The Human Adrenocorticotrophic Hormone (ACTH) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human Adrenocorticotrophic Hormone (ACTH) levels in plasma. The Eagle Biosciences Human Adrenocorticotrophic Hormone (ACTH) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: ACT31-K01 Categories: , ,


ACTH ELISA Assay Kit Developed and Manufactured in the USA

Size: 1×96 wells
Sensitivity: 1.0 pg/mL
Dynamic Range: 11 – 416 pg/mL
Incubation Time: 2.5 hours
Sample Type: Plasma
Sample Size: 200 µL
Alternative Names: Adrenocorticotrophic Hormone ELISA, Human ACTH ELISA
For Research Use Only

Controls Included

Expected Values:
EDTA plasma samples from normal healthy adults ages 20 – 60 were collected and measured with this ELISA. The recommended normal range for ACTH concentration by using this ELISA is between 1 – 72 pg/mL. We strongly recommend for each clinical laboratory to establish its own normal range by measuring EDTA plasma samples with this ELISA. Please note that sample collection time of the day may have impact on the ACTH normal range.

Assay Principle

The Human Adrenocorticotrophic Hormone (ACTH) ELISA Assay Kit is designed, developed and produced for the quantitative measurement of human ACTH in EDTA-plasma sample. The assay utilizes the two-site “sandwich” technique with selected antibodies that bind to N-terminal and C-terminal epitopes of ACTH.

Assay standards, controls and patient samples are added directly to wells of a microtiter plate that is coated with antibody to the N-terminal of human ACTH. Immediately, a horseradish peroxidase (HRP) conjugated anti C-terminal of human ACTH antibody is added to each well. After the first incubation period, a “sandwich” of solid-phase monoclonal antibody – human ACTH – HRP conjugated antibody” is formed. The unbound antibodies and buffer matrix are removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction and the absorbances are then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is directly proportional to the amount of human ACTH in the test sample. A standard curve is generated by plotting the absorbance versus the respective human ACTH concentration for each standard on a point-to-point or 4-parameter curve fitting. The concentration of human ACTH in test samples is determined directly from this standard curve.

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Additional Information

Assay Background

ACTH is a 39 amino acid polypeptide with a molecular weight of 4540 Dalton. ACTH is secreted from corticotropes in the anterior lobe (or adenohypophysis) of the pituitary gland in response to corticotropin-releasing hormone (CRH) released by the hypothalamus. ACTH is synthesized from pre-pro-opiomelanocortin (pre-POMC). The removal of the signal peptide during translation produces the 241-amino acid polypeptide POMC, which undergoes a series of post-translational modifications such as phosphorylation and glycosylation before it is proteolytically cleaved by endopeptidases to yield various polypeptide fragments with varying physiological activity.

ACTH is an important component of the hypothalamic-pituitary-adrenal axis and is often produced in response to biological stress. It stimulates secretion of glucocorticoid steroid hormones from adrenal cortex cells especially in the zona fasciculata of the adrenal. ACTH acts by binding to cell surface ACTH receptors, which are located primarily on adrenocortical cells of the adrenal cortex.

Assay Procedure

  1. Place a sufficient number of streptavidin coated microwell strips in a holder to run human Adrenocorticotrophic Hormone (ACTH) standards, controls and unknown samples in duplicate.
  2. Add 200 µl of Standards, Controls and patient samples into the designated microwells.
  3. Immediately add 25 µL of HRP Conjugated Anti-ACTH Antibody to each well.
  4. Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 2 hr. ± 5 minutes at 400 to 450 rpm.
  5. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  6. Add 200 µL of ELISA HRP Substrate into each of the wells.
  7. Cover the plate with aluminum foil to or other material to avoid exposure to light. Incubate plate static, at room temperature for 20 minutes.
  8. Immediately add 50 µL of ELISA Stop Solution into each of the wells. Mix gently.
  9. Read the absorbance at 450 nm with reference filter at 620 nm or 650 nm.

Typical Standard Curve

ACTH ELISA Assay Kit Standard Curve

Package Inserts


Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.

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