Human ABCD1 ELISA KIT Cat. No.:DEIA8716 Pkg.Size:96T Intended use The Human ABCD1 ELISA KIT is for the quantitative detection of human ABCD1 in serum and plasma. General Description ABCD1, also known as adrenoleukodystrophy protein (ALDP), is a member of the ATP-binding cassette (ABC) transporter superfamily. ALDP is a multi-pass membrane protein and localized at peroxisome membrane. Various mutations of ABCD1 gene cause X-linked adrenoleukodystrophy (X-ALD), an inherited neurodegenerative disease affecting the nervous system white matter and adrenal cortex. It results in mental deterioration, corticospinal tract dysfunction, and cortical blindness. ALDP is supposed to function as a homodimer allowing the entry of CoA-esters of very-long chain fatty acids into the peroxisome, the unique site of their β-oxidation. However, the defined function of ALDP has not been well investigated so far. Principle Of The Test This assay employs the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for ABCD1 has been pre-coated onto the wells of a microplate provided. Standards and samples are pipetted into the wells and the ABCD1 protein present in samples is captured by the coated antibody after incubation. After washing away any unbound substances, a monoclonal antibody specific for ABCD1 is added to the wells. After removal of excess antibody, a horseradish peroxidase (HRP)-conjugated anti-mouse antibody is added. After the third washing, the tetramethyl-benzidine (TMB) reagent is added to the wells and color develops in proportion to the amount of ABCD1 present in the original specimen. The color development is stopped and the intensity of the color is measured. Reagents And Materials Provided 1. Microplate: antibody coated 96-well microplate (8 wells x 12 strips), 1 plate 2. Standards: 2000 pg/vial, 2 vials 3. Detection antibody (100 ): 150 μl/vial, 1 vial 4. Anti-mouse IgG-Horseradish peroxidase (HRP) concentrate, (100 ): 150 μl/vial, 1 vial 5. Diluent Solution PT 1-a (1 ): 30 ml/bottle, 1 bottle 6. Diluent Solution PT 2 (1 ): 30 ml/bottle, 1 bottle 7. Washing Buffer Concentrate (20x): 30 ml/bottle, 1 bottle 8. Substrate, Tetramethylbenzidine (TMB): 12 ml/bottle, 1 bottle 9. Stop Solution: 12 ml/bottle, 1 bottle 10. Plate Covers, 3 pieces Do not use the kit after the expiration date. Note Dilution Solution PT 1-a is for Proteintech Standard and Sample; Dilution Solution PT 2 is for Detection antibody and Antimouse IgG-Horseradish peroxidase (HRP) concentrate. Materials Required But Not Supplied 1. Microtiter plate reader capable of measurement at or near 450 nm with the correction wavelength set at 630 nm. 1
2. Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.) 3. Distilled or deionized water. 4. Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.). 5. Data analysis and graphing software. Graph paper: linear (Cartesian), log-log, or semi-log, as desired. It is recommended to use the software which is capable of generating a four parameter logistic (4-PL) curve-fit. 6. Glass or plastic tubes for diluting and aliquoting standard. 7. Absorbent paper towels. 8. Calibrated beakers and graduated cylinders in various sizes. Storage Microplate and Standards can be store at -20 C for six months, but the other materials should be store at 2-8 C for six months. Specimen Collection And Handling 1. Serum: Allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Clear serum can be assayed immediately or aliquoted and stored at -20 C. Avoid repeated freeze-thaw cycles. 2. Plasma: Use EDTA or heparin as an anticoagulant to collect plasma. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. The plasma could be assayed immediately or aliquoted and stored at -20 C.Avoid repeated freeze-thaw cycles. Note: Do not use turbid or grossly hemolyzed samples. Mix thawed samples thoroughly before assaying. Caution: Handling of reagents, serum and plasma should be in accordance with local safety procedures. The serum or plasma samples may require proper dilution to fall within the range of the assay. A range of dilutions, like 1:2 or 1:4 is suggested according to the individual samples. Reagent Preparation Important Bring all reagents to room temperature prior to use. It normally takes 20-30 minutes for Microplate, Diluent solution PT 1-a, Diluent solution PT 2, Washing buffer, TMB and Stop solution to reach the room temperature. Standard, Detection antibody and HRP can be used immediately since little amount is needed. A. Diluent solution PT 1-a & Diluent solution PT 2 Allow the 1 Diluent solution PT 1-a and 1 Diluent solution PT 2 to reach room temperature. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Diluent solution PT 1-a is used to dilute Proteintech Standard and Sample. Diluent solution PT 2 is used to dilute Detection antibody and Anti-mouse IgG-HRP concentrate. B. Standards 1. Reconstitute the ABCD1 Standard (2000 pg) with 1 ml 1 Diluent solution PT 1-a, label it as 'sd7'. Do not substitute other diluent. This reconstitution gives a stock solution of 2000 pg/ml. Use this Standard within 30 minutes after reconstitution. (Note:Centrifuge the ABCD1 Standard briefly before reconstitution) 2. Make serial dilutions of standards as described in the following figure, i.e. Add 500 µl of standard made from last dilution into the tube containing 500 µl of 1 Diluent solution PT 1-a. Mix thoroughly between steps. This is a suggested serial dilution; further or less dilution could be applied depend on the individual case. 3. Set the Standard zero with 1 Diluent solution PT 1-a; label it as 'sd0'. Figure 1. 2
C. Detection Antibody The Detection Antibody solution should be diluted 100-fold in 1 Diluent solution PT 2 prior to assay. A suggested 100-fold dilution is 10 µl Detection Antibody + 990 µl 1 Diluent solution PT 2. The remaining diluted solution should be discarded. Note: The Detection Antibody (100 concentrate) MUST be stored at 4 C. Centrifuge the vial for seconds before use. Gently mix and slowly pipette the solution to ensure accurate dilution; Remove excess concentrate solution from the outer wall of pipette tips by wiping with clean absorbent tissues. D. Anti-mouse IgG-Horseradish Peroxidase (HRP) 1. Dilute Anti-mouse IgG-HRP (100 concentrate) solution to 1 Anti-mouse IgG-HRP with 1 Diluent solution PT 2 (see the table below). Label it as Anti-mouse IgG-HRP Working Solution. 2. Return the unused Anti-mouse IgG-HRP (100 concentrate) solution to the refrigerator (4 C) immediately. Table 1. Note: The Detection Antibody (100 concentrate) MUST be stored at 4 C. Centrifuge the vial for seconds before use. Gently mix and slowly pipette the solution to ensure accurate dilution; Remove excess concentrate solution from the outer wall of pipette tips by wiping with clean absorbent tissues. 3
E. Washing Buffer Allow the 20 Washing Buffer to reach room temperature before use. Dilute all 30ml 20 Washing Buffer with 570ml deionized or distilled water. If crystals are still in the concentrate, warm up to 37 C and mix gently until the crystals have completely dissolved. Label it as 1 Washing Buffer. Store it at 2-8 C, and use it within 15 days. Assay Steps Note: Please read this section before carrying out the assay. Bring all reagents and samples back to room temperature prior to use. Gently mix each reagent prior to use. It is recommended that all samples and Standards are assayed in duplicate. A standard curve must be run for each assay. 1. Prepare all reagents, samples, and working standards as instructed. 2. Take out the required number of microplate strips, and place the microwells in the strip holder. In the meantime, return the excess strips to the foil pouch containing the desiccant pack, and reseal; store them at 4 C immediately. Microplate strips should be used as soon as possible. 3. Add 100 µl of Standards and samples to the appropriate wells. Make sure sample addition is uninterrupted and completed within 5-10 minutes. 4. Seal with plate sealers and press firmly onto top of microwells. Incubate the plate for 1 hour at 37 C in a moisturized condition. 5. Wash wells: a. Gently remove the tape. b. Discard the liquid from wells by aspirating or decanting. c. Remove the residual solution by striking the plate hard enough on fresh towels at least 5 times, but do not allow wells to completely dry at any time. d. Wash 4 times with 1 Washing Buffer, at least 300 µl each time for each well. Remove the contents from wells by aspirating or decanting after each wash. In the last wash, strike plates on fresh towels hard enough at least 10 times to remove residual Washing Buffer. Avoid any towel fibers entering into the wells. 6. Add 100 µl of Detection Antibody to each well. Seal with plate sealers and incubate the plate for 1 hour at 37 C in a moisturized condition. 7. Repeat the wash as in Step 5. 8. Add 100 µl of Anti-mouse IgG-HRP antibody working solution to each well (including blank wells). Seal with plate sealers and incubate the plate for 40 minutes at 37 C in a moisturized condition. 9. Repeat the wash as in Step 5. 10. Develop color: Add 100 µl of TMB substrate solution to each well. Incubate for 20-30 minutes at room temperature, in the dark. For a positive reaction, the color should turn blue. The longer incubation or incubate at 37 C is suggested if the blue color appears very light. 11. Stop color development: Add 100 µl of Stop Solution to each well in the same order as adding the substrates. Mix gently by tapping side of the plate. The color of positive reactions in the wells should change from blue to yellow. 12. Read results: Read absorbance of each well at 450 nm by a plate reader. If wavelength correction is available, set to 630nm. If wavelength correction is not available, subtract readings at 630 nm from the readings at 450 nm.the absorbance should be read immediately after adding Stop solution. DO NOT exceed 5 minutes. Calculation Average the duplicate readings for each standard and sample and subtract the average zero standard absorbance. 4
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 ABCD1 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 the data. Note: If the samples have been diluted, the concentration read from the standard curve must be multiplied by the corresponding dilution factor. Typical Standard Curve These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed. Figure 2. Sensitivity The minimum detectable dose of human ABCD1 is 6 pg/ml. This was determined by adding two standard deviations to the concentration corresponding to the mean O.D. of 24 zero standard replicates. Linearity To assess the linearity of the assay, three samples were spiked with high concentrations of ABCD1 in human plasma and diluted with the Diluent solution PT 1-a to produce samples with values within the dynamic range of the assay. (The samples were 5
initially diluted 1:3) Table 2. Recovery The recovery of ABCD1 spiked to three different levels in four samples throughout the range of the assay in human plasma averaged 95%, ranging from 85%-118%. Reproducibility Intra-Assay (CV%): 3.4%-9.5% Inter-Assay (CV%): 4.0%-7.6% Limitations 1. Do not use the kit after the expiration date. 2. This product is for research use only and not for humans or animals therapeutic or diagnostic use. 3. Strictly follow the instructions; the reagents should not be mixed or substituted with those from other sources. 4. Samples may require further dilution if the values higher than the highest standard, dilute the samples with the appropriate diluent solution PT 1-a and be sure that their absorbance reading falls within those generated by standards. 5. Perform the assay in replicates as variations could be generated by the operator, pipetting technique, washing technique, incubation time or temperature. REFERENCES 1. Guimaraes C.P, et al. Characterisation of two mutations in the ABCD1 gene leading to low levels of normal ALDP. Hum. Genet. 109:616-622 (2001). 2. Wang Y., et al. X-linked adrenoleukodystrophy: ABCD1 de novo mutations and mosaicism. Mol. Genet. Metab. 104:160-166 (2011). 3. Genin EC., et al. Substrate specificity overlap and interaction between adrenoleukodystrophy protein (ALDP/ABCD1) and adrenoleukodystrophy-related protein (ALDRP/ABCD2). J Biol Chem. 286(10):8075-84 (2011). 6