture supernatant respectively, and the recovery rate was calculated.

Sample Type	Range (%)	Average Recovery Rate (%)
Serum (n=8)	84-101	96
Plasma (n=8)	93-104	102
Cell culture supernatant (n=8)	96-108	105

3. Linear dilution: High-concentration human trypsin was added into 4 batches of selected healthy human serum, plasma and cell culture supernatant. Samples were diluted within the dynamic range of standard curve to evaluate linearity.

Dilution Ratio	Recovery Rate (%)	Serum	Plasma	Cell Culture Supernatant
1:2	Range	84-95	88-97	90-110
1:2	Average Recovery Rate	91	94	96
1:4	Range	89-103	87-108	105-115
1:4	Average Recovery Rate	94	98	108

Troubleshooting:

If unsatisfactory experimental results occur, take photos of color reaction immediately, save experimental data, reserve used plate strips and unused reagents, then contact our technical support for solutions. You can also refer to the following contents including problem description, potential causes and corresponding solutions:

1. Poor standard curve linearity

① Incorrect standard dilution: Dissolve and dilute standards strictly in accordance with recommended protocols.

② Inaccurate pipetting: Calibrate pipettes regularly and check airtightness of pipette tips.

③ Evaporation of reaction solution: Seal the microplate with sealing film.

④ Inadequate washing: Ensure sufficient washing cycles and adequate volume of washing solution.

⑤ Foreign matters at well bottom: Clean the plate bottom before detection.

2. Weak or absent color development

① Insufficient incubation duration: Follow specified incubation time.

② Improper incubation temperature: Incubate at recommended temperature.

③ Insufficient reagent dosage: Verify pipettes and operate strictly as instructed.

④ Wrong dilution procedure: Recheck reagent dilution steps.

⑤ Inactivated enzyme conjugate: Mix enzyme conjugate with substrate and verify activity via color reaction.

3. Low OD value

① Incorrect microplate reader setting: Check detection wavelength.

② Termination solution not added: Add adequate termination solution.

③ Excess waiting time before reading: Measure absorbance promptly.

④ Excessively high sample concentration: Determine proper dilution ratio via preliminary test.

⑤ Excessively low sample concentration: Determine proper dilution ratio via preliminary test.

4. High background signal

① Contaminated chromogenic solution: Replace with fresh chromogenic solution.

② Excessive color developing time: Control reaction duration strictly.

③ Wrong dilution of detection antibody or enzyme conjugate: Adopt recommended dilution method.

④ Incomplete plate washing: Perform sufficient washing times and use enough washing buffer.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C

Enviado en

Wet ice

Estabilidad y almacenamiento

Store at 2-8℃ long term (6 months).

Contents & Storage

Item No.	Appearance	Components	48T	96T	Storage
EJ1514725A	—	Pre-coated Assay Plate	48 wells	96 wells	2-8℃.
EJ1514725B	Solid	Standard	1 vial	2 vials	2-8℃.
EJ1514725C	Liquid	Universal Diluent	1×20 mL	2×20 mL	2-8℃.
EJ1514725D	Liquid	Biotin-antibody (100×)	60 μL	120 μL	2-8℃.
EJ1514725E	Liquid	Streptavidin-HRP (100×)	60 μL	120 μL	2-8℃.
EJ1514725F	Liquid	Wash Buffer (20×)	1×10 mL	2×10 mL	2-8℃.
EJ1514725G	Liquid	TMB Substrate	5 mL	10 mL	2-8℃.
EJ1514725H	Liquid	Stop Solution	3 mL	6 mL	2-8℃.
EJ1514725I	—	Plate Sealer	4 pieces	4 pieces	2-8℃.

Note: EJ1514725B、EJ1514725D、EJ1514725E and EJ1514725F shall be diluted in accordance with the instructions.