Download biomedica product listMDA-oxLDL ELISA | BI-20022
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Method
Sandwich ELISA, HRP/TMB, 12×8-well detachable strips
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Sample type
Serum, EDTA plasma, heparın plasma, citrate plasma
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Sample volume
20 µl / well
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Assay time
90 min / 90 min / 30 min
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Sensitivity
0.05 µg/ml
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Standard range
0 – 10 µg/ml
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Specificity
Human MDA-oxLDL adducts
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Precision
In-between-run (n=4): ≤ 7 % CV
Within-run (n=3): ≤ 8 % CV
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Use
Research use only
MDA oxLDL ELISA Product Overview
The MDA oxLDL ELISA kit is a 4 hour, 96-well sandwich ELISA for the quantitative determination of MDA-oxLDL adducts in human serum and plasma (EDTA, heparın, citrate) samples. The oxldl assay employs human serum-based standards to ensure the measurement of biologically reliable data.
MDA oxLDL ELISA Assay Principle
The MDA-oxLDL ELISA kit is a sandwich enzyme immunoassay for the quantitative determination of human MDA-oxLDL adducts in serum and plasma (EDTA, heparın, citrate) samples.
The figure below explains the principle of the MDA-oxLDL sandwich ELISA:
Capture antibody: monoclonal mouse anti-human MDA-oxLDL antibody
Detection antibody: monoclonal mouse anti-human MDA-oxLDL antibody, HRP-labeled
Target antigen: MDA-oxLDL
In a first step, assay buffer and standard/control/sample are pipetted into the wells of the microtiter-strips, which are pre-coated with monoclonal mouse anti-human MDA-oxLDL antibody. MDA-oxLDL present in the standard/control/sample is captured by the pre-coated antibody in the well. In the washing step all non-specific unbound material is removed. In a second step, the conjugate (monoclonal mouse anti-human MDA-oxLDL-HRP) is pipetted into the wells and forms a sandwich with the MDA-oxLDL bound by the capture antibody on the plate. After another washing step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of MDA-oxLDL. This color change is detectable with a standard microtiter plate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) vs. standard concentration is generated, using the values obtained from the standard. The concentration of MDA-oxLDL in the sample is determined directly from the dose response curve.
MDA oxLDL ELISA Typical Standard Curve
The figure below shows a typical standard curve for the human oxldl ELISA kit.
MDA oxLDL ELISA Kit Components
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Contents |
Description |
Quantity |
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PLATE |
Mouse monoclonal anti-human MDA-oxLDL antibody pre-coated microtiter strips in strip holder, packed in aluminum bag with desiccant |
12 x 8 tests |
|
WASHBUF |
Wash buffer concentrate 20x, natural cap |
1 x 50 ml |
|
ASYBUF |
Assay buffer, red cap, ready to use |
1 x 13 ml |
|
STD |
Standards 1-6 ( 0; 0.62; 1.25; 2.5; 5; 10 μg/ml), MDA-oxLDL in human serum, white caps, lyophilized |
6 vials |
|
CTRL |
Control, white cap, lyophilized, exact concentration see label |
1 vial |
|
CONJ |
Conjugate (monoclonal mouse anti-human MDA-oxLDL antibody-HRP), amber cap, ready to use |
1 x 13 ml |
|
SUB |
Substrate (TMB solution), blue cap, ready to use |
1 x 13 ml |
|
STOP |
Stop solution, white cap, ready to use |
1 x 7 ml |
Storage instructions: All reagents of the MDA-oxLDL ELISA kit are stable at 4°C until the expiry date stated on the label of each reagent.
Serum, EDTA plasma, heparın plasma, citrate plasma, are suitable for use in this oxldl ELISA kit. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The listed sample collection and storage conditions are intended as general guidelines.
Serum & Plasma
Collect venous blood samples in standardized serum separator tubes (SST) or standardized blood collection tubes using EDTA, heparın or citrate as an anticoagulant. For serum samples, allow samples to clot for 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or haemolyzed samples may give erroneous results. Samples can undergo at least four freeze-thaw cyles.
Reagent Preparation
Wash Buffer
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1. |
Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature (18-26°C). |
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2. |
Dilute the WASHBUF concentrate 1:20, e.g. 50 ml WASHBUF + 950 ml distilled or deionized water. Only use diluted WASHBUF when performing the assay. |
The diluted WASHBUF is stable up to one month at 4°C (2-8°C).
Standards & Controls for Serum and Plasma Measurements
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1. |
Pipette 150 µl of distilled or deionized water into each standard (STD) and control (CTRL) vial. The exact concentration is printed on the label of each vial. |
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2. |
Leave at room temperature (18-26°C) for 15 min. Vortex gently. |
Reconstituted STDs and CTRLs are stable at -25°C or lower until expiry date stated on the label. STDs and CTRLs are stable for four freeze-thaw cycles.
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples.
Samples for which the OD value exceeds the highest point of the standard range can be diluted STD1 (standard 1) or oxLDL negative human serum. Dilutions up to 1:10 are recommended.
MDA oxLDL Assay Protocol
Read the entire protocol before beginning the assay.
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1. |
Bring samples and reagents to room temperature (18-26°C). |
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2. |
Mark positions for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet. |
|
3. |
Take microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until expiry date stated on the label. |
|
4. |
Add 100 μl ASYBUF (assay buffer) into each well. |
|
5. |
Add 20 μl STD/CTRL/SAMPLE in duplicates into respective wells, swirl gently. |
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6. |
Cover the strips tightly and incubate for 90 min at room temperature (18-26°C). |
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7. |
Aspirate and wash wells 5x with 300 μl diluted WASHBUF (wash buffer). Remove remaining WASHBUF by strongly tapping the plate against paper towel. |
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8. |
Add 100 μl CONJ (conjugate) into each well. |
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9. |
Cover the strips tightly and incubate for 90 min at room temperature (18-26°C). |
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10. |
Aspirate and wash wells 5x with 300 μl diluted WASHBUF. Remove remaining WASHBUF by strongly tapping the plate against a paper towel. |
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11. |
Add 100 μl SUB (Substrate) into each well. |
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12. |
Incubate for 30 min at room temperature (18-26°C) in the dark. |
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13. |
Add 50 μl STOP (stop solution) into each well, swirl gently. |
|
14. |
Measure absorbance immediately at 450 nm with reference 630 nm, if available. |
Calculation of Results
Read the optical density (OD) of all wells on a plate reader using 450 nm wavelength (reference wavelength 630 nm). Construct a standard curve from the absorbance read-outs of the standards using commercially available software capable of generating a four-parameter logistic (4-PL) fit. Alternatively, plot the standards’ concentration on the x-axis against the mean absorbance for each standard on the y-axis and draw a best fit curve through the points on the graph. Curve fitting algorithms other than 4-PL have not been validated and will need to be evaluated by the user.
Respective dilution factors have to be considered when calculating the final concentration of the sample.
The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each lot at production date. Data for OD obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life. However, this does not affect validity of results as long as an OD of 1.0 or more is obtained for the STD with the highest concentration and the value of the CTRL is in range (target range see label).
Oxidized low-density lipoproteins (oxLDLs) play an important role in the progression of atherosclerosis and coronary artery disease. Low-density lipoprotein (LDL), the main carrier of plasma cholesterol, consists of a hydrophobic core and a surface monolayer of polar lipids and Apolipoprotein-B (ApoB). Oxidative stress and the consequent formation of free radicals lead to the peroxidation of ApoB. Malondialdehyde (MDA) has been identified as one of the major lipid peroxidation products of LDL, thus playing an important role in the LDL oxidation.
The Biomedica MDA oxLDL ELISA specifically detects MDA-modified ApoB in human serum and plasma.
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Cardiovascular Disease
- Atherosclerosis
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