Ordering Information
- : ECM345
- : 1 kit
Human sCD26 ELISA Kit
Description:
Human sCD26 ELISA Kit
Trade Name:
Chemicon (Millipore)
Product Overview:
CD26, a T cell activation antigen, is a 110kDa cell surface glycoprotein with known dipeptidyl peptidase IV (DPPIV (Morimoto, C. et al., 1989); EC 3.4.14.5) activity in its extracellular domain, which is present on various cell types, including T cells and epithelial cells of the liver, kidney, and intestine (Hegen et al., 1990; Morimoto & Schlossman, 1994; Ulmer et al., 1990). Of the T cell antigens described to date, CD26 has proved to be one of the most intriguing in the scope of its functional associations. Considerable evidence suggests that CD26 can deliver a potent costimulatory signal to T-cells (Fleischer, 1987). This signal transducing property appears to be a property of its extracellular domain (Duke-Cohan et al., 1996). In addition, CD26 appears to be a functional collagen receptor (Dang et al., 1990) that may aid activated T-cells in localizing to inflammatory regions (Masuyama et al., 1992). It has also been demonstrated that CD26 not only acts as a functional dipeptidyl peptidase IV, but also binds strongly to adenosine deaminase (Dong et al., 1996).
Significant levels of DPPIV activity have been shown to exist in plasma, serum, and urine (Chikuma et al., 1990; Maes et al., 1994; Scharpe et al., 1988). The serum form of DPPIV is unique, with a high molecular weight of 175kDa, and is not a breakdown product of membrane CD26 (105-110kDa), nevertheless exposing significant structural similarity to the membrane form (Duke-Cohan et al., 1995, 1996).
Like many other T-cell molecules, CD26 is associated with HIV disease progression. There is a correlation of CD26 expression and HIV entry, replication and cytopathicity (Oravecz et al., 1995). CD26 has been identified as a key marker for monocytotropic HIV-1 infection, with a mechanism of early loss of CD26 - expressing cells in HIV-1 infected individuals described.
CD26 as an indicator of T-cell activation has been shown to fluctuate in parallel with several autoimmune diseases such as rheumatoid arthritis (Nakao et al., 1989) and autoimmune thyroiditis (Eguchi et al., 1989). CD26 has been described as a marker that correlates well with the level of activity of these diseases. It has furthermore been studied as an indicator of disease progression in chronic progressive multiple sclerosis (Constantinescu et al., 1995).
Test Principles:
An anti-sCD26 monoclonal coating antibody is adsorbed onto microwells.
Soluble CD26 (sCD26) present in the sample or standard binds to antibodies adsorbed to the microwells; a biotin?conjugated monoclonal anti-sCD26 antibody is added and binds to sCD26 captured by the first antibody.
Following incubation unbound biotin conjugated anti-sCD26 is removed during a wash step. Streptavidin-HRP is added and binds to the biotin-conjugated anti-sCD26. Following incubation unbound Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells.
A colored product is formed in proportion to the amount of sCD26 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from six sCD26 standard dilutions and sCD26 sample concentration determined.
Application:
The sCD26 ELISA is an enzyme-linked immunosorbent assay for quantitative detection of soluble human CD26 in cell culture supernatants, human serum, plasma or other body fluids. The sCD26 ELISA is for research use only. Not for use in diagnostic or therapeutic procedures.
Significant levels of DPPIV activity have been shown to exist in plasma, serum, and urine (Chikuma et al., 1990; Maes et al., 1994; Scharpe et al., 1988). The serum form of DPPIV is unique, with a high molecular weight of 175kDa, and is not a breakdown product of membrane CD26 (105-110kDa), nevertheless exposing significant structural similarity to the membrane form (Duke-Cohan et al., 1995, 1996).
Like many other T-cell molecules, CD26 is associated with HIV disease progression. There is a correlation of CD26 expression and HIV entry, replication and cytopathicity (Oravecz et al., 1995). CD26 has been identified as a key marker for monocytotropic HIV-1 infection, with a mechanism of early loss of CD26 - expressing cells in HIV-1 infected individuals described.
CD26 as an indicator of T-cell activation has been shown to fluctuate in parallel with several autoimmune diseases such as rheumatoid arthritis (Nakao et al., 1989) and autoimmune thyroiditis (Eguchi et al., 1989). CD26 has been described as a marker that correlates well with the level of activity of these diseases. It has furthermore been studied as an indicator of disease progression in chronic progressive multiple sclerosis (Constantinescu et al., 1995).
Test Principles:
An anti-sCD26 monoclonal coating antibody is adsorbed onto microwells.
Soluble CD26 (sCD26) present in the sample or standard binds to antibodies adsorbed to the microwells; a biotin?conjugated monoclonal anti-sCD26 antibody is added and binds to sCD26 captured by the first antibody.
Following incubation unbound biotin conjugated anti-sCD26 is removed during a wash step. Streptavidin-HRP is added and binds to the biotin-conjugated anti-sCD26. Following incubation unbound Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells.
A colored product is formed in proportion to the amount of sCD26 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from six sCD26 standard dilutions and sCD26 sample concentration determined.
Application:
The sCD26 ELISA is an enzyme-linked immunosorbent assay for quantitative detection of soluble human CD26 in cell culture supernatants, human serum, plasma or other body fluids. The sCD26 ELISA is for research use only. Not for use in diagnostic or therapeutic procedures.
Key Applications:
ELISA
Application Notes:
Abbreviated Procedure
1. Prepare reagents as described in Reagent Preparation.
2. Wash microwell strips twice with Wash Buffer
3. Add 100 μl Sample Diluent, in duplicate, to all standard wells except first (A1, A2)
4. Pipette 200 μl diluted sCD26 Standard into the first wells and create standard dilutions ranging from 500 to 15.6 ng/mL by transferring 100 μl from well to well. Discard 100 μl from the last wells
5. Add 100 μl Sample Diluent, in duplicate, to the blank wells
6. Add 80 μl Sample Diluent, in duplicate, to sample wells
7. Add 20 μl Sample, in duplicate, to designated wells
8. Prepare Biotin-Conjugate
9. Add 50 μl of diluted Biotin-Conjugate to all wells
10. Cover microwell strips and incubate 3 hours at room temperature (18° to 25°C)
11. Prepare Streptavidin-HRP solution
12. Empty and wash microwell strips 3 times with Wash Buffer
13. Add 100 μl of diluted Streptavidin-HRP to all wells
14. Cover microwell strips and incubate 1 hour at room temperature (18° to 25°C)
15. Prepare TMB Substrate Solution few minutes prior to use
16. Empty and wash microwell strips 3 times with Wash Buffer
17. Add 100 μl of mixed TMB Substrate Solution to all wells including blank wells
18. Incubate the microwell strips for about 10 minutes at room temperature (18°to 25°C).
19. Add 100 μl Stop Solution to all wells including blank wells
20. Blank microwell reader and measure colour intensity at 450 nm
21. Note: For samples which have been diluted 1:5, the concentration read from the standard curve must be multiplied by the dilution factor (x5). Calculation of samples with an O.D. exceeding 2.0 may result in incorrect, low sCD26 levels.
22. Such samples require further dilution with Sample Diluent in order to precisely quantitate the actual sCD26 level.
1. Prepare reagents as described in Reagent Preparation.
2. Wash microwell strips twice with Wash Buffer
3. Add 100 μl Sample Diluent, in duplicate, to all standard wells except first (A1, A2)
4. Pipette 200 μl diluted sCD26 Standard into the first wells and create standard dilutions ranging from 500 to 15.6 ng/mL by transferring 100 μl from well to well. Discard 100 μl from the last wells
5. Add 100 μl Sample Diluent, in duplicate, to the blank wells
6. Add 80 μl Sample Diluent, in duplicate, to sample wells
7. Add 20 μl Sample, in duplicate, to designated wells
8. Prepare Biotin-Conjugate
9. Add 50 μl of diluted Biotin-Conjugate to all wells
10. Cover microwell strips and incubate 3 hours at room temperature (18° to 25°C)
11. Prepare Streptavidin-HRP solution
12. Empty and wash microwell strips 3 times with Wash Buffer
13. Add 100 μl of diluted Streptavidin-HRP to all wells
14. Cover microwell strips and incubate 1 hour at room temperature (18° to 25°C)
15. Prepare TMB Substrate Solution few minutes prior to use
16. Empty and wash microwell strips 3 times with Wash Buffer
17. Add 100 μl of mixed TMB Substrate Solution to all wells including blank wells
18. Incubate the microwell strips for about 10 minutes at room temperature (18°to 25°C).
19. Add 100 μl Stop Solution to all wells including blank wells
20. Blank microwell reader and measure colour intensity at 450 nm
21. Note: For samples which have been diluted 1:5, the concentration read from the standard curve must be multiplied by the dilution factor (x5). Calculation of samples with an O.D. exceeding 2.0 may result in incorrect, low sCD26 levels.
22. Such samples require further dilution with Sample Diluent in order to precisely quantitate the actual sCD26 level.
Species Reactivity:
Human
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Kit or Assay Type:
ELISA Assays
Components:
- · 1 pouch with a Microwell plate coated with Monoclonal Antibody (murine) to human sCD26
- · 1 vial (100 μL) Biotin-Conjugate anti-sCD26 monoclonal (murine) antibody*
- · 1 vial (150 μL) Streptavidin-HRP*. Dilute 1:200 prior to use
- · 2 vials (0.050 mL) 5000 ng/mL sCD26 Standard concentrate*
- · 1 bottle (50 mL) Wash Buffer Concentrate 20x (PBS with 1% Tween 20)*
- · 1 vial (5 mL Assay Buffer Concentrate 20x (PBS with 1% Tween 20 and 10% BSA)*
- · 1 bottle (12 mL) Sample Diluent
- · 1 vial (7 mL) Substrate Solution I (tetramethyl-benzidine)
- · 1 vial (7 mL) Substrate Solution II (0.02 % buffered hydrogen peroxide)
- · 1 vial (12 mL) Stop Solution (1M Phosphoric Acid)
- · 4 adhesive Plate Covers
- · * reagents containing 0.01% thimerosal as preservative
Sensitivity:
The limit of detection of sCD26 defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus three standard deviations) was determined to be 11 ng/mL (mean of 6 independent assays).
Assay Specificity
The interference of circulating factors of the immune system was evaluated by spiking several of these proteins at physiologically relevant concentrations into a sCD26 positive serum. There was no detectable cross reactivity.
Typical Assay Values:
591 ng/mL ± 179 ng/mL.
Assay Specificity
The interference of circulating factors of the immune system was evaluated by spiking several of these proteins at physiologically relevant concentrations into a sCD26 positive serum. There was no detectable cross reactivity.
Typical Assay Values:
591 ng/mL ± 179 ng/mL.
Storage Conditions:
Store kit reagents between 2° and 8°C, as indicated. Immediately after use remaining reagents should be returned to cold storage (2° to 8°C or -20°C. Expiry of the kit and reagents is stated on labels.
Precautions:
- Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures.
- Do not mix or substitute reagents with those from other lots or other sources.
- Do not use kit reagents beyond expiration date on label.
- Do not expose kit reagents to strong light during storage or incubation.
- Do not pipette by mouth.
- Do not eat or smoke in areas where kit reagents or samples are handled.
- Avoid contact of skin or mucous membranes with kit reagents or specimens.
- Rubber or disposable latex gloves should be worn while handling kit reagents or specimens.
- Reagents containing thimerosal as preservative may be toxic if ingested.
- Avoid contact of substrate solutions with oxidizing agents and metal.
- Avoid splashing or generation of aerosols.
- In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test use disposable pipette tips and/or pipettes.
- Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagents.
- Exposure to acids will inactivate the conjugate.
- Glass-distilled water or deionized water must be used for reagent preparation.
- Substrate solutions must be at room temperature prior to use.
- Decontaminate and dispose specimens and all potentially contaminated materials as if they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121.5°C.
- Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0 % sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite.
Precautions:
- Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures.
- Do not mix or substitute reagents with those from other lots or other sources.
- Do not use kit reagents beyond expiration date on label.
- Do not expose kit reagents to strong light during storage or incubation.
- Do not pipette by mouth.
- Do not eat or smoke in areas where kit reagents or samples are handled.
- Avoid contact of skin or mucous membranes with kit reagents or specimens.
- Rubber or disposable latex gloves should be worn while handling kit reagents or specimens.
- Reagents containing thimerosal as preservative may be toxic if ingested.
- Avoid contact of substrate solutions with oxidizing agents and metal.
- Avoid splashing or generation of aerosols.
- In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test use disposable pipette tips and/or pipettes.
- Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagents.
- Exposure to acids will inactivate the conjugate.
- Glass-distilled water or deionized water must be used for reagent preparation.
- Substrate solutions must be at room temperature prior to use.
- Decontaminate and dispose specimens and all potentially contaminated materials as if they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121.5°C.
- Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0 % sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite.
Detection Methods:
Chromogenic
Gene Symbol:
- DPP4
- CD26
- ADCP2
- DPPIV
- ADABP
- TP103
- EC 3.4.14.5 [Contains: Dipeptidyl peptidase 4 membrane form (Dipeptidyl peptidase IV membrane form)
- Dipeptidyl peptidase 4 soluble form (Dipeptidyl peptidase IV soluble form)].
Materials Required but Not Delivered:
- 5 mL and 10 mL graduated pipettes
- 10 μL to 1,000 μL adjustable single channel micropipettes with disposable tips
- 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
- Multichannel micropipette reservoir
- Beakers, flasks, cylinders necessary for preparation of reagents
- Device for delivery of wash solution (multichannel wash bottle or automatic wash system)
- Microwell plate reader capable of reading at 450 nm (620 nm as optional reference wave length)
- Glass-distilled or deionized water
- Statistical calculator with program to perform linear regression analysis.
- 10 μL to 1,000 μL adjustable single channel micropipettes with disposable tips
- 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
- Multichannel micropipette reservoir
- Beakers, flasks, cylinders necessary for preparation of reagents
- Device for delivery of wash solution (multichannel wash bottle or automatic wash system)
- Microwell plate reader capable of reading at 450 nm (620 nm as optional reference wave length)
- Glass-distilled or deionized water
- Statistical calculator with program to perform linear regression analysis.