CD44 ELISA Kit

Catalog No. ABIN366268

Product Details CD44 ELISA Kit

Target: CD44

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 78-5000 pg/mL

Minimum Detection Limit: 78 pg/mL

Application: ELISA

Purpose: For the quantitative determination of human cluster of differentiation (CD44) concentrations in serum, plasma, cell culture supernates and tissue homogenates.

Sample Type: Serum, Plasma, Cell Culture Supernatant, Tissue Homogenate

Analytical Method: Quantitative

Specificity: This assay has high sensitivity and excellent specificity for detection of human CD44.

Cross-Reactivity (Details): Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between the target antigen and all analogues for other species. Therefore, cross reaction may still exist.

Sensitivity: 19.5 pg/mL

Components:

• Assay plate (12 × 8 coated Microwells)
• Standard (freeze dried)
• Biotin-antibody (100 × concentrate)
• HRP-avidin (100 × concentrate)
• Biotin-antibody Diluent
• HRP-avidin Diluent
• Sample Diluent
• Wash Buffer (25 × concentrate)
• TMB Substrate
• Stop Solution
• Adhesive Strip (for 96 wells)
• Instruction manual

Application Details

Application Notes:

• The supplier is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
• Samples to be used within 5 days may be stored at 2-8°C, otherwise samples must be stored at -20°C (≤ 1 month) or -80°C (≤ 2 months) to avoid loss of bioactivity and contamination.
• Grossly hemolyzed samples are not suitable for use in this assay.
• If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
• Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
• Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts of certain chemicals.
• Owing to the possibility of mismatching between antigens from another resource and antibodies used in this supplier's kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by this supplier's products.
• Influenced by factors including cell viability, cell number and cell sampling time, samples from cell culture supernatant may not be recognized by the kit.
• Fresh samples without long time storage are recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.

Comment:

Detection wavelength: 450 nm

Information on standard material:
Depending on the antigen to be detected, standards can be either native or recombinant protein. The recombinant proteins are being expressed in CHO cells in most cases. Please inquire for more information. The formulation of auxiliary material in the standard is considered proprietary information, however it does not contain any poisonous substance. Proclin 300 (1:3000) is used as preservative.

Information on reagents:
In most cases the stop solution provided is 1 N H2SO4. The formulation of wash solution is proprietary information. None of the components contain (sodium) azide, thimerosal, 2-mercaptoethanol (2-ME) or any other poisonous materials. For the sandwich method kits, the sample diluent, antibody diluent, enzyme diluent and standard all contain BSA.

Information on antibodies:
The antibodies provided in different kits vary in regards to clonality and host. Some antibodies are affinity purified, some are Protein A

Sample Volume: 100 μL

Assay Time: 1 - 4.5 h

Plate: Pre-coated

Protocol: This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for CD44 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any CD44 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for CD44 is added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of CD44 bound in the initial step. The color development is stopped and the intensity of the color is measured.

Reagent Preparation:

• Biotin-antibody (1×) - Centrifuge the vial before opening.
  Biotin-antibody requires a 100-fold dilution. The suggested dilution is 10µL of Biotin-antibody + 990µL of Biotin-antibody Diluent.
• HRP-avidin (1×) - Centrifuge the vial before opening.
  HRP-avidin requires a 100-fold dilution. The suggested dilution is 10µL of HRP-avidin + 990µL of HRP-avidin Diluent.
• Wash Buffer (1×) - If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20mL of Wash Buffer Concentrate (25×) into deionized or distilled water to prepare 500mL of Wash Buffer (1×).
• Standard - Centrifuge the standard vial at 6000-10000rpm for 30s.
  Reconstitute the Standard with 1ml of Sample Diluent. Do not substitute other diluents. This reconstitution produces a stock solution. Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
  Pipette 250µL of Sample Diluent into each tube. Use the stock solution to produce a 2-fold dilution series. Mix each tube thoroughly before the next transfer. The undiluted Standard serves as the high standard. Sample Diluent serves as the zero standard (0ng/mL).

Note:

• Kindly use graduated containers to prepare the reagent. Please don't prepare the reagent directly in the Diluent vials provided in the kit.
• Bring all reagents to room temperature (18-25°C) before use for 30 min.
• Prepare fresh standard for each assay. Use within 4 hours and discard after use.
• Making serial dilution in the wells directly is not permitted.
• Please carefully reconstitute Standards according to the instruction. Avoid foaming and mix gently until the crystals have completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10µL when pipetting.
• It is recommended to use distilled water to prepare reagents and samples. Using contaminated water or container for reagent preparation will influence detection result.

Assay Precision: Intra-assay precision (precision within an assay): Three samples of known concentration were tested twenty times on one plate to assess precision.
Inter-assay precision (precision between assays): Three samples of known concentration were tested in twenty assays to assess precision.

• Intra-assay: CV% less than 8%
• Inter-assay: CV% less than 10%

Restrictions: For Research Use only

Independent Validation (ELISA)

Validation #029852 (ELISA)

by: Affina Biotechnologies, Inc

No.: #029852

Date: 04/07/2016

Antigen: Human CD44 (cluster Of differentiation)

Lot Number: V15182623

Method validated: ELISA

Positive Control: Human pooled serum (Biochemed, 750-NS-FI-POM, Lot#BC033016HSPMG)

Negative Control: Chicken plasma (Biochemed, Lot#BC03316CSPMG)

Notes: CD44 was clearly detected in the positive sample serum. While calculated values for the chicken serum are high the reading of the chicken serum in this ELISA could not be distinguished from the blank and thus are actually very low. Spike recovery was >100% indicating that chicken serum did not interfere with the assay at 50-fold dilution.

Validation Images

Figure 1: Graph of corrected-average absorbance (OD 450 nm) readings plotted for standard curve samples.

Figure 2: Table of absorbance readings (OD 450 nm) for standard curve, spike controls and unknown control samples. Value for Average Reading is derived from the average of two readings (OD 450nm). The Average Reading for blank sample (no conjugate added) was subtracted from all Average Readings to yield Average Absorbance values for Standards, spike controls and control samples. Standard deviation is included for all samples. The values were generated from the logistic fit to the results (CurveExpert 1.4) for the standard curve and used to calculate CD44 concentrations shown in the Table.

Full Methods

Full Protocol:

• 1. 100 μL of standard and samples were added 96-well strip plates provided in the kit. All samples and standards were assayed in duplicate.
• 2. Removed liquid from wells without washing
• 3. 100 μl /well of Biotin-antibody conjugate added to each well and incubated at 37°C for 1 hr. Washed 3 times (200 μL/well, 2min soak),
• 4. 100 μL of HRP conjugate was added and contents in the wells were mixed.
• 5. The microplate was covered and incubated at 37°C for 1 hr.
• 6. Plate contents were discarded and wells were washed 5 times with 200 μl of 1x wash solution.
• 7. 90 μl of TMB susbtrate was added to each well. The plate was covered and incubated at 37°C for 10 min.
• 8. 50 μl of the Stop Solution was added per well.
• 9. The optical density (OD value) of each well was read immediately using a microplate reader set to 450 nm.
• 10. The duplicate readings for each sample were averaged and the average zero standard optical density subtracted. The corrected average-value was tabulated as Average Absorbance. A standard curve was generated by plotting the mean OD value for each standard on the x-axis against the concentration on the Y-axis using CurveExpert 1.4. A logistic fit through the points on the graph was used to calculate concentrations.
• 11. The Analyze feature of CurveFit expert was used to calculate CD44 concentrations of the samples based on their Average Absorbance values.

Experimental Notes: - The concentration of CD44 in human and chicken plasmas was measured according to the manufacturers directions.

Handling

Precaution of Use: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.

Handling Advice:

• 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 with Sample Diluent and repeat the assay.
• Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time/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.

Storage: 4 °C/-20 °C

Storage Comment: For unopened kit: All the reagents should be kept according to the labels on vials.

Expiry Date: 6 months

References for CD44 ELISA Kit (ABIN366268)

Scuruchi, DAscola, Avenoso, Zappone, Mandraffino, Campo, Campo: "miR9 inhibits 6-mer HA-induced cytokine production and apoptosis in human chondrocytes by reducing NF-kB activation." in: Archives of biochemistry and biophysics, Vol. 718, pp. 109139, (2022) (PubMed).

Avenoso, DAscola, Scuruchi, Mandraffino, Campo, Campo: "miR146a up-regulation is involved in small HA oligosaccharides-induced pro-inflammatory response in human chondrocytes." in: Biochimica et biophysica acta. General subjects, pp. 129731, (2020) (PubMed).

Target Details for CD44

Target: CD44

Alternative Name: Cluster Of differentiation (CD44) (CD44 Products)

Synonyms: CDW44 ELISA Kit, CSPG8 ELISA Kit, ECMR-III ELISA Kit, HCELL ELISA Kit, HUTCH-I ELISA Kit, IN ELISA Kit, LHR ELISA Kit, MC56 ELISA Kit, MDU2 ELISA Kit, MDU3 ELISA Kit, MIC4 ELISA Kit, Pgp1 ELISA Kit, AU023126 ELISA Kit, AW121933 ELISA Kit, AW146109 ELISA Kit, HERMES ELISA Kit, Ly-24 ELISA Kit, Pgp-1 ELISA Kit, CD44A ELISA Kit, METAA ELISA Kit, RHAMM ELISA Kit, XCD44 ELISA Kit, cd44 ELISA Kit, cdw44 ELISA Kit, cspg8 ELISA Kit, ecmr-iii ELISA Kit, hcell ELISA Kit, lhr ELISA Kit, mc56 ELISA Kit, mdu2 ELISA Kit, mdu3 ELISA Kit, mic4 ELISA Kit, mutch-i ELISA Kit, pgp1 ELISA Kit, CD44 molecule (Indian blood group) ELISA Kit, syndecan 4 ELISA Kit, CD44 antigen ELISA Kit, CD44 molecule (Indian blood group) S homeolog ELISA Kit, CD44 ELISA Kit, SDC4 ELISA Kit, Cd44 ELISA Kit, cd44.S ELISA Kit

Background: Synonyms: CDW44, CSPG8, ECMR-III, HCELL, IN, LHR, MC56, MDU2, MDU3, MGC10468, MIC4, MUTCH-I, Pgp1, CD44 antigen|CD44 antigen (homing function and Indian blood group system)|CDW44 antigen|GP90 lymphocyte homin

HGNC: 1681

UniProt: P16070

Pathways: Glycosaminoglycan Metabolic Process, Autophagy, Negative Regulation of intrinsic apoptotic Signaling