Instructions for use

Growth Hormone HGH ELISAME E-0200R

hGH ELISAINTENDED USE
For the direct quantitative determination of Growth Hormone by an enzyme immunoassay in human serum.
PRINCIPLE OF THE TESTThe principle of the following enzyme immunoassay test follows a typical one-step capture or ‘sandwich’ typeassay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for hGH
is immobilized onto the microplate and another monoclonal antibody specific for a different region of hGH isconjugated to horse radish peroxidase (HRP). hGH from the sample and standards are allowed to bindsimultaneously to the plate and to the HRP conjugate. The washing and decanting steps remove any unbound
HRP conjugate. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated byaddition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of thecolour formed by the enzymatic reaction is directly proportional to the concentration of hGH in the sample.
A set of standards is used to plot a standard curve from which the amount of hGH in samples and controls can bedirectly read.
PROCEDURAL CAUTIONS AND WARNINGS
1. Users should have a thorough understanding of this protocol for the successful use of this kit. Reliable
performance will only be attained by strict and careful adherence to the instructions provided.
2. Control materials or serum pools should be included in every run at a high and low level for assessing thereliability of results.
3. When the use of water is specified for dilution or reconstitution, use deionized or distilled water.
4. In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit
reagents and human specimens.
5. All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly
before use. Avoid repeated freezing and thawing of reagents and specimens.
6. A standard curve must be established for every run.
7. The controls should be included in every run and fall within established confidence limits.
8. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagentstorage may be indicated when assay values for the controls do not reflect established ranges.
9. When reading the microplate, the presence of bubbles in the wells will affect the optical densities (ODs).
Carefully remove any bubbles before performing the reading step.
10. The substrate solution (TMB) is sensitive to light and should remain colourless if properly stored. Instability
or contamination may be indicated by the development of a blue colour, in which case it should not beused.
11. When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will comeinto contact with any metal parts.
12. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent,sample, standard and control.
13. Do not mix various lot numbers of kit components within a test and do not use any component beyond theexpiration date printed on the label.
14. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
LIMITATIONS
1. All the reagents within the kit are calibrated for the direct determination of hGH in human serum. The kit isnot calibrated for the determination of hGH in saliva, plasma or other specimens of human or animalorigin.
2. Do not use grossly hemolyzed, grossly lipemic, icteric or improperly stored serum.
3. Any samples or control sera containing azide or thimerosal are not compatible with this kit, as they may
lead to false results.
4. Only Standard A may be used to dilute any high serum samples. The use of any other reagent may lead tofalse results.
SAFETY CAUTIONS AND WARNINGS
POTENTIAL BIOHAZARDOUS MATERIAL
Human serum that may be used in the preparation of the standards and controls has been tested and found to
be non-reactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCVand Human Immunodeficiency Virus (HIV) and found to be negative. No test method however, can offer
complete assurance that HIV, HCV and Hepatitis B virus or any infectious agents are absent. The reagents should
be considered as potential biohazard and handled with the same precautions as applied to any blood specimen.

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CHEMICAL HAZARDS
Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any of thesereagents, wash with plenty of water. TMB is a suspected carcinogen.
SPECIMEN COLLECTION AND STORAGE
Approximately 0.1 ml of serum is required per duplicate determination. Collect 4–5 ml of blood into an
appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C forup to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human specimens
as possible biohazardous materials and take appropriate precautions when handling.
SPECIMEN PRETREATMENT
This assay is a direct system; no specimen pretreatment is necessary.
REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED
1. Precision pipettes to dispense 25, 50, 100 and 300 μl
2. Disposable pipette tips
3. Distilled or deionized water
4. Plate shaker
5. Microplate reader with a filter set at 450nm and an upper OD limit of 3.0 or greater*(see assay procedure step 10)
REAGENTS PROVIDED
Wash Buffer Concentrate – Requires Preparation X10

1. AA E-0030
Contents:One bottle containing buffer with a non-ionic detergent and a non-mercury preservative.

Volume:50 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

Preparation:

Dilute 1:10 in distilled or deionized water before use. If the whole plate is to be used dilute
50 ml of the wash buffer concentrate in 450 ml of water.
TMB Substrate - Ready To Use.

2. AA E-0055
Contents:

One bottle containing tetramethylbenzidine and hydrogen peroxide in a non-DMF or DMSO
containing buffer.

Volume:16 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

3. AA E-0080

Stopping Solution - Ready To Use.

Contents:

One bottle containing 1M sulfuric acid.

Volume:6 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.

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4. Standards and Controls- Ready To Use.
Listed below are approximate concentrations, please refer to vial labels for exact concentrations:Cat. no.

SymbolME E-0201

Standard A

Concentration
0 ng/ml

ME E-0202

Standard B

1 ng/ml

0.5 mlME E-0203

Standard C

5 ng/ml

0.5 mlME E-0204

Standard D

10 ng/ml

0.5 mlME E-0205

Standard E

25 ng/ml

0.5 ml
0.5 mlME E-0251

Standard F
Control 1

50 ng/ml

ME E-0252

Control 2

ME E-0206

StandardRefer to vial labels for expectedvalue and acceptable range!

Volume/Vial
2.0 ml

0.5 ml
0.5 mlContents:

hGH in a serum-based buffer with a non-mercury preservative. Prepared by spiking serumwith a defined quantity of hGH. Calibrated against World Health Organization (WHO) 1st IS80/505.

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months in unopened vials or as indicated on label. Once opened, the standards should be
used within 14 days or aliquoted and stored frozen. Avoid multiple freezing and thawing
cycles.
Assay Buffer - Ready To Use.

5. ME E-0213
Contents:

One bottle containing a protein-based buffer with a non-mercury preservative.

Volume:15 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
Mouse Anti-hGH Antibody-Coated Break-Apart Wells Microplate- Ready To Use.

6. ME E-0231
Contents:

One 96 well (12x8) monoclonal antibody-coated microplate in a resealable pouch withdesiccant.

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
Mouse Anti-hGH Antibody-Horseradish Peroxidase (HRP)
Conjugate Concentrate – Requires Preparation X100

7. ME E-0240
Contents:

Anti-hGH monoclonal antibody-HRP conjugate in a protein-based buffer with a non-mercury
preservative.

Volume:200 μl /vial

Storage:

Refrigerate at 2 - 8 oCStability:

12 months or as indicated on label.

Preparation:

Dilute 1:100 in assay buffer before use (eg. 20 μl of HRP in 2 ml of assay buffer). If thewhole plate is to be used dilute 120 μl of HRP in 12 ml of assay buffer. Discard any that is leftover.

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ASSAY PROCEDURE
Specimen Pretreatment: None
All reagents must reach room temperature before use. Standard, controls and specimen samples should beassayed in duplicate. Once the procedure has been started, all steps should be completed without interruption.
1.

Prepare working solutions of the anti-hGH-HRP - conjugate and wash buffer.

2.

Remove the required number of well strips. Reseal the bag and return any unused strips to therefrigerator.

3.

Pipette 25 μl of each standard, control and specimen sample into correspondingly labelled wells induplicate.

4.

Pipette 100 μl of the conjugate working solution into each well.
(We recommend using a multichannel pipette.)

5.

Incubate on a plate shaker (approximately 200 rpm) for 1 hour at room temperature..

6.

Wash the wells 3 times with 300 µl of diluted wash buffer per well and tap the plate firmly against
absorbent paper to ensure that it is dry. (The use of a washer is recommended.)7.

Pipette 100 μl of TMB substrate into each well at timed intervals.

8.

Incubate the plate on a plate shaker for 10-15 minutes at room temperature.
(or until Standard F attains dark blue colour for desired OD).

9.

Pipette 50 μl of stopping solution into each well at the same timed intervals as in step 7.

10.

Read the plate on a microplate reader at 450 nm within 20 minutes after addition of the stopping solution.
If the OD exceeds the upper limit of detection or if a 450 nm filter is unavailable, a 405 or 415 nm filter may
be substituted. The optical densities will be lower, however, this will not affect the results of donor/control
samples.

CALCULATIONS
1. Calculate the mean optical density of each standard duplicate.
2. Calculate the mean optical density of each unknown duplicate.
3. Subtract the mean absorbance value of the “0” standard from the mean absorbance values of the standards,
controls and serum samples.
4. Draw a standard curve on log-log paper with the mean optical densities on the Y-axis and the standard
concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve isrecommended.
5. Read the values of the unknowns directly off the standard curve.
6. If a sample reads more than 50 ng/ml then dilute it with Standard A at a dilution of no more than 1:10. The
result obtained should be multiplied by the dilution factor.
TYPICAL TABULATED DATA
Sample data only. Do not use to calculate results.

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Standard

OD 1

OD 2

Mean OD

A

0.074

0.072

0.073

Value(ng/ml)
0B

0.158

0.159

0.159

1C

0.574

0.580

0.577

5D

0.997

1.014

1.006

10E

2.021

2.009

2.015

25F

2.809

2.773

2.791

50

Unknown

0.549

0.561

0.555

5.0

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TYPICAL STANDARD CURVE
Sample curve only. Do not use to calculate results.

OD 450nm

101

0.1

15

1025

50

hGH (ng/ml)

PERFORMANCE CHARACTERISTICSSENSITIVITY
The lower detection limit is calculated from the standard curve by determining the resulting concentration of themean OD of Standard A (based on 10 replicate analyses) plus 2 SD. Therefore, the sensitivity of the Direct hGH
ELISA kit is 0.2 ng/ml.
SPECIFICITY (CROSS REACTIVITY)
The specificity of the Direct hGH ELISA kit was determined by measuring the apparent hGH value of Standard A
spiked with various levels of prolactin.
Substance
Prolactin
Calibrated against
WHO 3rd IS 84/500

Concentration
Range (ng/ml)
50100500
1000

Apparent hGHValue (ng/ml)
Not Detected
Not Detected
Not Detected
Not Detected

INTRA-ASSAY PRECISION
Three samples were assayed ten times each on the same standard curve. The results (in ng/ml) are tabulated
below:
Sample
123

Mean
1.46
12.33
41.87

SD
0.09
0.68
0.97

CV%
5.8
5.5
2.3

INTER-ASSAY PRECISION
hree samples were assayed ten times over a period of four weeks. The results (in ng/ml) are tabulated below:
Sample123

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Mean
2.95
19.29
36.06

Effective: 2019-06-06

SD
0.27
0.86
1.72

CV%
9.0
4.4
4.7

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RECOVERY
Spiked samples were prepared by adding defined amounts of hGH to three serum samples.
The results (in ng/ml) are tabulated below:
Sample1 Unspiked+1.0+5.0+50
2 Unspiked+1.0+5.0+50
3 Unspiked+1.0+5.0+50

Obs. ResultND
0.96
5.6
49
0.7
1.5
6.6
53
1.0
1.7
6.8
48.8

Exp. Result
1.0
5.0
50
1.7
5.7
50.7
2.0
6.0
51

Recovery %
96.0
112.0
98.0
88.2
115.8
104.5
85.0
113.3
95.7

LINEARITY
Three serum samples were diluted with Standard A. The results (in ng/ml) are tabulated below:Sample
1
1:2
1:5
1:10
2
1:2
1:5
1:10
3
1:2
1:5
1:10

Obs. Result
6.44
3.12
1.15
0.59
16.60
7.97
2.82
1.59
33.00
16
6.4
3.3

Exp. Result
3.22
1.29
0.64
8.30
3.32
1.66
16.5
6.6
3.3

Recovery %
96.9
89.1
92.2
96.0
84.9
95.8
97.0
97.0
100.0

HIGH DOSE HOOK EFFECTThe Direct hGH ELISA kit did not experience any high dose hook effect.
EXPECTED VALUESEach laboratory should collect data and establish their own range of expected normal values.

Version: 9.0-rGroupNMalesFemales
Premenopausal
Postmenopausal

120

95% Confidence Range(ng/ml)
ND-3.7120
120

ND-8.71
ND-3.09

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REFERENCES
1. Beck P, et al. Correlative Studies of Growth Hormone and Insulin Plasma Concentrations With Metabolic
Abnormalities in Acromegaly. J Lab Clin Med. 1965; 66(3):366–79.
2. Chochinov RH, Daughaday WH. Current Concepts of Somatomedin and Other Biologically Related GrowthFactors. Diabetes. 1976; 25(10):994–1004.
3. Chawla RK, Parks JS, Rudman D. Structural Variants of Human Growth Hormone: Biochemical, Genetic, andClinical Aspects. Ann Rev Med. 1983; 34:519–47.
4. Celniker AC, et al. Variability in the Quantitation of Circulating Growth Hormone Using CommercialImmunoassays. J Clin Endocrinol Metab. 1989; 68(2):469–76.
5. Daughaday WM, Cryer PE. Growth Hormone Hypersecretion and Acromegaly. Hosp Pract. 1978 Aug;13(8):75–80.
6. Engvall E. Enzyme Immunoassay ELISA and EMIT. Methods Enzymol. 1980; 70(A):419–39.
7. Eddy R L, et al. Human G rowth H ormone Release. Comparison of Provocative Test Procedures. Am J Med.
1974; 56(2):179–85.
8. Frasier SD. A Preview of Growth Hormone Stimulation Tests in Children. Pediatrics. 1974; 53(6):929–37.
9. Goldfine ID. Medical Treatment of Acromegaly. Ann Rev Med. 1978; 29:407–15.

Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2019-06-06

Contains sufficient for<n> tests

For research use
only!

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