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ACTH [Adrenocorticotropic Hormone]

 ELISA [Enzyme-Linked ImmunoSorbent Assay]

 

Specific quantitative assay for the determination of adrenocorticotropic hormone in plasma

 

 

Catalog # RDI-SDX018

$562.00/kit  -10% 2-5 kits -15% 6-9    -25% on 10+

 

 

 

 

For Research Use Only

 

 

 

 

Order through:  Research Diagnostics Inc

                          San Jose, 95123 CA Snell ave 658

                          Flanders NJ 07836              

                          (phone) 973-584-7093

                          (fax)      973-584-0210

                          web: http://www.researchd.com


INTENDED USE

 

This ACTH ELISA is intended for the quantitative determination of ACTH (Adrenocorticotropic Hormone) in human plasma.

 

SUMMARY AND EXPLANATION

 

ACTH (Adrenocorticotropic hormone) or corticotropin is a 39-amino acid peptide hormone (MW=4500) secreted by the pituitary to regulate the production of steroid hormones by the adrenal cortex.  ACTH secretion from the anterior pituitary is controlled by both a classical negative feedback control mechanism and CNS-stress mediated control system.1 Various types of stress or pain perceived in higher levels of the brain modulate secretion of the hypothalamic neurosecretory hormone, corticotropin releasing hormone (CRH), a 41-amino acid peptide.  CRH stimulates pituitary ACTH secretion.  The second peptide that modulates ACTH secretion is vasopressin (AVP).  AVP secretion is also stimulated by stress and acts synergistically with CRH to increase ACTH secretion in the pituitary portal circulation.  ACTH increases the synthesis and release of all adrenal sterioids, aldosterone, cortisol and adrenal androgens.  It is the principal modulator of cortisol, the most important glucocorticoid in man.  As the cortisol level in blood increases, release of ACTH is inhibited directly at the pituitary level.  Through this same mechanism, decreasing cortisol levels lead to elevated ACTH levels. 2,3,4,5

 

Biologically active ACTH results from enzymatic cleavage of a large precursor molecule, pro-opiomelanocortin (POMC).  This molecule contains within its structure the amino acid sequences of ACTH, Pro-ACTH, ß-melanocyte stimulating hormone, lipotropin, as well as endorphin and the enkephalins.  Because the reaction in immunoassays is determined by antigenic structure, not biological function, the usual ACTH RIA reacts with POMC, Pro-ACTH, ACTH and some fragments of the ACTH.5 

 

Like other pituitary hormones, ACTH is secreted in a pulsatile manner. These small pulses are superimposed on a characteristic diurnal fluctuation of greater amplitude.  In healthy individuals, ACTH reaches a peak in the early morning (6:00 - 8:00 hour) and levels become lowest late in the day and near the beginning of the sleep period.  Because of this diurnal rhythm it is customary to draw plasma ACTH samples between 8:00 and 10:00 hour.  However, differentiation of patients with Cushing’s disease from normal individuals may be best achieved on samples obtained in the evening (16:00 - 18:00 hour).  In Cushing’s disease and in ectopic ACTH syndromes, the diurnal pattern of ACTH secretion is generally absent.  Stress may also override the diurnal variation.

 

CLINICAL SIGNIFICANCE

 

Plasma ACTH assays are useful in the differential diagnosis of pituitary Cushing’s disease, Addison’s disease, autonomous ACTH producing pituitary tumors (e.g. Nelson’s syndrome), hypopituitarism with ACTH deficiency and ectopic ACTH syndrome. 5,6,7,8,9,10

 

Cushing’s syndrome is caused by the effects of excess glucocorticoid actions. All causes of Cushing’s syndrome, with the exception of glucocorticoid medication, are associated with incresed 24-hour urinary cortisol. The most common cause of Cushing’s syndrome is bilateral adrenal hyperplasia, due to pituitary ACTH hypersecretion (Cushing’s disease) from a pituitary adenoma or corticotroph hyperplasia.5,6,7,8,9,10 Laboratory diagnosis of Cushing’s disease is supported by the following: (1) suppression of plasma ACTH and cortisol concentrations, by  high-dose (2.0 mg q 6h x 8) dexamethasone administration, (2) absence of ACTH and cortisol suppression with low-dose (0.5 mg q 6h x 8 or 1 mg given at 23:30 hour) dexamethasone, (3) larger than normal response to metyrapone (Metopirone ) stimulation and normal or elevated plasma ACTH levels.4 

 

When Cushing’s syndrome is caused by primary adrenal abnormality (adenoma or carcinoma), the adrenal gland acts independently of ACTH and pituitary ACTH secretion is suppressed. 5,6,7,8,9,10 Hence, there is no response to dexamethasone suppresion or metyrapone stimulation.  This type of Cushing’s syndrome is characterized by very low, or undetectable levels of ACTH.

 

Therefore, measurement of plasma ACTH is helpful in differential diagnosis of pituitary Cushing’s syndrome.  In patients with adrenal tumors, ACTH levels are low.  High levels of ACTH are seen in patients with ectopic ACTH syndrome.  Patients with bilateral adrenal hyperplasia will have ACTH levels inappropriately elevated for their degree of hypercortisolism, which should suppress ACTH.  However, in most cases the ACTH concentration will be within the normal range.

 

Adrenocortical insufficiency or inadequate cortisol production can be due to destruction of the adrenal cortex or to abnormalities of the pituitary or hypothalamus, which result in inadequate ACTH production of release. 5,6,7,8,9,10 Primary adrenocortical insufficiency, Addison’s disease, is characterized by markedly elevated plasma ACTH levels and adrenal unresponsiveness to stimulation with exogenous ACTH.  Hypopituitarism with ACTH deficiency, which is secondary adrenocortical insufficiency, is characterized by low plasma ACTH and cortisol concentrations, and a subnormal, but usually distinct adrenal response to stimulation with synthetic ACTH (Cortrosyn®).  If hypoglycemic stress or metyrapone stimulation is required for diagnosis, ACTH and cortisol responses are less than normal. 

 

Aggressive and invasive ACTH producing pituitary tumors occurring before or following bilateral adrenalectomy for Cushing’s disease (Nelson’s syndrome) are characterized by the development of Addisonian pigmentation, often in an adrenalectomized patient who is taking adequate glucocorticoid replacement therapy.  In these patients, plasma ACTH levels are markedly elevated and do not respond well to dexamethasone suppression.

 

PRINCIPLE OF THE TEST

 

This ACTH Immunoassay is a two-site ELISA [Enzyme-Linked ImmunoSorbent Assay] for the measurement of the biologically active 39 amino acid chain of ACTH.  A goat polyclonal antibody to human ACTH, purified by affinity chromatography, and a mouse monoclonal antibody to human ACTH are specific for well defined regions on the ACTH molecule.  One antibody is prepared to bind only the C-terminal ACTH 34-39 and this antibody is biotinylated. The other antibody is prepared to bind only the mid-region and N-terminal ACTH 1-24 and this antibody is labeled with horseradish peroxidase [HRP] for detection.

Streptavidin Well  -  Biotinylated Anti-ACTH (34-39)  -- ACTH  -- HRP conjugated Anti-ACTH (1-24)

 

 

 

 


In this assay, calibrators, controls, or patient samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidin-coated microplate well.  At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB).  An acidic stopping solution is then added to stop the reaction and converts the color to yellow.  The intensity of the yellow color is directly proportional to the concentration of ACTH in the sample.    A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of ACTH present in the controls and patient samples are determined directly from this curve.

 

KIT COMPONENTS

 

Catalog

Number

Kit Components

Description

Quantity

30-1801

 

Reagent 1

Biotinylated ACTH Antibody [affinity purified goat anti human ACTH]

1 x 2.5 mL

30-1802

Reagent 2

Peroxidase (Enzyme) labeled ACTH Antibody [mouse monoclonal anti human ACTH]

1 x 2.5 mL

30-1613

ELISA Reagent A

ELISA Wash Concentrate [Saline with surfactant]

1 x 30 mL

30-1611

ELISA Reagent B

TMB Substrate [tetramethylbenzidine]

1 x 15 mL

30-1612

ELISA Reagent C

ELISA Stop Solution [1 N sulfuric acid]

1 x 10 mL

30-1601

Microplate

One holder with Streptavidin Coated Strips.

12 x 8-well strips

 

Refer to vial labels for exact concentrations

 

30-1803

30-1804

30-1805

30-1806

30-1807

30-1808

Calibrators

A:      0 pg/mL

B:   

C:   

D: 

E: 

F:

Lyophilized [except zero calibrator] synthetic h-ACTH.  Zero calibrator [BSA/equine serum solution] is in liquid form, ready to use.  All other calibrators consist of synthetic

h-ACTH (1-39) in BSA/equine serum solution

1 x 4 mL for  the

zero calibrator

 

1 x 2 mL for all

other calibrators

 

Refer to vial labels for exact ranges

 

30-1809

30-1810

Controls 1 & 2

 

Lyophilized.  2 Levels. Synthetic h-ACTH (1-39) in BSA/equine serum solution.

1 x 2 mL per level

 


MATERIAL AND Equipment REQUIRED BUT NOT PROVIDED

 

·        Microplate reader.

·        Microplate washer [if washer is unavailable, manual washing may be acceptable].

·        Precision Pipettors to deliver 25, 200, 100 and 150 µL.

·         (Optional): A multi-channel dispenser or a repeating dispenser for 25, 100 and 150 µL.

 

WARNINGS AND PRECAUTIONS FOR USERS

 

Although the reagents provided in this kit has been specifically designed to contain no human blood components, the human patient samples, which might be positive for HBsAg, HBcAg or HIV antibodies, must be treated as potentially infectious biohazard.  Common precautions in handling should be exercised, as applied to any untested patient sample.

 

ELISA Reagent C, Stop Solution, consists of 1 N Sulfuric Acid.  This is a strong acid.  Although diluted, it still must be handled with care.  It can cause burns and should be handled with gloves and eye protection, with appropriate protective clothing.  Any spill should be wiped immediately with copious quantities of water.  Do not breath vapor and avoid inhalation. 

 

SAMPLE COLLECTION AND STORAGE

 

The determination of  ACTH should be performed on EDTA plasma  To assay the specimen in duplicate, 400 µL of EDTA plasma is required.  Collect whole blood in a lavender [EDTA] tube.  After allowing blood to clot, the plasma should be promptly separated, preferably in a refrigerated centrifuge, and stored at -20oC or lower. EDTA plasma samples may be stored up to 8 hours at 2-8°C.  EDTA plasma samples frozen at -20°C are stable for up to 4 months.

 

REAGENT PREPARATION AND STORAGE

 

Store all kit components at 2-8 oC except Wash Concentrate and Stop Solution

 

1.     All reagents except the non-zero calibrators, kit controls and the Wash Concentrate are ready-to-use.  Store all reagents at 2-8 oC, except the Wash Concentrate, which should be kept at room temperature until dilution to avoid precipitation.

 

2.     For each of the non-zero calibrators (Calibrator B through F) and kit controls 1 and 2, reconstitute each vial with 2 mL of distilled or deionized water and mix.  Allow the vial to stand for 10 minutes and then mix thoroughly by gentle inversion to insure complete reconstitution.  Use the calibrators and controls as soon as possible upon reconstitution.  Freeze (-20oC) the remaining calibrators and controls as soon as possible after use.  Standards and controls are stable at -20 oC for 6 weeks after reconstitution with up to 3 freeze thaw cycles when handled as recommended in “Procedural Notes” section.

 

3.     ELISA Reagent A: Wash Concentrate: Mix contents of wash concentrate thoroughly.  Add wash concentrate (30 mL) to 570 mL of distilled or deionized water and mix.


ASSAY PROCEDURE

 

1.      Place sufficient Streptavidin Coated Strips in a holder to run all six (6) ACTH calibrators, A - F of the  ACTH CALIBRA­TORS [Exact concentration is stated on the vial label], Quality Control Plasma and patient samples. 

 

2.      Pipet 200 µL of sample into the designated or mapped well.  Freeze (-20oC) the remaining calibrators and controls as soon as possible after use.

 

3.      Add or dispense 25 µL of Reagent 1 (Biotinylated Antibody) into each of the wells which already contain the sample.

 

4.      Add or dispense 25 µL of Reagent 2 (Enzyme Labeled Antibody) into each of the same wells.

 

Cover the microplate(s) with aluminum foil or a tray to avoid exposure to light, And place it on an orbital shaker or rotator set at 170 + 10 rpm for 4 hours + 30 minutes at room temperature (22­o28oC).

 

5.      Aspirate and wash each well five (5) times with the Working Wash Solution (prepared from ELISA Reagent A), using an automatic microplate washer.  Blot dry by inverting the plate on an absorbent material.  The wash solution volume should be set to dispense 0.35 mL into each well.

 

6.      Add or dispense 150 µL of the ELISA Reagent B  (TMB Substrate) into each of the wells.

 

7.      With appropriate cover to avoid light exposure, place the microplate(s) on an orbital shaker or rotator set at 170 + 10 rpm for 30 +5 minutes at room temperature (22­o28oC).

 

8.      Add or dispense 100 µL of the ELISA Reagent C (Stop Solution) into each of the wells.  Mix gently.

 

9.      Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to 450 nm against 250 µL of distilled or deionized water.  Read the plate again with the reader set to 405 nm against distilled or deionized water.

 

Note: The second reading is designed to extend the analytical validity of the calibration curve to the value represented by the highest calibrator, which is approximately 500 pg/mL. Hence, patient samples with ACTH > 150 pg/mL can be quantified against a calibration curve consisting of the readings all the way up to the concentration equivalent to the highest calibrator using the 405 nm reading, away from the wavelength of maximum absorbance.  In general, patient and control samples should be read using the 450 nm for ACTH concentrations up to 150 pg/mL.  ACTH concentrations above 150 pg/mL should be interpolated using the 405 nm reading.

 

10.   By using the final absorbance values obtained in the previous step, construct a calibration curve via cubic spline, 4 parameter logistics, or point-to-point interpolation to quantify the concentration of the  ACTH.


PROCEDURAL NOTES

 

·        ACTH 1-39 is a very labile molecule.  Set up the assay immediately upon the reconstitution or the thawing of all calibrators, controls, and patient samples.

 

·        It is recommended that all calibrators, controls, and patient samples are assayed in duplicate.  The average absorbance units of duplicate sets should then be used for reduction of data and the calculation of results. 

 

·        The samples should be pipetted into the well with minimum amount of air-bubble. To achieve this, “reverse pipet” described in the package insert of the manufacturers of Pipettors is recommended.

 

·        Patient samples with values greater than the highest calibrator (Calibrator F), which is approximately 500 pg/mL (see exact concentration on vial label), may be diluted with Calibrator A (Zero Calibrator) and reassayed.  Multiply the result by the dilution factor.

 

·        Reagents from different lot numbers must not be interchanged.

 

·        If preferred, mix in equal volumes, in sufficient quantities for the assay, Reagent 1 (Biotinylated Antibody) and Reagent 2 (Enzyme Labeled Antibody) in a clean amber bottle, Then use 50 µL of the mixed antibody into each well.  This alternative method should replace Step (3) and (4), to be followed with the incubation with orbital shaker.

 

Assay Protocol Flow-Diagram

 

Microplate

Well Position

Sample

CalibratorsControls

OR

Patients

Reagent 1

Biotinylated Antibody

Solution

Reagent 2

Enzyme Conjugate Antibody

Incubate at room temperature

 

Working Wash Solution

 

ELISA Reagent B TMB Substrate

Incubate

At room temp.

 

ELISA Reagent C Stop (Acid) Solution

Read Aborbance

At 450 nm and 405 nm

A1

 

 

B1

C1

D1

E1

F1

G1

H1

A2

B2

C2

D2

E2

F2

Distilled Water

 

Calibrator A

Calibrator B

Calibrator C

Calibrator D

Calibrator E

Calibrator F

Control 1

Control 2

Patient 1

Patient 2

ETC.

ETC.

ETC.

250 µL

 

 

200 µL

 

 

 

 

25 µL

 

 

 

 

25 µL

 

 

 

 

 

4 + ½ hours

 

@ 170 +

10 rpm

 

 

 

 

350 µL

 

 

 

 

Wash 5 times

Aspirate

 

 

 

 

150 µL

 

 

 

 

 

30 + 5 minutes

 

@ 170 +

10 rpm

 

 

 

 

100 µL

 

Read against distilled or deionized water

 

CALCULATION OF RESULTS

 

Manual Method

 

1.      For the 450 nm readings, construct a dose response curve (calibration curve) using the first five calibrators provided, i.e. Calibrators A, B, C, D and E.  For the 405 nm readings, construct a second dose response curve using the three calibrators with the highest concentrations, i.e. Calibrators D, E and F.

 

2.      Assign the concentration for each calibrator stated on the vial in pg/mL.  Plot the data from the calibration curve on linear graph paper with the concentration on the X-axis and the corresponding A.U. on the Y-axis.

 

3.      Draw a straight line between 2 adjacent points.  This mathematical algorithm is commonly known as the "point-to-point" calculation.  Obtain the concentration of the sample by locating the absorbance unit on the Y-axis and finding the corresponding concentration value on the X-axis. Patient and control samples should be read using the 450 nm for ACTH concentrations up to 150 pg/mL.  ACTH concentrations above 150 pg/mL should be interpolated using the 405 nm reading.

4.       

Automated Method:

 

Computer programs using cubic spline or 4 PL [4 Parameter Logistics] can generally give a good fit.

 

 

 

 

Sample Data at 450 nm [raw A.U. readout against distilled or deionized water]

 

Microplate Well

1st Reading Absorbance Unit

2nd Reading Absorbance Unit

 

 Average Absorbance Unit

 

 

 ACTH  pg/mL

 

 ACTH  pg/mL –Result to report

Calibrator A

0.020

0.018

0.019

 

0

Calibrator B

0.077

0.074

0.076

 

5

Calibrator C

0.221

0.229

0.225

 

18

Calibrator D

0.624

0.692

0.685

 

55

Calibrator E

1.802

1.934

1.868

 

165

Control 1

0.417

0.398

0.408

   33.5

  33.5

Control 2

2.868

2.774

      2.821

    > 150

*

Patient Sample 1

0.072

0.078

0.075

  4.9

 4.9

Patient Sample 2

0.185

0.177

0.181

  14.0

 14.0

Patient Sample 3

0.495

.491

.493

40.8

40.8

Patient Sample 4

2.090

2.122

2.106

> 150

*

 

* Because the concentration readout is > 150 pg/mL, it is recommended to use the data obtained at 405 nm as shown in Sample Data at 405 nm in the table below.

 

Sample Data at 405 nm [raw A.U. readout against distilled or deionized water]

 

Microplate Well

1st Reading Absorbance Unit

2nd Reading Absorbance Unit

 

 Average Absorbance Unit

 

 

 

 ACTH  pg/mL

 

 ACTH  pg/mLResult to report

Calibrator A

0.011

0.008

0.0095

 

  0

Calibrator D

0.032

0.032

0.032

 

 55

Calibrator E

0.074

0.081

0.078

 

165

Calibrator F

1.838

1.817

1.828

 

500

Control 1

0.138

0.132

0.135

< 150

Control 2

0.921

0.894

      0.908

   256

256

Patient Sample 1

0.030

0.032

0.031

< 150

Patient Sample 2

0.068

0.062

0.065

< 150

Patient Sample 3

0.165

0.159

      0.162

< 150

Patient Sample 4

0.663

.677

0.670

188

188

 

For samples with readout < 150 pg/mL, it is recommended to use the data obtained at 450 nm as shown in Sample Data at 450 nm in the table above. This practice should give the results with optimum sensitivity of the assay.

 

NOTE:    The data presented are for illustration purposes only and must not be used in place of data generated at the time of the assay.

 

 

QUALITY CONTROL

 

Control plasma or plasma pools should be analyzed with each run of calibrators and patient samples. Results generated from the analysis of the control samples should be evaluated for acceptability using appropriate statistical methods.  In assays in which one or more of the quality control sample values lie outside the acceptable limits, the results for the patient sample may not be valid.

 

LIMITATIONS OF THE PROCEDURE

 

This ACTH ELISA kit has exhibited no “high dose hook effect” with samples spiked with 20,000 pg/mL of ACTH.  Samples with ACTH levels greater than the highest calibrator, however, should be diluted and reassayed for correct values.

 

Like any analyte used as a diagnostic adjunct, ACTH results must be interpreted carefully with the overall clinical presentations and other supportive diagnostic tests.

 

EXPECTED VALUES

 

ACTH levels were measured in eighty-three (83) apparently normal individuals in the U.S. with this ACTH ELISA.  The values obtained ranged from 7.9to 66.1 pg/mL.  Based on statistical tests on skewness and kurtosis, the population, when transformed logarithmically, follows the normal or Gaussian distribution as shown in the histograms.  The geometric mean + 2 standard deviations of the mean were calculated to be 8.3to 57.8 pg/mL

 ACTH Histograms

 

 

 


Raw ACTH Results

 

Logarithmically Transformed

 

.

 



PERFORMANCE CHARACTERISTICS

 

Accuracy

 

One hundred seventeen (117) patient samples, with ACTH values ranging from 1.5 to  1045

pg/mL were assayed by this ELISA procedure and the Nichols IRMA (immunoradiometric assay) ACTH kit.  Linear regression analysis gives the following statistics:

 

ELISA = 0.976   IRMA Kit + 4.2 pg/mL            r = 0.995          N = 117

 

 

 

 


 

 

Sensitivity

 

The sensitivity, or minimum detection limit, of this assay is defined as the smallest single value, which can be distinguished from zero at the 95% confidence limit.   This ACTH ELISA has a calculated sensitivity of 0.46 pg/mL.


Precision and Reproducibility

 

The precision (intra-assay variation) of this ACTH ELISA Test was calculated from 21 replicate determinations on each of the two samples. 

 

Intra-Assay Variation

 

   Mean Value                                      Coefficient

Sample                   (pg/mL)                             N        of Variation

                                                                                       %                 

    A                                  35.7                           21                    3.1

  B                                255                              21                    4.2

             

 

The total precision (inter-assay variation) of this ACTH ELISA Test was calculated from data on two samples obtained in 35 different assays, by three technicians on three different lots of reagents, over a nine-week period

 

Inter-Assay Variation

 

   Mean Value                                       Coefficient

Sample                        (pg/mL)                        N        of Variation

                                                                                       %                 

    A                              35.2                             35                    5.8

                B                            230                                35                    6.2

 

Specificity and Cross-Reactivity

 

Cross-reactivity in the ACTH was studied by the addition of various materials to an ACTH standard.  The results are as follows:

 

Cross-reactant

Concentration

ACTH

ACTH

Change in

%

 

 

of

without

With

ACTH

Cross-reactivity

 

 

Cross-reactant

Cross-reactant

Cross-reactant

[pg/mL]

 

 

 

 

[pg/mL]

[pg/mL]

 

 

 

ACTH (1-24)

100,000 pg/mL

74.5

3.1

-71.4

-0.07%

 

 

10,000 pg/mL

74.5

17.1

-57.4

-0.57%

 

 

1,000 pg/mL

74.5

60.9

-13.6

-1.36%

 

 

200 pg/mL

74.5

68

-6.5

-3.25%

 

 

 

 

 

 

 

ACTH (18-39)

5,000 pg/mL

67

19

-48

-0.96%

 

 

2,000 pg/mL

67

26.8

-40.2

-2.01%

 

 

500 pg/mL

67

43.3

-23.7

-4.74%

 

 

 

 

 

 

 

 

a-MSH

100,000 pg/mL

72.3

1.3

-71

-0.07%

 

 

10,000 pg/mL

72.3

9.8

-62.5

-0.63%

 

 

1,000 pg/mL

72.3

44.5

-27.8

-2.78%

 

 

200 pg/mL

72.3

61

-11.3

-5.65%

 

 

 

 

 

 

 

 

b-ENDORPHIN

100,000 pg/mL

76.3

69.3

-7

-0.01%

 

 

50,000 pg/mL

76.3

73.5

-2.8

-0.01%

 


Recovery

 

Various amounts of ACTH were added to four different patient plasma to determine the recovery.  The results are described in the following table:

 

Plasma

Sample

Endogenous

ACTH

Expected

Measured

Recovery

 

ACTH

added

Value

Value

(%)

 

(pg/mL)

(pg/mL)

(pg/mL)

(pg/mL)

 

A

23.3

--

--

--

--

 

21.0

50.0

71.0

75.0

105.6%

 

18.6

100.0

118.6

126.0

106.2%

B

28.1

--

--

--

--

 

25.3

50.0

75.3

80.7

107.2%

 

22.5

100.0

122.5

142.0

115.9%

C

21.8

--

--

--

--

 

19.6

50.0

69.6

67.6

97.1%

 

17.4

100.0

117.4

125.0

106.4%

D

9.8

--

--

--

--

 

8.8

50.0

58.8

51.6

87.7%

 

7.8

100.0

107.8

96.4

89.4%

 

Kinetic Effect of the Assay

 

To determine whether there is any systematic kinetic effect between the beginning of the run and the end of the run, three spiked patient pools, selected to represent a good cross section of the ACTH concentration, were placed in sequence throughout the run of one microplate or 96 wells [with twelve 8-well strips].  The results, displayed in the following graphs, show no significant assay drift.

 


Linearity of Patient Sample Dilutions:  Parallelism

 

Five patient plasma samples were diluted with Calibrator A (Zero Calibrator).  Results in pg/mL are shown below:

 

 

REFERENCES:

1.    Ryan, WG: Endocrine Disorders – A Pathophysiiologic Approach, 2nd Edition Year Book Medical Publishers, Inc. 1980.

2.    Watts,  N.B.,  J.H. Keffer: Practical Endocrine Diagnosis, Third Edition, Lea and Febioer, 1982.

3.    Ganong, WF. L.D. Alber, TC Lee: ACTH  and the Regulation of Adrenocorticol Secretion, N. Engl. J. Med. 290 : 1006, 1974.

4.    Tepperman, J: Metabolic and Endocrine Physiology, 4th Edition, Year Book Medical Publishers, Inc., 1981.

5.    Odell, W.D., R. Horton, M.R. Pandian,  J. Wong: The Use of ACTH and Cortisol Assays in the Diagnosis of Endocrine Disorders. Nichols Institute Publication, 1989.

6.    Radioimmunoassay Manual, Edited by  A.L. Nichols and J.C. Nelson, 4th Edition Nichols Institute, 1977.

7.    Gold, E.M.: The Cushing’s Syndromes: Changing Views of Diagnosis and Treatment. Ann Intern. Med. 90:829, 1979.

8.    Plasma Cortisol, RIA for Physicians, Edited by J.C. Travis, 1:8, Scientific Newsletter, Inc. 1976.

9.    Krieger, D.T.: Physiopathology of Cusihing’s Disease, Endocrine Review 4:22-43, 1983.

10. Krieger, D.T., A.S. Liotta,  T. Suda, A Goodgold, and E. Condon: Human Plasma Immunoreactive Lipotropin and Adrenocorticotropin in Normal Subjects and in Patients with Pituitary-Adrenal Disease, J. Clin. Endocrinol Metab. 48:566-571, 1979.

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