Download biomedica product listoLAB (Anti-Oxidized LDL Autoantibodies) ELISA | BI-20032
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Method
Indirect ELISA, HRP/TMB, 12×8-well detachable strips
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Sample type
Serum
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Sample volume
50 µl / well
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Assay time
1.5 h / 30 min / 15 min
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Sensitivity
48 mU/ml
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Standard range
0 – 1,200 mU/ml
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Specificity
Anti-oxidized LDL autoantibodies
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Precision
In-between-run (n=5): ≤ 8 % CV
Within-run (n=8): ≤ 4 % CV
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Use
Research use only
Anti-Oxidized LDL Autoantibodies Product Overview
The anti-oxidized LDL autoantibodies (oLAB) immunoassay is a 2 hour 15 minutes, 96-well indirect ELISA for the quantitative determination of anti-oxidized LDL autoantibodies in serum.
Anti-Oxidized LDL Autoantibodies Principle of the Assay
The anti-oxldl antibody ELISA kit is an indirect enzyme immunoassay for the quantitative determination of anti-oxidized LDL autoantibodies in serum samples.
In a first step, prediluted standard/control/sample are pipetted into the wells of the microtiter strips, which are pre-coated with oxidized LDL antigen. Anti-oxidized LDL autoantibodies present in the standard/control/sample bind to the pre-coated antigen in the well. After a washing step, which removes all non-specific unbound material, the conjugate (monoclonal anti-human IgG-HRP) is pipetted into the wells and reacts with the anti-oxidized LDL autoantibodies.
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 anti-oxidized LDL autoantibodies present in the sample. This color change is detectable with a standard microplate reader. The concentration of anti-oxidized LDL autoantibodies in the sample is determined directly from the dose response curve.
Anti-Oxidized LDL Autoantibodies Typical Standard Curve
The figure below shows a typical standard curve for oxldl assay kit.
Anti-Oxidized LDL Autoantibodies ELISA Kit Components
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Contents |
Description |
Quantity |
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PLATE |
Oxidized LDL precoated microtiter strips in strip holder, packed in aluminum bag with desiccant |
12 x 8 tests |
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DILPLATE |
Uncoated microtiter plate for sample pre-treatment |
12 x 8 wells |
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WASHBUF |
Wash buffer concentrate 20x, natural cap |
1 x 50 ml |
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STD |
Standards 1-6 ( 0; 0.62; 1.25; 2.5; 5; 10 μg/ml), anti-oxidized LDL IgG antibodies, white caps, ready to use |
6 x 500 µl |
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CTRL |
Control A and B, yellow caps, ready to use, exact concentrations see labels |
2 x 500 µl |
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ASYBUF |
Assay buffer, red cap, ready to use |
1 x 60 ml |
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CONJ |
Conjugate, (monoclonal anti-human IgG-HRP), amber cap, ready to use |
1 x 13 ml |
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SUB |
Substrate (TMB solution), blue cap, ready to use |
1 x 13 ml |
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STOP |
Stop solution, white cap, ready to use |
1 x 7 ml |
Serum is suitable for use in this oxldl assay kit. We recommend duplicate measurements for all samples, standards and controls. The sample collection and storage conditions listed are intended as general guidelines.
Serum
Collect venous blood samples in standardized serum separator tubes (SST). 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. Do not freeze-thaw samples more than four times.
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).
Sample Preparation
Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples.
Anti-Oxidized LDL Autoantibodies Assay Protocol
Read the entire protocol before beginning the assay.
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1. |
Bring samples and reagents to room temperature (18-24°C). |
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2. |
Mark positions for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet. |
In the Predilution Plate
Note: All STD /CTRL/SAMPLE (standard/control/sample) must be used in 1:55 end-dilution in the assay (pre-dilution 1:5 + assay-dilution 1:11). Use the enclosed DILPLATE (uncoated microtiter plate) for the 1:5 predilution step.
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1. |
Pipette 200 µl ASYBUF (assay buffer) into the appropriate wells of the uncoated microtiter plate. . |
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2. |
Add 50 µl STD/CTRL/SAMPLE (standard/control/sample) into the respective wells, mix well (= 1:5 dilution). |
In the Pre-Coated Plate
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1. |
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. |
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2. |
Pipette 200 µl ASYBUF (assay buffer, red cap) into each well, including blank. |
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3. |
Add 20 µl 1:5 prediluted STD/CTRL/SAMPLE into the respective wells. Swirl gently. Note: The transfer of the prediluted STD /CTRL/SAMPLE into the precoated microtiter strips must be completed within 15 minutes. Use a multichannel pipette. |
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4. |
Cover the plate tightly and incubate for 1.5 hours at 37°C. |
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5. |
Aspirate and wash wells 4 x with 300 µl diluted WASHBUF (wash buffer). After the final wash, remove the remaining WASHBUF by strongly tapping plate against a paper towel. |
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6. |
Add 100 µl CONJ (conjugate, amber cap) into each well except blank. |
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7. |
Cover tightly and incubate for 30 minutes at room temperature (18-24°C). |
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8. |
Aspirate and wash wells 4 x with 300 µl diluted WASHBUF. After the final wash, remove remaining WASHBUF by strongly tapping plate against a paper towel. |
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9. |
Add 100 µl SUB (substrate, blue cap) into each well. |
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10. |
Incubate for 15 min at room temperature in the dark. |
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11. |
Add 50 µl STOP (stop solution, white cap) into each well. Swirl gently. |
| 12. |
Measure absorbance immediately at 450 nm with reference 630 nm, if available. |
Calculation of Results
Subtract the absorbance read-out obtained for the blank from all other values. Construct a standard curve from the values 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.
Obtain sample concentrations from the standard curve. Samples for which the optical density (OD) value exceeds the highest point of the standard range can be diluted further. Sample predilutions other than 1:5 have to be considered when calculating the final concentration of the sample.
The quality control protocol supplied with the kit shows the results of the final release QC for each kit. Data for optical density obtained by customers may differ due to various influences including the normal decrease of signal intensity throughout shelf life. However, this does not affect validity of results as long as an optical density of 1.00 or higher is obtained for the standard with the highest concentration and the values of the CTRLs are within the target range (see labels).
Anti-Oxidized LDL Autoantibodies
Oxidized low density lipoprotein (oxLDL) is believed to play a critical role in the development and progression of atherosclerosis. Accumulation of oxLDL in macrophages and smooth muscle cells causes foam cell formation, an initial step in the disease. Autoantibodies against oxidatively modified LDL can be used as a parameter that consistently mirrors the occurrence of oxidation processes taking place in vivo. In fact, elevated levels of autoantibodies against oxLDL have been detected in the blood stream of patients with coronary artery disease. Moreover, recent studies indicate a correlation between autoantibodies against oxLDL and the progression of carotid atherosclerosis. Increased serum concentrations of oLAB have also been described in various diseases such as pre-eclampsia and systemic lupus erythematosus. Decreased oLAB titers were observed during septicemia and myocardial infarction.
An overview on the clinical applications of oLAB has been published.
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Cardiovascular Disease
- Artherosclerosis (Bornaun et al., 2017; de Freitas et al., 2018; Pawlak et al., 2012; Shoji et al., 2003; Stanek et al., 2018)
- Coronary artery disease (Faviou et al., 2005; Miller et al., 2003)
- Myocardial infarction (Huang et al., 2013)
- Stroke (Ciancarelli et al., 2012)
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Other
- Acute septicaemia (Al-Banna and Lehmann, 2013)
- Preeclampsia (Arifin et al., 2017)
Literature
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de Freitas, A.C.P., Torres, L.C., Duarte, M. do C.M.B., da Matta, M.C., Casarini, D.E., Schor, N., 2018. Urolithiasis.
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Increased circulating oxidised low-density lipoprotein and antibodies to oxidised low-density lipoprotein in preeclampsia.
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Oxidized LDL and LOX-1 in Experimental Sepsis.
Al-Banna, N., Lehmann, C., 2013. Mediators Inflamm.PMID: 24000272
Using Oxidized Low-Density Lipoprotein Autoantibodies to Predict Restenosis after Balloon Angioplasty in Patients with Acute Myocardial Infarction.
Huang, C.-H., Chang, C.-C., Huang, C.-S., Kuo, C.-L., Chen, C.-P., Hsia, C.-H., Chang, Y.-M., Chen, H.-T., Feng, C.-C., Lin, L.-S., Yang, P.-T., Tsai, C.-D., Lin, C.-S., Liu, C.-S., 2013. PLoS ONE 8, e74726.
Oxidative stress in post-acute ischemic stroke patients after intensive neurorehabilitation.
Ciancarelli, I., De Amicis, D., Di Massimo, C., Carolei, A., Ciancarelli, M.G.T., 2012. Curr. Neurovasc. Res. 9, 266–273PMID: 22873723
Oxidized LDL to autoantibodies against oxLDL ratio – The new biomarker associated with carotid atherosclerosis and cardiovascular complications in dialyzed patients.
Pawlak, K., Mysliwiec, M., Pawlak, D., 2012. Atherosclerosis 224, 252–257.
Circulating oxidized low density lipoprotein, autoantibodies against them and homocysteine serum levels in diagnosis and estimation of severity of coronary artery disease.
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- The impact of antihypertensive pharmacotherapy on interplay between protein-bound uremic toxin (indoxyl sulfate) and markers of inflammation in patients with chronic kidney disease.
Kaminski, T.W., Pawlak, K., Karbowska, M., Znorko, B., Mor, A.L., Mysliwiec, M., Pawlak, D., 2019. Int Urol Nephrol.
- Novel approaches for the assessment of relative body weight and body fat in diagnosis and treatment of anorexia nervosa: A cross-sectional study.
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- Upregulation of the Nitrosylome in Bipolar Disorder Type 1 (BP1), but not BP2, and Major Depression: Increased IgM Antibodies to Nitrosylated Conjugates are Associated with Indicants of Leaky Gut.
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- Is oxidized low-density lipoprotein the connection between atherosclerosis, cardiovascular risk and nephrolithiasis?
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PMID:30302491 - Metabolic disorders and inflammation are associated with familial combined hyperlipemia.
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PMID:29531242 PMCID: PMC5847532 - Whole-Body Cryotherapy Decreases the Levels of Inflammatory, Oxidative Stress, and Atherosclerosis Plaque Markers in Male Patients with Active-Phase Ankylosing Spondylitis in the Absence of Classical Cardiovascular Risk Factors.
Stanek, A., Cholewka, A., Wielkoszyński, T., Romuk, E., Sieroń, A., 2018. Mediators of Inflammation 2018, 1–11.
- Increased circulating oxidised low-density lipoprotein and antibodies to oxidised low-density lipoprotein in preeclampsia.
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PMID:28358592 - Assessment of lipid profile and some risk factors of atherosclerosis in children whose parents had early onset coronary artery disease.
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