Apr 08, 2026
Competitive ELISA Protocol for Quantification of Rat Testosterone Using CUSABIO CSB-E05100r ELISA Kit
- Rosie Liu1
- 1CUSABIO
- CUSABIO TECHNOLOGY LLC
Protocol Citation: Rosie Liu 2026. Competitive ELISA Protocol for Quantification of Rat Testosterone Using CUSABIO CSB-E05100r ELISA Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbj8zovpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 08, 2026
Last Modified: April 08, 2026
Protocol Integer ID: 314681
Keywords: quantitative detection of endogenous testosterone, cusabio rat testosterone, testosterone in the sample, unlabeled testosterone in the sample, rat testosterone, endogenous testosterone, unlabeled testosterone, conjugated testosterone, testosterone, competitive elisa protocol for quantification, competitive elisa protocol, e05100r elisa kit this protocol, elisa protocol, rat serum, e05100r elisa kit, competitive inhibition enzyme immunoassay technique, specific antibody, using cusabio csb, immunoassay technique, limited amount of specific antibody, cusabio csb, antibody, elisa kit, assay
Abstract
This protocol details the quantitative detection of endogenous testosterone in rat serum, plasma, cell culture supernatants, and tissue homogenates using the CUSABIO Rat Testosterone (T) ELISA kit (Catalog No. CSB-E05100r). This assay employs the competitive inhibition enzyme immunoassay technique in which the HRP-conjugated testosterone and unlabeled testosterone in the sample compete for a limited amount of specific antibody. The resulting color development is inversely proportional to the concentration of testosterone in the sample.
Guidelines
- Principle: Competitive inhibition enzyme immunoassay. A pre-coated plate (goat-anti-rabbit antibody) binds an antibody specific for testosterone. Competition occurs between HRP-conjugated testosterone and sample testosterone for antibody binding sites. Color development is inversely proportional to sample testosterone concentration.
- Detection Range: 0.13 ng/ml – 25.6 ng/ml.
- Sensitivity (LLD): c 0.06 ng/ml.
- Specificity: High specificity for rat testosterone. No significant cross-reactivity observed with tested analogues (note: comprehensive cross-reactivity testing not performed).
- Precision:**
- Intra-assay: CV% c 15% (tested 20 times on one plate).
- Inter-assay: CV% c 15% (tested in 20 assays).
- Sample Types: Serum, plasma, cell culture supernatants, tissue homogenates.
- Sample Storage:**
- Short term (≤5 days): 2-8°C.
- Long term: ≤1 month at -20°C; ≤2 months at -80°C. Avoid repeated freeze-thaw cycles.
- Reagent Storage:**
- Unopened Kit: 2-8°C (use before expiration date).
- Opened Kit: Components may be stored at 2-8°C for up to 1 month (within kit expiration).
- Calculation: Average duplicate OD readings. Subtract Blank OD. Generate a standard curve using a four-parameter logistic (4-PL) curve fit (software recommended). Multiply the concentration read from the curve by the sample dilution factor.
- Low Sensitivity/OD: Confirm reagent expiration. Check reagent preparation accuracy (use distilled water and clean containers). Verify incubation times/temperatures. Ensure proper storage of samples/reagents (avoid freeze-thaw). Check pipette calibration.
- Poor Precision: Ensure consistent pipetting technique and tip changes between samples/reagents. Verify washing consistency and completeness. Control incubation conditions (time, temperature, plate sealing).
- Standard Curve Issues: Confirm standard reconstitution/storage. Ensure accurate pipetting of standards. Check the microplate reader calibration.
- Color Development Issues: Observe color change after adding Substrates; stop reaction early if color develops too rapidly. Ensure Stop Solution is added in the same order as Substrates and mixed thoroughly (green color indicates incomplete mixing).
- Samples > Highest Standard: Dilute samples appropriately and repeat the assay.
- Unexpected Results: Predict sample concentration to determine optimal dilution. Preliminary experiments are necessary for sample types not explicitly listed. Consider potential interference or sample degradation.
Materials
**Provided Reagents:**
- Pre-coated Microplate (96 wells)
- Testosterone Standard (6 vials, 0.5 ml each: 0, 0.13, 0.5, 2, 6.4, 25.6 ng/ml)
- Anti-Testosterone Antibody (1 x 6 ml)
- HRP-Conjugated Testosterone (1 x 6 ml)
- Wash Buffer Concentrate (20xconcentrate) (1 x 15 ml)
- Substrate A (1 x 7 ml)
- Substrate B (1 x 7 ml)
- Stop Solution (1 x 7 ml)
- Adhesive Strips (for 96 wells) (4 strips)
**Required Equipment/Supplies (Not Provided):**
- Microplate reader (measure absorbance at 450nm with 600-630 nm correction wavelength)
- Incubator (provide stable incubation conditions up to 37°C ± 0.5°C)
- Plate washer (squirt bottle, dispenser, or automated washer)
- Absorbent paper for blotting the microtiter plate
- Graduated cylinders (100 mL, 500 mL)
- Deionized/Distilled Water
- Pipettes and pipette tips
- Test tubes for dilutions
- Curve-fitting software
Safety warnings
Avoid repeated freeze-thaw cycles to preserve sample integrity.
- Intended Use: For research use only. Not approved for clinical diagnosis.
- Expiration: Do not use the kit beyond the expiration date on the label.
- Reagents: Do not mix or substitute reagents from other lots or sources.
- Sample Limitations: Grossly hemolyzed samples are unsuitable. Samples generating values e the highest standard require dilution and re-assay.
- Variability: Results can vary due to operator technique, pipetting, washing, incubation time/temperature, and kit age.
- Interference: While designed to eliminate interference, the possibility cannot be entirely excluded until all factors are tested.
- Stop Solution: Contains acid. Wear appropriate personal protective equipment (eye, hand, face, clothing).
- Tissue/Cell Extracts: Chemical lysis buffers may cause unexpected results.
- Antigen-Antibody: A potential mismatch exists between this antibody and antigens from other sources/manufacturers.
- Cell Culture Supernates: Detectability depends on cell viability, number, and sampling time; may not be detected.
- Sample Freshness: Fresh samples recommended; degradation can lead to erroneous results.
Ethics statement
This protocol involves the use of animal blood samples. Users must obtain prior approval from their Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee before performing this protocol. All procedures must comply with applicable institutional and governmental regulations regarding the ethical use of animals.
Before start
The CUSABIO Rat Testosterone (T) ELISA Kit is suitable for the detection of testosterone from the following samples:
- Serum
- Plasma
- Cell culture supernatants
- Tissue homogenates
Pre-experiment Sample Processing
Serum collection: Collect rat blood in a serum separator tube (SST) and allow it to clot completely at room temperature for 2 hours or at 4 °C overnight. Following clotting, centrifuge the sample at 1000 × g for 15 minutes. Carefully remove the separated serum and proceed to immediate analysis. Alternatively, aliquot the serum and store it at −20 °C or −80 °C. Avoid repeated freeze-thaw cycles to preserve sample integrity.
Plasma collection: Use EDTA or heparin as the anticoagulant to collect plasma. Centrifuge the sample at 1000 × g for 15 minutes at 2–8°C within 30 minutes of collection. Following centrifugation, promptly transfer the plasma supernatant. The plasma can either be assayed immediately or aliquoted and stored at −20°C or −80°C. Repeated freeze-thaw cycles should be avoided to preserve sample integrity.
Cell Culture Supernatant collection: Clarify cell culture supernatant by centrifugation at 1000 × g for 15 minutes at 2–8°C. The resulting supernatant should be analyzed immediately or aliquoted and stored at −20°C or −80°C. Repeated freeze-thaw cycles should be avoided to ensure sample integrity.
Tissue Homogenate collection: Rinse approximately 100 mg of tissue with 1X PBS, then homogenize it in 1 mL of 1X PBS. Store the homogenate overnight at −20°C. Perform two freeze-thaw cycles to lyse the cells, then centrifuge the homogenate at 5000 × g for 5 minutes at 2–8°C. Collect the resulting supernatant for immediate analysis. Alternatively, aliquot and store the supernatant at −20°C or −80°C. If frozen samples are used, centrifuge them again after thawing and before analysis. Avoid repeated freeze-thaw cycles to maintain sample integrity.
Reagent Preparation
Bring all reagents to room temperature (18-25°C) for 30 min before use.
Use graduated containers to prepare the reagent.
Wash Buffer (1×): Dilute 15 ml of 20× concentrate to 300 ml with distilled water. Warm to dissolve crystals if present.
Use clean containers and distilled water to prevent contamination.
Assay Procedure
Preparation: Equilibrate reagents/samples to room temperature. Centrifuge thawed samples.
Plate Setup: Designate wells for standards (duplicate), samples, and one Blank well (no solution). Seal any unused wells with the desiccant in the original pouch and store at 4°C.
Loading:
Add 50 µl standard/sample per well. Standard needs test in duplicate.
Add 50 µl HRP-conjugate to all wells except Blank.
Add 50 µl Antibody to all wells.
Incubation: Mix gently, incubate 60 min at 37°C.
Washing: Perform three wash cycles. For each wash, add 200 µL Wash Buffer to each well, let stand for 10 seconds, and then completely remove all liquid. After the final wash, remove any residual buffer by aspiration or decanting, then invert and blot the plate on clean paper towels.
Substrate Reaction:
Add 50 µl Substrate A + 50 µl Substrate B to each well, mix well.
Incubate 15 min at 37°C in the dark.
Termination: Add 50 µl Stop Solution to each well, gently tap the plate to ensure thorough mixing.
Reading: Measure the optical density (OD) at 450 nm (with 600–630 nm correction) within 10 min using a microplate reader.
Result Analysis
Download the professional software "Curve Expert" from CUSABIO's Web to make a standard curve.
Average the duplicate readings for each standard and sample, and subtract the average OD of the blank.
Construct a standard curve by plotting standard concentration against corrected OD values. A four-parameter logistic (4PL) curve fit is recommended for best accuracy.
Determine the sample concentration from the curve. Multiply by the dilution factor if applicable.
Notes
Washing: Complete aspiration is critical. Insufficient washing causes a high background.
Timing: Add all reagents within 10 min to ensure consistent incubation.
Substrates: Protect from light; contamination causes aberrant results.
Color Development: Stop reaction early if color develops rapidly (green wells indicate incomplete mixing).
Dilution: Samples exceeding 25.6 ng/ml require dilution and re-assay.
Warnings
Intended Use: For research use only. Not approved for clinical diagnosis.
Expiration: Do not use the kit beyond the expiration date on the label.
Reagents: Do not mix or substitute reagents from other lots or sources.
Sample Limitations: Grossly hemolyzed samples are unsuitable. Samples generating values e the highest standard require dilution and re-assay.
Variability: Results can vary due to operator technique, pipetting, washing, incubation time/temperature, and kit age.
Interference: While designed to eliminate interference, the possibility cannot be entirely excluded until all factors are tested.
Stop Solution: Contains acid. Wear appropriate personal protective equipment (eye, hand, face, clothing).
Tissue/Cell Extracts: Chemical lysis buffers may cause unexpected results.
Antigen-Antibody: A potential mismatch exists between this antibody and antigens from other sources/manufacturers.
Cell Culture Supernates: Detectability depends on cell viability, number, and sampling time; may not be detected.
Sample Freshness: Fresh samples recommended; degradation can lead to erroneous results.
Ethical statements: This protocol involves the use of animal blood samples. Users must obtain prior approval from their Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee before performing this protocol. All procedures must comply with applicable institutional and governmental regulations regarding the ethical use of animals.
Troubleshooting
High Background/OD: Ensure thorough washing (complete aspiration essential). Check wash buffer preparation and dispenser function. Verify incubation times/temperatures. Protect Substrates from light/contamination.
Low Sensitivity/OD: Confirm reagent expiration. Check reagent preparation accuracy (use distilled water and clean containers). Verify incubation times/temperatures. Ensure proper storage of samples/reagents (avoid freeze-thaw). Check pipette calibration.
Poor Precision: Ensure consistent pipetting technique and tip changes between samples/reagents. Verify washing consistency and completeness. Control incubation conditions (time, temperature, plate sealing).
Standard Curve Issues: Confirm standard reconstitution/storage. Ensure accurate pipetting of standards. Check the microplate reader calibration.
Color Development Issues: Observe color change after adding Substrates; stop reaction early if color develops too rapidly. Ensure Stop Solution is added in the same order as Substrates and mixed thoroughly (green color indicates incomplete mixing).
Samples e Highest Standard: Dilute samples appropriately and repeat the assay.
Unexpected Results: Predict sample concentration to determine optimal dilution. Preliminary experiments are necessary for sample types not explicitly listed. Consider potential interference or sample degradation.