Osteomodulin ELISA Kit

Catalog No. ABIN4884045

Product Details Osteomodulin ELISA Kit

Target: Osteomodulin (OMD)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: Custom Human Osteoadherin (Osteomodulin) ELISA Kit.

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

Analytical Method: Quantitative

Specificity: This ELISA antibody pair recognizes human Osteoadherin. Other species not yet determined.

Characteristics:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Components:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Material not included:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Restrictions: For Research Use only

Independent Validation (ELISA)

Validation #103081 (ELISA)

by: Bone and Cartilage Research Unit, University of Liège

No.: #103081

Date: 10/04/2018

Antigen: Osteomodulin

Lot Number: 070218 2424

Method validated: ELISA

Positive Control:

ELISA using diluent A recommended for serum:

Recombinant full length Osteomodulin (R&D systems, 2884-AD-050, lot NFV031506A) at the same concentration as the standard curve

Two human serum samples in duplicate, diluted 1:2, 1:4, 1:8, and 1:16 (suggested 1:2)

ELISA using diluent C recommended for culture supernatant:

Four supernatants of osteoblasts culture, in duplicate, diluted 1:1, 1:3, and 1:9

Negative Control:

Sample diluent

one guinea pig serum sample in duplicate, diluted 1:2, 1:4, 1:8, and 1:16 (cross-reactivity unknown)

Notes:

ABIN4884045 recognizes human osteomodulin in culture supernatant and recombinant human osteomodulin but not in human serum.

Validation Images

A. Standard curve (std), human (serum) and guinea pig (guinea pig) serum samples, recombinant Osteomodulin (control), and spike control (spike) measurements using diluent A. B. The standard curve for diluent A was fitted with a 4-Parameter Logistic model (R2=0.975). C. Standard curve (std), human osteoblast serum free supernatant (T0: pre-osteoblasts, T2: mature osteoblasts) measurements using diluent C. D. Standard curve for diluent C fitted with a 4-Parameter Logistic model (R2=0.999).

Full Methods

Full Protocol:

• Reagent preparation and the assay are carried out according to the manufacturer’s instructions.
• For the spike control prepare a 40ng/ml solution of standard protein using diluent A. Dilute standard twofold in undiluted, 1:2, 1:4 and 1:8 diluted serum to achieve a final serum dilution 1:2, 1:4, 1:8, and 1:16 in 100µl total volume using diluent A.
• Add 100μl of the standard or the sample to each well.
• Incubate for 2.5h at RT shaking at 500rpm.
• Aspirate the solution in the wells and wash wells 4x with 350µl wash buffer.
• Add 100μl of the biotinylated detection antibody to each well and incubate for 1h at RT shaking at 500rpm.
• Aspirate the solution in the wells and wash wells 4x with 350µl wash buffer.
• Add 100μl of the HRP conjugate to each well and incubate for 45min at RT shaking at 500rpm.
• Aspirate the solution in the wells and wash wells 4x with 350µl wash buffer.
• Add 100µl of the substrate reagent in dim light and incubate plate for 30min at RT protected from light.
• Add 50µl of the stop solution to each well in the same order the substrate reagent was added.
• Read OD₄₅₀ immediately using a Multiskan GO (Thermofisher Scientific).

Experimental Notes:

• The standard curves in the different diluents don’t look like the figure in the kit insert: OD₄₅₀ were lower, but are good, with good duplicate reproducibility (A).

• The curve with recombinant full length Osteomodulin from R&D Systems gives higher ODs than the standard protein from the kit.

• ABIN4884045 allows detection of Osteomodulin in culture supernatants, in undiluted wells with values around 3-12ng/ml. This concentration appears very low but possible.

• Serum values remain below the blank values and are not detected by the detecting antibody as confirmed with western blot, contrarily to standard protein, recombinant protein and culture supernatant (C).

• The ABIN4884045 detection antibody recognizes osteomodulin from the kit standard and human recombinant osteomodulin which in a western blot. It also detects osteomodulin from human osteoblast culture supernatant but the signal is weak. However, in serum there is no signal after immunodetection although we can see that there is the correct transfer of total protein on the blot. The same western blot procedure with a different commercial antibody (R&D systems, BAF2884) gives good results in these human serum samples (Sanchez C (2018)).

• Furthermore, a substantial matrix effect of serum makes the spiking of 20ng/ml recombinant Osteomodulin undetectable, even in 1:16 serum dilution with diluent A before adding the Osteomodulin. Recovery of Osteomodulin in serum samples is 0% even at a 1:16 dilution.

Handling

Storage: -20 °C

Storage Comment: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Expiry Date: 6 months

Target Details for Osteomodulin

Target: Osteomodulin (OMD)

Alternative Name: Osteoadherin (OMD Products)

Synonyms: si:ch211-103f16.5 ELISA Kit, OMD ELISA Kit, OSAD ELISA Kit, SLRR2C ELISA Kit, osteomodulin ELISA Kit, omd ELISA Kit, OMD ELISA Kit, Omd ELISA Kit

Gene ID: 4958

UniProt: Q99983

Pathways: Glycosaminoglycan Metabolic Process