ADAM8 ELISA Kit

Catalog No. ABIN415444

Product Details ADAM8 ELISA Kit

Target: ADAM8 (ADAM Metallopeptidase Domain 8 (ADAM8))

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 78 pg/mL - 5000 pg/mL

Minimum Detection Limit: 78 pg/mL

Application: ELISA

Purpose: The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of ADAM8 in Serum,Plasma,Tissue Homogenate,Biological Fluids

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

Analytical Method: Quantitative

Specificity:

This assay has high sensitivity and excellent specificity for detection of A Disintegrin And Metalloprotease 8 (ADAM8).
No significant cross-reactivity or interference between A Disintegrin And Metalloprotease 8 (ADAM8) and analogues was observed.

Cross-Reactivity (Details): No significant cross-reactivity or interference between A Disintegrin And Metalloprotease 8 (ADAM8) and analogues was observed.

Sensitivity: 32 pg/mL

Components:

• Pre-coated, ready to use 96-well strip plate, flat buttom
• Plate sealer for 96 wells
• Reference Standard
• Standard Diluent
• Detection Reagent A
• Detection Reagent B
• Assay Diluent A
• Assay Diluent B
• Reagent Diluent (if Detection Reagent is lyophilized)
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

Application Details

Application Notes:

• Limited by the current condition and scientific technology, we cannot completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.
• The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
• Kits from different batches may be a little different in detection range, sensitivity and color developing time.
• Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
• Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism.
• There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed.
• Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment.
• Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
• Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
• Kits from different manufacturers for the same item might produce different results, since we have not compared our products with other manufacturers.

Comment:

Information on standard material:
The standard might be recombinant protein or natural protein, that will depend on the specific kit. Moreover, the expression system is E.coli or yeast or mammal cell. There is 0.05% proclin 300 in the standard as preservative.

Information on reagents:
The stop solution used in the kit is sulfuric acid with concentration of 1 mol/L. And the wash solution is TBS. The standard diluent contains 0.02 % sodium azide, assay diluent A and assay diluent B contain 0.01% sodium azide. Some kits can contain is BSA in them.

Information on antibodies:
The provided antibodies and their host vary in different kits.

Sample Volume: 100 μL

Assay Time: 3 h

Plate: Pre-coated

Protocol: The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to A Disintegrin And Metalloprotease 8 (ADAM8). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to A Disintegrin And Metalloprotease 8 (ADAM8). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain A Disintegrin And Metalloprotease 8 (ADAM8), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of A Disintegrin And Metalloprotease 8 (ADAM8) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reagent Preparation:

1. Bring all kit components and samples to room temperature (18-25 °C) before use. If the kit will not be used up in one time, please only take out strips and reagents for present experiment, and leave the remaining strips and reagents in required condition.
2. Standard - Reconstitute the Standard with 1.0 mL of Standard Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). The concentration of the standard in the stock solution is 5,000pg/mL. Prepare 7 tubes containing 0.5 mL Standard Diluent and produce a double dilution series. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 5,000pg/mL, 2,500pg/mL, 1,250pg/mL, 625pg/mL, 312pg/mL, 156pg/mL, 78pg/mL, and the last microcentrifuge tube with Standard Diluent is the blank as 0pg/mL.
3. Detection Reagent A and Detection Reagent B - If lyophilized reconstitute the Detection Reagent A with 150μL of Reagent Diluent, keep for 10 minutes at room temperature, shake gently (not to foam). Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute them to the working concentration 100-fold with Assay Diluent A and B, respectively.
4. Wash Solution - Dilute 20 mL of Wash Solution concentrate (30x) with 580 mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
5. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.

Note:

1. Making serial dilution in the wells directly is not permitted.
2. Prepare standards within 15 minutes before assay. Please do not dissolve the reagents at 37 °C directly.
3. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are 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 for one pipetting.
4. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
5. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
6. Contaminated water or container for reagent preparation will influence the detection result.

Assay Precision:

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level A Disintegrin And Metalloprotease 8 (ADAM8) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level A Disintegrin And Metalloprotease 8 (ADAM8) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Restrictions: For Research Use only

Independent Validation (ELISA)

Validation #101187 (ELISA)

by: Department of Neurosurgery, Philipps-University Marburg

No.: #101187

Date: 07/05/2017

Antigen: ADAM8

Lot Number: L170502272

Method validated: ELISA

Positive Control:

Adam8 wild type (wt) C57BL/6 mice serum (ABWT serum)

Adam8 wt bone marrow transplant C57BL/6 mice serum (ABWT BM)

Negative Control:

Adam8 gene mutation knock-out (ko) C57BL/6 mice serum (ABKO serum)

Blank samples is Reagent Diluent A only

Recombinant human ADAM8 diluted with Reagent Diluent A

Adam8 ko bone marrow transplant C57BL/6 mice serum (ABKO BM)

CX3CL1 WT murine thymus

Notes:

Passed. The Murine ADAM8 ELISA kit (ABIN415444) specifically detects Adam8 in mouse serum.

Validation Images

A. Layout of standards, controls and samples on the assay plate. B. Standard curve for ABIN415444, generated from the average absorbance measured at 450nm. C. Absorbance values as 450nm after subtraction of the average reading from two blank controls (see text for sample nomenclature). D. Calculated sample concentrations. E. Average measured Adam8 concentrations in the different samples. See the 'Experimental Notes' section for details.

Full Methods

Full Protocol:

• Reagent Preparation
• • Bring all kit components and samples to RT before use.
  • Reconstitute the standard with 1ml of standard diluent to a 1000pg/ml stock solution. Keep 10min at RT.
  • Shake gently to avoid foaming.
  • Use 7 tubes containing 0.5ml standard diluent to produce a 1:2 serial dilution series (Fig. A and B).
  • Dilute 6ml of assay Diluent A or B concentrate (2x) with 6ml deionized H₂O to prepare 12ml of Assay Diluent A or B.
  • Dilute Detection Reagent A and B 1:100 with working Assay Diluent A and B respectively.
  • Dilute 20ml of Wash Solution 30x 1:30 with 580ml deionized H₂O.
  • Pipette the necessary amount of the TMB substrate.
• Assay Procedures
• • Pipette 100ul of the standards, controls, and samples as indicated in Fig. A.
  • Seal the plate and incubate for 2h at 37^oC.
  • Carefully pipette the liquid out of the wells.
  • Wash the wells 3x with 350ul 1x Wash Solution using a multi-channel pipette; let the plate sit for 2min at RT.
  • Remove the remaining liquid by gently tapping the plate on blotting paper.
  • Pipette 100ul of Detection Reagent B working solution to each well.
  • Seal the plate and incubate for 30min at 37^oC.
  • Wash the wells 5x with 350ul 1x Wash Solution using a multi-channel pipette; let the plate sit for 2min at RT.
  • Remove the remaining liquid by gently tapping the plate on blotting paper.
  • Pipette 90ul of the TMB Substrate solution to each well; the liquid will turn blue.
  • Seal the plate and incubate for 10-20min at 37^oC protected from light.
  • Pipette 50ul of the Stop Solution to each well; the liquid will turn yellow. Gently tap the plate to mix the liquid and verify homogenous coloration of the liquid.
  • Verify that the bottom of the plate is clean and no bubbles are in the liquid. Then read the absorbance at 450nm in a filter-based multi-mode microplate reader (BMG LABTECH GmbH).

Experimental Notes:

• Both standards and some of the samples were prepared in duplicates. However because we have too many samples, for some of them only a single sample was measured (Fig. A).
• In Adam8 wt mice serum we measure on average approximately 235pg/ml Adam8 (Fig. E). In the Adam8 ko mice, the value is clearly lower with on average of 49pg/ml in the experimental background (unfortunately, the variation between both values is strong so that we get either a value of 12g/ml or 86pg/ml). We see a comparable concentration of 49pg/ml in the serum mice transplanted with bone marrow of Adam8 ko mice (A8KO BM). In contrast, the signal from Adam8 ko mice transplanted with bone marrow from wt mice (A8WT BM) is in the range of 83.75pg/ml, so significantly higher.
• Detection of human recombinant ADAM8 failed, indicating no cross-reactivity with human ADAM8.

Handling

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

Handling Advice: The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Storage: 4 °C

Storage Comment:

• For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4°C.
• For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
  Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
• For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C.

Expiry Date: 6 months

References for ADAM8 ELISA Kit (ABIN415444)

Schick, Babendreyer, Wozniak, Awan, Noels, Liehn, Bartsch, Vlacil, Grote, Zayat, Goetzenich, Ludwig, Dreymueller: "Elevated expression of the metalloproteinase ADAM8 associates with vascular diseases in mice and humans." in: Atherosclerosis, Vol. 286, pp. 163-171, (2019) (PubMed).

Target Details for ADAM8

Target: ADAM8 (ADAM Metallopeptidase Domain 8 (ADAM8))

Alternative Name: ADAM8 (ADAM8 Products)

Synonyms: CD156 ELISA Kit, MS2 ELISA Kit, CD156a ELISA Kit, E430039A18Rik ELISA Kit, adam8 ELISA Kit, reprolysin ELISA Kit, si:zc217g15.7 ELISA Kit, zgc:64059 ELISA Kit, ADAM8 ELISA Kit, RGD1566394 ELISA Kit, ADAM metallopeptidase domain 8 ELISA Kit, a disintegrin and metalloproteinase domain 8 ELISA Kit, a disintegrin and metallopeptidase domain 8 ELISA Kit, ADAM metallopeptidase domain 8a ELISA Kit, ADAM8 ELISA Kit, LOC100304737 ELISA Kit, Adam8 ELISA Kit, adam8a ELISA Kit, adam8 ELISA Kit

UniProt: Q05910

Pathways: Activation of Innate immune Response, M Phase