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Amplite® Human Apolipoprotein A1 (ApoA1) Kit *Optimized For ELISA Development with HRP*
Example protocol
AT A GLANCE
AT A GLANCE
Intended use: For quantitative determination of human Apolipoprotein A1 (apoA1) in serum/ plasma samples and cell culture supernatants. 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, but not bovine serum, as HDL 44 also binds bovine apoA1.
Serum/plasma samples: When analyzing human serum/plasma samples it is recommended to use Apo ELISA buffer for dilution of samples, standard and detection an- tibody. The buffer prevents false positive read-outs which may be caused by interference of hetero- philic antibodies commonly found in human plasma and serum. Triton X-treatment of samples, necessary for apoB analysis, will not interfere with apoA1 analysis. It is recommended to dilute se- rum/plasma samples 150,000x to 200,000x. Avoid repeated freezing-thawing cycles and do not store samples in -20°C. Samples stored in -20°C will give false high apoA1 values.
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: 0.6-40 ng/ml
Intended use: For quantitative determination of human Apolipoprotein A1 (apoA1) in serum/ plasma samples and cell culture supernatants. 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, but not bovine serum, as HDL 44 also binds bovine apoA1.
Serum/plasma samples: When analyzing human serum/plasma samples it is recommended to use Apo ELISA buffer for dilution of samples, standard and detection an- tibody. The buffer prevents false positive read-outs which may be caused by interference of hetero- philic antibodies commonly found in human plasma and serum. Triton X-treatment of samples, necessary for apoB analysis, will not interfere with apoA1 analysis. It is recommended to dilute se- rum/plasma samples 150,000x to 200,000x. Avoid repeated freezing-thawing cycles and do not store samples in -20°C. Samples stored in -20°C will give false high apoA1 values.
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: 0.6-40 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 apoA1 standard by reconstituting contents of vial 4 in 1 ml PBS with 1% BSA, do not stir and leave at room temperature for 15 minutes followed by vortex for 3 sek. This gives a stock solution of 4 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 apoA1 standard by reconstituting contents of vial 4 in 1 ml PBS with 1% BSA, do not stir and leave at room temperature for 15 minutes followed by vortex for 3 sek. This gives a stock solution of 4 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 HDL 110, 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 apoA1 standard by reconstituting contents of vial 4 in 1 ml PBS with 1% BSA, do not stir and leave at room temperature for 15 minutes followed by vortex for 3 sek. This gives a stock solution of 4 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 stand- ard range as a guideline.
- Prepare apoA1 standard by reconstituting contents of vial 4 in 1 ml PBS with 1% BSA, do not stir and leave at room temperature for 15 minutes followed by vortex for 3 sek. This gives a stock solution of 4 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 stand- ard range as a guideline.
- Wash as in step 4.
- Add 100 μl/well of mAb HDL 44-biotin at 0.5 μg/ml in incubation buffer or Assay 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, available from Mabtech product code 3652-F10.
- Measure the optical density in an ELISA reader after suitable developing time. If required stop the reaction first.
Page updated on December 17, 2024
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