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Amplite™ Mouse Apolipoprotein A1 (ApoA1) Kit *Optimized For ELISAPro Automated ELISA Processing*

Ordering information
Price ()
Catalog NumberV101070
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Platform


Absorbance microplate reader

Recommended plateClear bottom

Components


Adhesive plate covers3
Biotinylated detection mAb mHDL931 vial (25 uL, 0.5 mg/mL)
Dilution buffer1 bottle (60 mL)
Pre-coated ELISA strip plate (Anti-apoA1 mAb mHDL93)1x96 wells
Purified mouse apoA1 ELISA standard1 vial
Standard reconstitution buffer A81 vial (1 mL)
Stop solution1 bottle (15 mL)
Streptavidin-HRP (1000x)1 vial (15 uL)
TMB substrate1 bottle (15 mL)
Wash buffer concentrate (20X)1 bottle (120 mL)
x Sample diluent1 bottle (60 mL)

Example protocol


AT A GLANCE

AT A GLANCE
ELISAPRO kits provide all the necessary reagents to conveniently quantify analytes in serum, plasma, and cell culture supernatants in a robust, sensitive, and specific manner.

ELISA assay principle
ELISAPRO kits are supplied with ELISA strip plates precoated with monoclonal antibody (mAb). Analyte in the sample is captured by the coated mAb and detected by the biotinylated detection mAb followed by Streptavidin-HRP (SA-HRP). Addition of TMB substrate will result in a colored substrate product. The reaction is stopped with sulfuric acid and the optical density can be quantified using an ELISA plate reader. The concentration of analyte is determined by comparison to a serial dilution of the ELISA standard analyzed in parallel.

Materials required but not supplied
  1. Microplate reader capable of reading at 450 nm
  2. ELISA plate washer; automated or manual (e.g., multipipette or squirt bottle)
  3. Precision pipettes, tips, and graduated cylinders
  4. Tubes for standard and sample dilutions
  5. Distilled or deionized water 

Safety information
The Stop solution, 0.18 M H2SO4 (< 1%), is irritating to eyes and skin and should be handled with care. The standard should also be handled carefully as the effects of exposure are unknown. Buffers and reagents in solution contain the preservative Kathon CG (0.002%), a potential allergen that may cause sensitization through skin contact. Human and animal samples should be treated as potentially hazardous biologic material. All material should be disposed of in accordance with local regulations. For further information please consult the Safety Data Sheet on our website.

Preparation
Allow the plates and assay reagents to reach room temperature before starting the assay (except for the TMB substrate which should preferably be used cold). Plan the plate layout to include a standard curve, samples, and an assay background control all in duplicate. The volume per well should not exceed 100 μl. Plate blanks (only substrate and Stop solution) can be included for subtraction by the reader software.

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.

1. Wash buffer
Add 50 ml Wash buffer concentrate to 950 ml distilled or deionized water (sufficient for all washing steps of 1 plate). If crystals have formed in the 20x concentrate, bring to room temperature and mix gently to dissolve.

2. Sample diluent
Prepare the required volume of Sample diluent by diluting Sample diluent concentrate 5-fold with distilled or deionized water. For each plate, add 30 ml Sample diluent to 120 ml water.

3. Samples
All samples should be diluted at least 2-fold in Sample diluent. Remove visible precipitates and dilute in polypropylene tubes/plates, buffer should be added prior to the samples. Strongly hemolyzed and hyperlipemic samples may result in inaccurate quantifications. Samples containing high levels of analyte exceeding the standard range of the assay will require further dilution.

4. Dilution guidelines for mouse serum/plasma
We recommend a dilution factor of 200,000X based on repeated analyses of BALB/c and C57BL/6 samples. Precise pipetting is important, change tips between dilution steps and use freshly made dilutions. Indicated volumes are sufficient for duplicates.

5. ELISA standard
Reconstitute the ELISA standard to a stock solution of 1 μg/ml by adding 1 ml Standard reconstitution buffer. Do not stir. Allow the standard to dissolve for 20 minutes and mix thoroughly. The standard should be kept in aliquots at -20 °C. Avoid repeated freeze-thaw cycles.

6. Preparation of standard curve
Dilute the standard stock solution to create a standard curve as shown. The indicated volumes are sufficient for duplicates. The last vial is used as an assay background control, i.e., the standard should be omitted. Prepare the standard curve within 30 minutes of use.

PREPARATION OF WORKING SOLUTION

1. Detection antibody
Dilute the detection antibody in Dilution buffer to a concentration of 1 μg/ml within 15 minutes of use. For each plate, dilute 24 μl detection antibody in 12 ml Dilution buffer.

2. Streptavidin-HRP
Dilute the Streptavidin-HRP 1000-fold in Streptavidin-HRP diluent within 15 minutes of use. For each plate, dilute 12 μl Streptavidin-HRP in 12 ml Streptavidin-HRP diluent.

SAMPLE EXPERIMENTAL PROTOCOL

Prepare the reagents, standard curve, and samples as described in the Preparation section. Assemble the required number of strips in the plate frame and label the top of each strip. Store the remaining strips in the foil bag containing the desiccant at 4-8 °C.
  1. Add samples (diluted at least 2-fold), standard, and assay background control (100 μl/well). Mix by tapping the plate. Cover the plate with an adhesive plate cover and incubate at room temperature for 1 hour.
  2. Wash the plate as described above.
  3. Add detection antibody (100 μl/well). Cover the plate and incubate at room temperature for 1 hour.
  4. Wash the plate as described above.
  5. Add Streptavidin-HRP (100 μl/well). Cover the plate and incubate at room temperature for 1 hour.
  6. Wash the plate as described above.
  7. Add TMB substrate (100 μl/well). Incubate at room temperature protected from direct light for 15 minutes.
  8. Add Stop solution to all wells (100 μl/well) to stop the color development.
  9. Measure absorbance at 450 nm within 15 minutes. If possible, use a reader capable of subtracting a reference wavelength of between 570 and 650 nm.