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Amplite® Human Apolipoprotein E (ApoE) Kit *Optimized For ELISA Development with HRP*
Example protocol
AT A GLANCE
AT A GLANCE
Intended use: For quantitative determination of human Apolipoprotein E (apoE) in serum/plasma samples and cell culture supernatants. The mAbs detect the three apoE isoforms apoE2, apoE3 and apoE4. Please note that wash, block and incubation buffers should contain detergent. Tween 20, Triton X-100 or NP40 can be used at a concentration of 0.05-0.5%. In block and incubation buffers it is recommended to use 0.1% BSA.
Serum/plasma samples: When analyzing human serum/plasma samples it is recommended to use Apo ELISA buffer for dilution of samples, standard and detection antibody. The buffer prevents false positive read-outs which may be caused by interference of heterophilic antibodies commonly found in human plasma and serum. The Apo ELISA buffer has been validated using serum/plasma from normal healthy human blood donors. Please note that heterophilic antibody interference in samples from human subjects with various diseases or other conditions has not been assessed. Serum and plasma samples containing EDTA, citrate or heparin may be used. However, heparin containing samples will give higher apoE values due to displace- ment of proteoglycan bound apoE. Triton X-treatment of samples, necessary for apoB analysis, will not interfere with apoE analysis. It is recommended to dilute serum/plasma samples 10,000x to 20,000x, see dilution guidelines at https://www.aatbio.com/tools/serial-dilution/. Avoid repeated freezing-thawing cycles.
Note Apo ELISA buffer is not provided in this kit. It can be purchased from MabTech (product code: 3652-M2).
Reagents: Antibodies are supplied in sterile-filtered (0.2 μm) PBS with sodium azide (0.02%). Streptavidin-HRP is supplied in PBS with 1% BSA and 0.002% Kathon CG.
Standard range: 0.1-10 ng/ml
Intended use: For quantitative determination of human Apolipoprotein E (apoE) in serum/plasma samples and cell culture supernatants. The mAbs detect the three apoE isoforms apoE2, apoE3 and apoE4. Please note that wash, block and incubation buffers should contain detergent. Tween 20, Triton X-100 or NP40 can be used at a concentration of 0.05-0.5%. In block and incubation buffers it is recommended to use 0.1% BSA.
Serum/plasma samples: When analyzing human serum/plasma samples it is recommended to use Apo ELISA buffer for dilution of samples, standard and detection antibody. The buffer prevents false positive read-outs which may be caused by interference of heterophilic antibodies commonly found in human plasma and serum. The Apo ELISA buffer has been validated using serum/plasma from normal healthy human blood donors. Please note that heterophilic antibody interference in samples from human subjects with various diseases or other conditions has not been assessed. Serum and plasma samples containing EDTA, citrate or heparin may be used. However, heparin containing samples will give higher apoE values due to displace- ment of proteoglycan bound apoE. Triton X-treatment of samples, necessary for apoB analysis, will not interfere with apoE analysis. It is recommended to dilute serum/plasma samples 10,000x to 20,000x, see dilution guidelines at https://www.aatbio.com/tools/serial-dilution/. Avoid repeated freezing-thawing cycles.
Note Apo ELISA buffer is not provided in this kit. It can be purchased from MabTech (product code: 3652-M2).
Reagents: Antibodies are supplied in sterile-filtered (0.2 μm) PBS with sodium azide (0.02%). Streptavidin-HRP is supplied in PBS with 1% BSA and 0.002% Kathon CG.
Standard range: 0.1-10 ng/ml
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Prepare apoE standard by reconstituting contents of vial 4 in 1 ml PBS with 0.5 mM DTT and 0.1% BSA, do not stir and leave at room temperature for 20 minutes. This gives a stock solution of 5 μg/ml which should be used immediately or stored in aliquots at -20°C for future use. We recommend the aliquots not to be refrozen after initial use. For the test, prepare dilutions of the stock using the standard range as a guideline.
Prepare apoE standard by reconstituting contents of vial 4 in 1 ml PBS with 0.5 mM DTT and 0.1% BSA, do not stir and leave at room temperature for 20 minutes. This gives a stock solution of 5 μg/ml which should be used immediately or stored in aliquots at -20°C for future use. We recommend the aliquots not to be refrozen after initial use. For the test, prepare dilutions of the stock using the standard range as a guideline.
SAMPLE EXPERIMENTAL PROTOCOL
- Coat a high protein binding ELISA plate with mAb E276, diluted to 2 μg/ml in PBS, pH 7.4, by adding 100 μl/well. Incubate overnight at 4-8°C.
- Wash twice with PBS (200 μl/well).
- Block plate by adding 200 μl/well of PBS with 0.05% Tween 20 containing 0.1% BSA (incubation buffer). Incubate for 1 hour at room temperature.
- Wash five times with PBS containing 0.05% Tween.
- Prepare apoE standard by reconstituting contents of vial 4 in 1 ml PBS with 0.5 mM DTT and 0.1% BSA, do not stir and leave at room temperature for 20 minutes. This gives a stock solution of 5 μg/ml which should be used immediately or stored in aliquots at -20°C for future use. We recommend the aliquots not to be refrozen after initial use. For the test, prepare dilutions of the stock using the standard range as a guideline.
- Add 100 μl/well of samples or standards diluted in incubation buffer or Apo ELISA buffer for serum/plasma samples and incubate for 1 to 2 hours at room temperature. Dilution recommendations for serum/plasma samples can be found at https://www.aatbio.com/tools/serial-dilution/.
- Wash as in step 4.
- Add 100 μl/well of mAb E887-biotin at 1 μg/ml in incubation buffer or Apo ELISA buffer for serum/plasma samples. Incubate for 1 hour at room temperature.
- Wash as in step 4.
- Add 100 μl/well of Streptavidin-HRP diluted 1:1000 in incubation buffer. Incubate for 1 hour at room temperature. Please note that sodium azide used in buffers will inhibit HRP activity.
- Wash as in step 4.
- Add 100 μl/well of appropriate substrate solution e.g. TMB.
Note available from AAT Bioquest, Cat# 11012 - Measure the optical density in an ELISA reader after suitable developing time. If required stop the reaction first.
Page updated on November 21, 2024
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