cathepsin D (cath-D) ELISA Kit

Antigen: Cathepsin D (CTSD)

Synonyms: CPSD, CLN10, MGC2311, CD, CatD, catD, fb93e11, fj17b09, wu:fb93e11, wu:fj17b09, CTSD, LOC398557, Cathd, LOC100136761, DKFZp469J0315

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN367008

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of human cath-D concentrations in cell culture supernates, serum, plasma and other biological fluids.

Alternative name: cathepsin D (cath-D)

Description: Cathepsin D is peptidase belonging to the family of aspartic peptidases. Major function of cathepsin D is the digestion of proteins and peptides within the acidic compartment of lysosome1. This function is allowed by the catalytic mechanism given by two aspartate residues in the deep cleft of the active side. The common catalytic mechanism, together with low optimal pH for their catalytic function and high level of similarity of both primary and tertiary structures, are the reasons why cathepsin D belongs to the aspartic peptidase A1 family. It is widely accepted that the major function of cathepsin D is the intracellular catabolism within the lyso-somal compartment. Cathepsin D is apparently also involved in the processing of antigens, hormones, and neuropeptides. Of interest, a relatively high concentration of procathepsin D was found in human breast milk suggesting as yet unknown function. Tissue remodeling represents another involvement of cathepsin D in mammal physiology. Procathepsin D was also suggested to take part in programmed cell death – apoptosis. While majority of cathepsin D is found in soluble parts of most of human cells, about 20 % is apparently membrane bounded. It was shown that cathepsin D can serve as an independent prognostic factor in many types of cancers. A strong predictive value was found for cathepsin D concentrations in breast cancer as well as many other tumor types.

Specificity: This assay recognizes human cath-D. No significant cross-reactivity or interference was observed.

Sensitivity: The minimum detectable dose of human cath-D is typically less than 16.65 pg/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of human cath-D is typically less than 16.65 pg/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to cath-D. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP) -conjugated antibody preparation specific for cath-D and incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of cath-D in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well. 6 1. Set a Blank well without any solution. Add 50μl of Standard or Sample per well. Standard need test in duplicate. Incubate for 30mins at 37° C. 2. Aspirate each well and wash, repeating the process five times for a total of five washes. Wash by filling each well with Wash Buffer (200μl) using a squirt bottle, multi -channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 3. Add 50μl of HRP-conjugate to each well (not to Blank well). Mix well and then incubate for 20mins at 37°C. 4. Fill each well with Wash Buffer (about 200μl), stay for 10 seconds and Spinning. Repeat the process for a total of five washes. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 7 5. Add 50μl of Substrate A and Substrate B to each well, mix well. Incubate for 8-15 minutes at room temperature. Keeping the plate away from drafts and other temperature fluctuations in the dark. 6. Add 50μl of Stop Solution to each well. 7. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.
Calculation of Results:
Using the professional soft ",Curve Exert 1.3", to make a standard curve is recommended, which can be downloaded from our web. Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the cath-D concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of 8 the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Limitations of the Procedure:
The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources.If samples generate values higher than the highest standard, dilute the samples and repeat the assay.Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Sample collection and storage:
Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay.

Application Notes: Centrifuge vials before opening to collect contents. 9 When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless or light blue until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless or light blue to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Components: Reagent (Quantity): Assay plate (1), Standard 5x1ml HRP-conjugate (1x6ml), Substrate A (1x7ml), Substrate B (1x7ml), Stop Solution (1x7ml), Wash Buffer (20×concentrate) (1x15ml)

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer. An incubator which can provide stable incubation conditions up to 37°C±0.5°C.

Storage: 1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit, provided it is stored as prescribed above. Refer to the package label for the expiration date. 2. Opened test plate should be stored at 2-8 C in the aluminum foil bag with desiccants to minimize exposure to damp air. The kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Restrictions: For Research Use only