General Assay Protocols & Reagents

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Steroid Fecal Extraction

An organic phase extraction is recommended to extract steroids from non-liquid matrices, such as dried solids or other organic matter. We use ethanol or ethyl acetate as a safer alternative to diethyl ether or methylene chloride used in standard protocols. We recommend centrifugal vacuum devices, like a SpeedVac, to remove the solvent completely and safely. We also recommend the use of ethanol as a means to completely solubilize the dried steroid because certain steroids have limited aqueous solubility.

Recovery of the desired substance is likely to be incomplete. Therefore, both optimization and efficiency of the extraction procedure are recommended for accurate determinations.

Materials Needed:

Procedure:

  1. Weigh out ≥ 0.2gm of dried fecal solid into tube.
  2. Add 1 mL of Ethanol (or Ethyl Acetate) for every 0.1 gm of solid.
  3. Shake vigorously for at least 30 minutes.
  4. Centrifuge samples at 5,000 rpm for 15 minutes. Transfer measured volume of supernatant to a clean tube for evaporation.
  5. Evaporate supernatant solution to dryness in a SpeedVac or under nitrogen. Keep dried extracted samples fronzen < -20˚C in a desiccator. (Note: if only a portion of the organic solvent is being evaporated, ensure final amounts of measured steroid per gm solid accounts for volume of solution evaporated.)
  6. Dissolve extracted sample with 100uL ethanol, followed by at least 400uL Assay Buffer (AB). Vortex well and allow to sit 5 minutes at room temperature. Vortex and sit for 5 minutes twice more to ensure complete steroid solubility. For immunoassays, ethanol content in the sample well should be below 5%. Dilute the ethanol:AB mixture ≥ 1:10 with AB, or as directed in the kit manual.
  7. Run reconstituted diluted samples in assay immediately according to kit manual directions.

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Steroid Serum/Plasma Extraction

Assays will yield the total steroid concentration in serum or plasma when samples are treated with a supplied Dissociation Reagent. Some assays require extraction of steroids in serum and plasma samples because Dissociation Reagent is not suitable for use in the assay and/or there are interfering substances, such as bulk proteins and lipids. One researcher using an Arbor Assay kit saw a need for extraction due to the high level of carotenoids present in Northern Cardinal plasma. Extraction may also be necessary to concentrate the sample to within an assay's measurement range. If the steroid to be measured and the assay requires samples to be concentrated, we would recommend using at least 1-2 mL of serum or plasma.

Recovery of the desired substance is likely to be incomplete. Therefore, both optimization and efficiency of the extraction procedure are recommended for accurate determinations.

Solid Phase Extraction

Materials Needed:

Procedure:

  1. Condition 200 mg C18 solid phase columns on a vacuum manifold by passing 5-10 mL of 100% methanol through the columns, followed by 5-10 mL of water.
  2. Apply serum and plasma samples to individual washed columns.
  3. Wash columns with 5-10 mL of water. Allow water to drain completely from columns until dry.
  4. Elute samples by addition of 2 mL of diethyl ether to the individual columns.
  5. Dry samples down in a SpeedVac for 2-3 hrs. If samples need to be stored, they should be kept desiccated at -20˚C.
  6. Rehydrate samples at room temperature in Assay Buffer (AB). A minimum of 125 uL AB is recommended for reconstitution to allow for duplicate sample measurements.

Liquid Extraction

Materials Needed:

Procedure 1: Allows maximum recovery of steroid but takes longer to complete.

  1. Add diethyl ether to serum or plasma samples at a 5:1 (v/v) ether:sample ratio.
  2. Mix solutions by vortexing for 2 minutes. Allow ether layer to separate for 5 minutes.
  3. Freeze samples in a dry ice/ethanol bath and pour ether solution from the top of the sample into a clean tube. Repeat steps 1-3 for maximum extraction efficiency, combining top layer of ether solutions.
  4. Dry pooled ether samples down in a SpeedVac for 2-3 hrs. If samples need to be stored they should be kept desiccated at -20˚C.
  5. Redissolve samples at room temperature in Assay Buffer (AB). A minimum of 125 uL AB is recommended for reconstitution to allow for duplicate sample measurement.

Procedure 2: Better suited for larger number of samples.

  1. Add five parts of diethyl ether to each part of serum or plasma samples.
  2. Nutate solutions for 5 minutes or vortex for 2 minutes.
  3. Allow phases to separate for 5 minutes.
  4. Transfer the top organic ether phase to a clean glass test tube containing 1 mL water.
  5. Nutate the ether/water solution for 5 minutes or vortext the mixture for 2 minutes.
  6. Allow phases to separate for 2 minutes.
  7. Transfer the top organic ether layer to a clean glass test tube. Steps 1-6 can be repeated for maximum extraction efficiency, combining top layers of ether solutions.
  8. Dry pooled ether samples down in a SpeedVac for 2-3 hrs. If samples need to be stored they should be kept desiccated at -20˚C.
  9. Redissolve samples at room temperature in Assay Buffer (AB). A minimum of 125 uL AB is recommended for reconstitution to allow for duplicate sample measurement.

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Plate Coating

Coating solid-phase plates with a secondary antibody against an assay's primary antibody will allow for conservation of a very critical aspect of any competitive immunoassay, as the amount of primary antibody to be used as a solution added to a "generic" antibody plate is significantly less. Also, coating and blocking using specially formulated buffers will allow for plates to be prepared in advance and stored long-term as a dried, desiccated product at 4˚C.

Materials Needed:

Procedure:

  1. DAY 1: Prepare enough 1x Coating Buffer with Coating Antibody at 10ug/mL to pipet at 0.15 mL per well of solid phase plate.
    • # of plates * 96wells/plate * 0.15mL/well= Minimum 1x Coating Buffer Volume (in mL)
    • Volume 1x Coating Buffer ÷ 20 = Volume 20x Coating Buffer needed to make Volume 1x Coating Buffer.
    • Volume 1x Coating Buffer (mL) * 0.01 mg Coating Antibody/mL ÷ Concentration of Coating Antibody (in mg/mL) = Volume of Antibody to add, after removing equivalent volume from 1x Coating Buffer.
  2. Using an 8-channel pipet or an automated filling instrument (ie. MultiDrop), add 0.15mL Coating Solution to each well.
  3. Cover coated plates in plastic wrap and leave at room temperature (RT) overnight (15-24 hours).
  4. DAY 2: Prepare enough 1x Blocking Buffer to pipet at 0.25 mL per well of solid phase plate.
    • # of plates coated * 96wells/plate * 0.25mL/well = Minimum 1x Blocking Buffer Volume (in mL)
    • Volume 1x Blocking Buffer ÷ 10 = Volume 10x Blocking Buffer needed to make Volume 1x Blocking Buffer.
  5. Using an aspirator, or just dumping into a sink, remove coating solution from plates. Blot upside-down onto clean paper toweling to remove excess moisture, in the same order the plates were coated.
  6. Using an 8-channel pipet or an automated filling instrument, add 0.25mL Blocking Solution to each well.
  7. Cover blocked plates in plastic wrap and leave at RT 6-24 hours.
  8. Using an aspirator, or just dumping into a sink, remove blocking solution from plates. Blot upside-down onto clean paper toweling to remove excess moisture, in the same order the plates were blocked.
  9. Place plates upside-down in a desiccated drying cabinet and allow to dry until moisture content falls below 20% (and at least overnight).
  10. Once dry, package plates individually inside foil ziploc bags, along with a ≥ 1g desiccant pack. Heat seal bag closed and store plates at 4˚C until needed.

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General Assay Reagents

Note: Clicking Catalog No. links below will redirect page to Arbor Assays' product details
Reagent Arbor Assays Catalog No.
Solid Phase Coating Buffer, 20x Conc. X108-10ML (10 mL fill), X108-100ML (100 mL fill)
Blocking Buffer, 10x Conc. X109-25ML (25 mL fill), X109-250ML (250 mL fill)

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