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Human cd36 elisa 196-02

HUMAN SOLUBLE CD36 ELISA KIT PAGE
HUMAN SOLUBLE CD36
ELISA KIT
PRODUCT INFORMATION:

ELISA NAME
HUMAN SOLUBLE CD36 ELISA
FOR THE QUANTITATIVE DETERMINATION OF
HUMAN SOLUBLE CD36 CONCENTRATIONS IN
PLASMA AND CELL CULTURE SUPERNATES
1.95 - 250 ng/mL 8-16 for plasma samples (Optimal dilutions should be
determined by each
laboratory for each
application)
EDTA Plasma, Cell Culture Human sCD36 Recombinant This kit contains sufficient materials to run 35 samples duplicated provided that assay is run according to protocol. Order Contact:
AVISCERA BIOSCIENCE, INC.
2348 Walsh Ave., Suite C
ALWAYS REFER TO LOT SPECIFIC PROTCOL
Santa Clara, CA 95051
PROVIDED WITH EACH KIT FOR
INSTRUCTIONS. PROTOCOL MUST BE
Tel: (408) 982 0300
READ BEFORE USING THIS PRODUCT.
Fax: (408) 982 0301
Email: [email protected]
FOR RESEARCH USE ONLY.NOT FOR USE IN
[email protected]
DIAGNOSTIC PROCEDURES.
HUMAN SOLUBLE CD36 ELISA KI
HUMAN SOLUBLE CD36 ELISA KIT PAGE
DESCRIPTION
MATERIALS PROVIDED
This Human sCD36 ELISA Kit contains the necessary DESCRIPTION
QUANTITY
components required for the quantitative measurement of recombinant and/or natural human sCD36 Microplate - 96 well 196-02-01
sCD36 from cell culture supernates and plasma in a polystyrene microplate (12 strips of 8 wells) coated with sandwich ELISA format. an antibody against sCD36. sCD36 Standard – 500
This immunoassay contains recombinant human 196-02-02
ng/vial of recombinant sCD36 and antibodies raised against this protein. sCD36 in a buffered protein Results from this immunoassay have shown to base with preservative; accurately quantify recombinant and natural sCD36 Detection Antibody
196-02-03
Concentrate – 1.05 mL/vial,
ASSAY OVERVIEW
10-fold concentrate of This assay employs the quantitative sandwich ELISA biotinylated antibody against format. The plate is pre-coated with an antibody sCD36 with preservative; lyophilized. specific for human sCD36. The capture antibody can Positive Control – one vial
bind to the human sCD36 in the standard and 196-02-04
of recombinant human samples. After washing the plate of any unbound sCD36; lyophilized. substances, a biotinylated antibody against human sCD36 is added to the wells. After another washing Conjugate – 60 L/vial,
of the plate, Streptavidin-HRP Conjugate is added. 200-fold concentrated After the last wash to remove any unbound enzyme, solution of Streptavidin-HRP a substrate solution (TMB) is added to the wells and color develops in direct proportion to the amount of Dilution Buffer – 60 mL of
human sCD36 bound in the standard solutions or buffered protein based samples. A standard curve can be established and solution with preservative. sample values can be read off the standard curve. HRP Diluent Solution - 12
mL of buffered protein based PROCEDURAL LIMITATIONS
solution with preservative. _FOR RESEARCH USE ONLY. NOT FOR USE IN Wash Buffer - 50 mL of 10-
DIAGNOSTIC PROCEDURES. fold concentrated buffered surfactant, with preservative. _This ELISA kit should not be used beyond the TMB Substrate Solution -
expiration date on the kit label. 11 mL of TMB Substrate _Do not mix reagents with those from other lots or Stop Solution – 11 mL of
_It is important that the Dilution Buffer selected for the standard curve be consistent with the samples Plate Sealer
_Each laboratory must determine the optimal Plastic Pouch
dilution factors for the samples being assayed with a pretest. If samples generate values that are not within the dynamic range of the standard curve, further concentrate or dilute the samples as Unopened Kit: Store at 2 – 8° C for up to 8 months.
required with Dilution Buffer and repeat the assay. For longer storage, unopened Standard, Positive _Any modifications in buffers, pipetting technique, Control and Detection Antibody Concentrate should washing technique, incubation time or temperature, be stored at -20° C or -70° C. Do not use kit past as well as kit age can cause a change in signal. expiration date. _Not all interfering factors have been tested in the Opened / Reconstituted Reagents: Reconstituted
immunoassay, therefore the possibility of Standard (stock) solution and Detection Antibody interference cannot be excluded. concentrated solution SHOULD BE STORED at -20° C or -70° C for up to one month. Streptavidin-HRP HUMAN SOLUBLE CD36 ELISA KI
HUMAN SOLUBLE CD36 ELISA KIT PAGE
Conjugate 200-fold concentrated solution and TMB Optional: Use Aprotinin (enzyme inhibitor) for ALL
Substrate Solution can be stored at 2 – 8° C for up to sample collection to prevent sample degradation.
8 months (DO NOT FREEZE and PROTECT FROM
0.5 TIU per ml of sample solution.
LIGHT). All other components may be stored at 2 –
8° C for up to 8 months. SAMPLE PREPARATION
Microplate Wells: Return unused strips to the plastic
Plasma samples may require 8 16 fold dilution. A pouch with the desiccant pack. Microplate may be suggested 8-fold dilution is 40 L sample + 280 L stored for up to 6 months at 2 – 8° C after opening. Dilution Buffer. A suggested 16-fold dilution is 20 L sample + 300 L Dilution Buffer. ADDITIONAL MATERIALS REQUIRED
Serum samples may require 32 64 fold dilution. A  Microplate reader capable of absorbance suggested 32-fold dilution is 10 L sample + 310 L measurement at 450 nm. Dilution Buffer. Then, to make a final dilution of 64-  Microplate shaker (250 – 300 rpm). fold is 150 L 32-fold diluted sample + 150 L  Microplate washer or manifold dispenser. Dilution Buffer.  100 mL and 500 mL graduated cylinders. Optimal dilutions should be determined by each
 Multi-channel Pipette, Pipettes and pipette tips. laboratory for each application with a pretest.
 Deionized or distilled water. Use polypropylene test tubes.
PRECAUTIO
REAGENT PREPARATION
This kit should be handled by those persons who Bring all reagents to room temperature before use.
have been trained in and can follow the principles of good laboratory practice. Wear protective clothing Wash Buffer - If crystals have formed in the
such as laboratory overalls, safety glasses and concentrate, warm to room temperature and mix gloves. Care should be taken while handling gently until the crystals have completely dissolved. solutions in this kit to avoid contact with skin or Dilute 50 mL of Wash Buffer Concentrate into eyes, especially with the stop solution because it deionized or distilled water (450 mL) to prepare 500 contains diluted hydrochloric acid. Wash mL of 1x Wash Buffer. immediately with water in case of contact on skin or Dilution Buffer (DB09) - If Dilution Buffer is highly
viscous, warm in 27 - 30° C water bath until liquid SAMPLE COLLECTION AND STORAGE
flows more freely. Cell Culture Supernates - Remove particulates by
centrifugation and assay immediately or aliquot and sCD36 Standard - Reconstitute the sCD36 standard
store samples at ≤ -20° C. Avoid repeated freeze- with 1.0 mL of Dilution Buffer. This reconstitution produces a stock solution of 500 ng/mL. Allow the Plasma - Collect plasma using EDTA, heparin, or
standard to sit for a minimum of 15 minutes with citrate as an anticoagulant. Centrifuge for gentle agitation prior to making dilutions. Pipette 15 minutes at 1000 x g within 30 minutes of 250 L of Dilution Buffer into tubes #1 to #8. Use the collection. Assay immediately or aliquot and store stock solution to produce a dilution series (below). samples at ≤ -20° C. Avoid repeated freeze-thaw Mix each tube thoroughly before the next transfer. The 250 ng/mL standard serves as the high standard.
Serum - Use a serum separator tube (SST) and allow
The Dilution Buffer serves as the zero standard (0 samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20° C.
Avoid repeated freeze-thaw cycles.
Note: CD36 was expressed in platelets. Activation of
platelets may increase sCD36 release. Serum samples
may have high levels of sCD36.

HUMAN SOLUBLE CD36 ELISA KI
HUMAN SOLUBLE CD36 ELISA KIT PAGE
STANDARD
DILUTION
1. Prepare all reagents and working standards as directed in the previous sections. 2. Remove excess microplate strips from the plate 250l of stock frame, return them to the plastic pouch (P01) with the desiccant pack. 3. Add 100L per well of Dilution Buffer to Blank 4. Add 100 L of standard dilutions in reverse order of serial dilution, samples, or positive control per well. Cover with plate sealer. Incubate for 2 hours on microplate shaker at room temperature. 5. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with 1x Wash Buffer (300L) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 6. Add 100L of Detection Antibody working solution to each well. Cover with plate sealer. Incubate for Positive Control - Reconstitute the Positive Control
2 hours on microplate shaker at room temperature. with 1.0 mL of Dilution Buffer. Note: Positive
7. Repeat the aspiration/wash as in step 5. Control solution could be reused within a few days if 8. Add 100 L of Streptavidin-HRP Conjugate stored at -20° C or -70° C. working solution to each well. Incubate for 1 hour on microplate shaker at room temperature. Detection Antibody Concentrate – Reconstitute the
Protect from light.
Detection Antibody Concentrate with 1.05 mL of 9. Repeat the aspiration/wash as in step 5. Dilution Buffer to prepare a 10-fold concentrated 10. Add 100 L of Substrate Solution to each well. solution. Pipette 9.45 mL of Dilution Buffer into a 15 Incubate 1-10 minutes on microplate shaker at mL centrifuge tube and transfer the 1.05 mL of 10- room temperature. Protect from light.
fold concentrated solution to the tube to make 1x 11. Add 100L of Stop Solution to each well. The color in the wells should change from blue to yellow. If working solution. the color in the wells is green, or if the color change does not appear uniform, gently tap the Streptavidin-HRP Conjugate - Pipette 11.94 mL of
plate to ensure thorough mixing. HRP Diluent Solution (DB08) into a 15 mL centrifuge
12. Determine the optical density of each well within tube and transfer 60 L of 200-fold concentrated 15 minutes, using a microplate reader set to stock solution to prepare working solution. Note: 1x
working solution of Streptavidin-HRP Conjugate should be used within a few days (protect from
light).
ELISA PROTOCOL
Bring all reagents and samples to room
temperature before the start of the assay. Blank,
standard dilutions, positive control and samples
should be assayed in duplicate. ELISA Protocol may
need further optimization.
HUMAN SOLUBLE CD36 ELISA KI
HUMAN SOLUBLE CD36 ELISA KIT PAGE
CALCULATION OF RESULTS
SPECIFICITY
Average the duplicate readings for each standard, PROTEIN NAME
positive control and sample, and subtract the Human CD36 ECD/Fc (Sf21 average zero standard optical density. Create a standard curve by reducing the data using computer Human CD36 ECD, His Tag software capable of generating a log-log curve fit. As Human CD320, ECD 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 sCD36 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. LINEARITY
To assess the linearity of the assay, pooled human If samples have been diluted, the concentration read EDTA plasma samples were diluted with Dilution from the standard curve must be multiplied by the Buffer (DB09) and assayed. dilution factor. DILUTION
RECOVERY (%)
Calculation of samples with a concentration exceeding that of standard 250 ng/ml may result in inaccurate, low human sCD36 levels. Such samples require further external pre-dilution according to expected human sCD36 values with Dilution Buffer in order to precisely quantify the actual human To assess the linearity of the assay, pooled human serum samples were diluted with Dilution Buffer TYPICAL DATA
(DB09) and assayed. This standard curve is provided for demonstration only. A new standard curve should be generated for DILUTION
RECOVERY (%)
each set of samples assayed. STANDARD (NG/ML)
CORRECTED
Positive Control: HUMAN SOLUBLE CD36 ELISA KI
HUMAN SOLUBLE CD36 ELISA KIT PAGE
REFERENCES
SUMMARY OF ASSAY PROCEDURE
PREPARE REAGENTS, SAMPLES AND STANDARDS
1. Masson CJ, et al. Fatty acid- and cholesterol transporter protein expression along the Add 100 l of standard dilutions, samples, or positive human intestinal tra Apr control to each well. Incubate 2 hours on the plate 2. Marecki JC, et al. Hyperinsulinemia and ectopic fat deposition can develop in the Aspirate and wash 4 times. face of hyperadiponectinemia in young obese rats. J Nutr Biochem. 2010 Apr 30. Add 100 l Detection Antibody working solution to [Epub ahead of print] each well. Incubate 2 hours on the plate shaker at 3. Bell JA, et al. Lipid partitioning, incomplete fatty acid oxidation, and insulin signal transduction in primary human muscle cells: Aspirate and wash 4 times. effects of severe obesity, fatty acid incubation, and fatty acid translocase/CD36 Add 100 l Streptavidin-HRP conjugate working overexpression. J Clin Endocrinol Metab. solution to each well. Incubate 1 hour on plate 2010 Jul;95(7):3400-10. Epub 2010 Apr 28. shaker at RT. Protect from light.
4. Sandoval JC, et al. Fenofibrate reduces postprandial hypertriglyceridemia in CD36 Aspirate and wash 4 times. knockout mice. J Atheroscler Thromb. 2010 Jun 30;17(6):610-8. Epub 2010 Mar 30. 5. Abe T, et al. Key role of CD36 in Toll-like Add 100 l Substrate Solution to each well. Incubate receptor 2 signaling in cerebral ischemia. 1-10 min on plate shaker at RT. Protect from light.
Stroke. 2010 May;41(5):898-904. Epub 2010 Add 100 l Stop Solution to each well. Read at 450 6. Steinbusch LK, et al. Differential regulation nm within 15 min. of cardiac glucose and fatty acid uptake by endosomal pH and actin filaments. Am J Physiol Cell Physiol. 2010 Jun;298(6):C1549-59. Epub 2010 Apr 7. HUMAN SOLUBLE CD36 ELISA KI

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