DiaMetra HGH Elisa Kit

PRINCIPLE OF THE METHOD
In this method, the calibrators and the patient specimens and/or controls (containing the native HGH antigen) are first added to streptavidin coated wells. Biotinylated monoclonal and enzyme labelled antibodies are then added: these antibodies have high affinity and specificity and are directed against distinct and different epitopes of HGH. Reaction between the various HGH antibodies and native HGH occurs in the microwells without competition or steric hindrance, forming a soluble sandwich complex.

CLINICAL SIGNIFICANCE
Growth hormone is a polypeptide hormone synthesised and secreted by the anterior pituitary gland which stimulates growth and cell reproduction in humans and other vertebrate animals.
. GH is released from the pituitary into the bloodstream in a pulsatile manner under the regulatory control of hypothalamic somatostatin (SS) and GH-releasing factor (GHRF).
Stimulators of GH secretion include, exercise, hypoglycemia, dietary protein, and estradiol. Inhibitors of GH secretion include dietary carbhoydrate and glucocorticoids.
Almost 50% of GH in circulation is bound to a high affinity GH binding protein (GHBP), which represents the extracellular domain of the GH receptor.
The plasma concentration of GH during these peaks may range from 5 to 30 ng/mL or more. Peaks typically last from 10 to 30 minutes before returning to basal levels. The largest and most predictable of these GH peaks occurs about an hour after onset of sleep. Otherwise there is wide variation between days and individuals. Between the peaks, basal GH levels are low, usually less than 3 ng/mL for most of the day and night.
The amount and pattern of GH secretion change throughout life. Basal levels are highest in early childhood. The amplitude and frequency of peaks is greatest during the pubertal growth. Healthy children and adolescents average about 8 peaks per 24 hours. Adults average about 5 peaks. Basal levels and the frequency and amplitude of peaks decline throughout adult life.

PRECAUTIONS
Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use. 
All reagents should be stored refrigerated at 2-8°C in their original container. Any exceptions are clearly indicated. The reagents are stable until the expiry date when stored and handled as indicated.
Allow all kit components and specimens to reach room temperature (22-28°C) and mix well prior to use.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date.
If you use automated equipment, the user has the responsibility to make sure that the kit has been appropriately tested.
The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase the background. To improve the performance of the kit on automatic systems, it is recommended to increase the number of washes.
It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed, follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose response curve in each plate
Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition of the Stop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same sequence to eliminate any time deviation during the reaction.
Observe the guidelines for performing quality control in medical laboratories by assaying controls and/or pooled sera.
Maximum precision is required for reconstitution and dispensation of the reagents.
Samples microbiologically contaminated, highly lipemeic or haemolysed should not be used in the assay.
Plate readers measure vertically. Do not touch the bottom of the wells.

QUALITY CONTROL
Each laboratory should assay controls at levels of a low, normal, and high range for monitoring assay performance.
These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents.
Pertinent statistical methods should be employed to ascertain trends. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents.
Fresh reagents should be used to determine the reason for the variations.

RESULTS
Note
Optical densities (O.D.) higher than 2.0 could be out of the measurement range of some microplate readers. It is therefore necessary, for O.D.s higher than 2.0, to perform a reading at 405 nm (=wavelength of peak shoulder) in addition to 450 nm (peak wavelength) and 620 (reference filter for the subtraction of interferences due to the plastic).
For microplate readers unable to read the plate at 3 wavelengths at the same time,
it is advisable to proceed as follows:
Read the microplate at 450 nm and at 620 nm.
Read again the plate at 405 nm and 620 nm.
Find out the wells whose ODs at 450 nm are higher than 2.0
Select the corresponding ODs read at 405 nm and multiply these values at 405 nm by the conversion factor 3.0 (where OD 450/OD 405 = 3.0), that is: OD 450 nm = OD 405 nm x 3.0
Warning: The conversion factor 3.0 is suggested only. For better accuracy, the user is advised to calculate the conversion factor specific for his reader.