Instructions for use

IGF-1 ELISAME E-0500R

IGF-1 ELISA
1 INTRODUCTION
1.1 Intended Use
The IGF-1 ELISA is an enzyme immunoassay for the quantitative measurement of Insulin-like Growth Factor
1 (IGF-1) in serum.
2 PRINCIPLE OF THE TESTThe IGF-1 ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA), based on the principle ofcompetitive binding.
Samples, standards and controls are acidified and neutralized prior to the assay procedure.
The microtiter wells are coated with a monoclonal antibody directed towards an antigenic site on the IGF-1
molecule.
The pre-treated sample is incubated at room temperature with Conjugate (biotinylated IGF-1). The wells are
washed and then incubated with Enzyme Complex (Streptavidin-HRP-Complex).
After addition of the substrate solution, the intensity of colour developed is reverse proportional to theconcentration of IGF-1 in the sample.
3 WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.

This kit is for research use only.
All reagents of this test kit which contain human serum or plasma have been tested and confirmed
negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should betreated as potential biohazards in use and for disposal.
Before starting the assay, read the instructions completely and carefully. Use the valid version of the
package insert provided with the kit. Be sure that everything is understood.
The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch and used in the frame provided.
Pipetting of samples and reagents must be done as quickly as possible and in the same sequence foreach step.
Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using areservoir for dispensing a substrate solution that had previously been used for the conjugate solution
may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reusemicrowells.
Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
Allow the reagents to reach room temperature (21 °C to 26 °C) before starting the test. Temperature
will affect the absorbance readings of the assay. However, values for the samples will not be affected.
Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
Wear disposable latex gloves when handling specimens and reagents. Microbial contamination ofreagents or specimens may give false results.
Handling should be done in accordance with the procedures defined by an appropriate national
biohazard safety guideline or regulation.
Do not use reagents beyond expiry date as shown on the kit labels.
All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiter plate readers.
Do not mix or use components from kits with different lot numbers. It is advised not to exchange wellsof different plates even of the same lot. The kits may have been shipped or stored under different
conditions and the binding characteristics of the plates may result slightly different.
Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes orskin, flush immediately with water.
TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them. If inhaled, take the person to open air.
Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline or regulation.
For information on hazardous substances included in the kit please refer to Safety Data Sheets.
Safety Data Sheets for this product are available upon request directly from the manufacturer.

Version: 11.0b-r

Effective 2019-10-28

2/10

4 REAGENTS
4.1 Reagents provided
0.2 M HCl - ready to use

ME E-0519
Volume:2 x 3 mlfor sample acidification

Hazards
identification:
H290 May be corrosive to metals.
Neutralization Buffer - ready to use

ME E-0587
Volume:1 x 3 mlfor neutralization of samples

ME E-0531

Microtiterwells
12x8 (break apart) strips, 96 wells; Wells coated with anti-IGF-1 antibody(monoclonal)

Contents:

Standards and Controls - ready to use
Cat. no.

Component

ME E-0501

Standard

Concentration

Volume/Vial

Standard A

0 ng/ml

1 ml

ME E-0502

Standard B

10 ng/ml

1 ml

ME E-0503

Standard C

50 ng/ml

1 ml

ME E-0504

Standard D

150 ng/ml

1 ml

ME E-0505

Standard E

300 ng/ml

1 ml

ME E-0506

Standard F

600 ng/ml

1 ml

ME E-0551

Control Low

1 ml

ME E-0552

Control HighFor control values andranges please refer tovial label or QC-Report.

1 ml

Conversion:

1 ng/ml x 0.13 = nmol/lContent:

Contain non-mercury preservative
The standards are calibrated against the International Reference Reagent for IGF-1,
NIBSC code: 02/254.

ME E-0540

Enzyme Conjugate - ready to use

Content:

biotinylated IGF-1
contains non-mercury preservative.

Volume:1 x 14 ml

ME E-0515

Enzyme Complex - ready to use

Content:

Streptavidin HRP Complex
contains non-mercury preservative

Volume:1 x 20 ml

FR E-0055

Substrate Solution - ready to use

Content:

Tetramethylbenzidine (TMB)

Volume:1 x 14 ml

FR E-0080

Stop Solution - ready to use

Content:

contains 0.5 M H2SO4

Volume:1 x 14 ml

Avoid contact with the stop solution. It may cause skin irritations and burns.
Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
Version: 11.0b-r

Effective 2019-10-28

3/10

FR E-0030
Volume:Wash Solution - 40x concentrated
1 x 30 ml

see „Preparation of Reagents“.
Note: Additional Standard A for sample dilution is available upon request.
4.2 Materials required but not provided








A microtiter plate calibrated reader (450 nm, with reference wavelength at 620 nm - 630 nm)
Calibrated variable precision micropipettes
Absorbent paper
Distilled water
1.5 ml Reaction Cups (e.g. from Eppendorf) for Sample Preparation (Acidification and Neutralization)
Timer
Semi logarithmic graph paper or software for data reduction

4.3 Storage Conditions
When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not use
reagents beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once thefoil bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for 8 weeks if stored as described above.
4.4 Reagent Preparation
Bring all reagents and required number of strips to room temperature prior to use.
Wash Solution
Add deionized water to the 40X concentrated Wash Solution.
Dilute 30 ml of concentrated Wash Solution with 1170 ml distilled water to a final volume of 1200 ml.
The diluted Wash Solution is stable for 2 weeks at room temperature.
4.5 Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for thisproduct is given in the Safety Data Sheet.
4.6 Damaged Test Kits
In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing,at the latest, one week after receiving the kit. Severely damaged single components should not be used fora test run. They have to be stored until a final solution has been found. After this, they should be disposed
according to the official regulations.
5 SPECIMEN COLLECTION AND PREPARATION
Serum can be used in this assay.
NOTE: In plasma significantly reduced values were observed.
Note: Samples containing sodium azide should not be used in the assay.
In general it should be avoided to use haemolytic, icteric or lipaemic specimens. For further informationrefer to chapter “Interfering Substances”.
5.1 Specimen Collection
Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum bycentrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Donors
receiving anticoagulant therapy may require increased clotting time.
5.2 Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 24 hours at 2 °C to 8 °C prior to assaying
Specimens held for a longer time (at least one year) should be frozen only once at -20 °C prior to assay.
Thawed samples should be inverted several times prior to testing.

Version: 11.0b-r

Effective 2019-10-28

4/10

5.3 Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be
diluted with Standard A and re-assayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.
Example:a) dilution 1:2:b) dilution 1:10:

50 µl Serum + 50 µl Standard A (mix thoroughly)
10 µl Serum + 90 µl Standard A (mix thoroughly)

6 ASSAY PROCEDURE
6.1 General Remarks






All reagents and specimens must be allowed to come to room temperature before use. All reagents must
be mixed without foaming.
Once the test has been started, all steps should be completed without interruption.
Use new disposal plastic pipette tips for each standard, control or sample in order to avoid crosscontamination.
Absorbance is a function of the incubation time and temperature. Before starting the assay, it isrecommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will
ensure equal elapsed time for each pipetting step without interruption.
As a general rule the enzymatic reaction is linearly proportional to time and temperature.

6.2 Acidification and Neutralization of Samples, Standards and Controls
1.

Pipette 50 µl Sample, Standard and Control in 1.5 ml-Reaction caps (e.g. Eppendorf-Caps).
Please note: The standards should be acidified and neutralized too, according to the proceduredescribed below.

2.

Add 50 µl 0.2 M HCl.

3.

Mix and incubate for 30 minutes.

4.

For Neutralization add 10 µl Neutralization Buffer to all caps and mix the solution.
A pH check and correction of pH is not necessary.
Immediately (within 10 minutes) continue with the test procedure in chapter 6.3.

6.3 Test Procedure
Each run must include a standard curve
1.

Secure the desired number of Microtiter wells in the frame holder.

2.

Dispense 20 µl of each acidified and neutralized Standard, Control and samples with new disposabletips into appropriate wells.

3.

Dispense 100 µl Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4.

Incubate for 120 minutes at room temperature.

5.

Briskly shake out the contents of the wells.
Rinse the wells 3 times with diluted Wash Solution (400 µl per well). Strike the wells sharply on
absorbent paper to remove residual droplets.
Important note:
The sensitivity and precision of this assay is markedly influenced by the correct performance of thewashing procedure!

6.

Dispense 150 µl Enzyme Complex into each well.

7.

Incubate for 30 minutes at room temperature.

8.

Briskly shake out the contents of the wells.
Rinse the wells 3 times with diluted Wash Solution (400 µl per well). Strike the wells sharply on
absorbent paper to remove residual droplets.

9.

Add 100 µl of Substrate Solution to each well.

10.

Incubate for 15 minutes at room temperature.

11.

Stop the enzymatic reaction by adding 100 µl of Stop Solution to each well.

12.

Determine the absorbance (OD) of each well at 450 nm (reading) and at 620 - 630 nm(background subtraction, recommended) with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

Version: 11.0b-r

Effective 2019-10-28

5/10

6.4 Calculation of Results
1. Calculate the average absorbance values for each set of standards, controls and samples.
2. Using semi-logarithmic graph paper, construct a standard curve by plotting the mean absorbanceobtained from each standard against its concentration with absorbance value on the vertical (Y) axis
and concentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a
4 Parameter curve fit (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods).
Other data reduction functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples withconcentrations higher than that of the highest standard have to be further diluted or reported as> 600 ng/ml. For the calculation of the concentrations this dilution factor has to be taken into account.
6.4.1 Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time of
assay.
Standard

Optical Units (450 nm)

Standard A (0 ng/ml)

2.01

Standard B (10 ng/ml)

1.76

Standard C (50 ng/ml)

1.21

Standard D (150 ng/ml)

0.64Standard E (300 ng/ml)

0.41Standard F (600 ng/ml)

0.23

Version: 11.0b-r

Effective 2019-10-28

6/10

7 EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently normal healthy subjects, using the IGF-1 ELISA the following values
are observed:

101

2.5th
Percentile(ng/ml)65

50th
Percentile(ng/ml)94

97.5thPercentile(ng/ml)
172

Min.
Value
(ng/ml)
65

10

75

45

77

115

44

119

10

91

67

92

120

64

126

8

143

103

133

204

103

206

Age
(years)nMean(ng/ml)Newborn55

1
2
3

Max. Value(ng/ml)
184

4

11

121

71

119

195

65

208

5

10

165

130

160

232

126

248

6

11

162

80

170

231

62

236

7

10

171

102

169

236

95

242

8

12

200

156

199

249

150

251

9

13

191

61

207

270

44

275

10

11

227

132

237

273

108

278

11

13

227

139

246

308

136

315

12

9

284

174

279

372

158

375

13

3

295

203

265

411

200

419

14

6

269

153

255

416

151

424

15

11

269

119

303

375

111

386

16

9

229

106

228

379

88

407

17

6

251

175

243

349

174

355

18

4

244

186

222

338

184

346

19

5

256

168

246

387

163

402

20

3

317

246

346

363

240

363

21 - 25

23

191

97

175

304

92

304

26 - 30

7

211

137

218

278

133

284

31 - 35

8

184

120

193

229

115

229

36 - 40

5

232

190

220

294

188

300

41 - 45

14

154

107

156

216

105

228

46 - 50

8

150

86

164

196

84

200

51 - 55

17

148

102

140

212

100

214

56 - 60

18

151

96

141

217

91

218

61 - 65

11

130

81

124

216

79

217

66 - 70

10

119

80

117

176

79

183

71 - 75

15

124

57

124

199

46

212

76 - 80

14

122

74

115

192

73

205

Version: 11.0b-r

Effective 2019-10-28

7/10

8 QUALITY CONTROLGood laboratory practice requires that controls be run with each calibration curve. A statistically significant
number of controls should be assayed to establish mean values and acceptable ranges to assure proper
performance.
It is recommended to use control samples according to state and federal regulations. The use of control
samples is advised to assure the day to day validity of results. Use controls at both normal and pathologicallevels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC Report added to thekit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for
direct comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order toensure the accuracy of the results.
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do
not fit to the established acceptable ranges of control materials results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration
dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or the
manufacturer directly.
9 PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
The range of the assay is between 9.75 ng/ml – 600 ng/ml.
9.2 Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity of the assay:
Compound
IGF-1
IGF-2
Insulin% Cross reactivity
100
1.02
3.3

9.3 Sensitivity
The analytical sensitivity of the ELISA was calculated by subtracting 2 standard deviations from the mean of
20 replicate analyses of the Standard A and was found to be 9.75 ng/ml.
9.4 Reproducibility
9.4.1 Intra Assay
The within assay variability is shown below:
SamplenMean (ng/ml)

CV (%)

1
2

20

89.34

7.39

20

227.39

6.93

3

20

390.82

6.39

9.4.2 Inter Assay
The between assay variability is shown below:
SamplenMean (ng/ml)

CV (%)

1

40

14.50

14.84

2

40

66.26

10.34

3

40

125.3012.63

Version: 11.0b-r

Effective 2019-10-28

8/10

9.5 Recovery
Samples have been spiked by adding IGF-1 solutions with known concentrations in a 1:1 ratio.
The % recovery has been calculated by multiplication of the ratio of the measurements and the expectedvalues with 100 (expected value = (endogenous IGF-1 + added IGF-1) / 2; because of a 1:2 dilution ofserum with spike material).
Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Concentration (ng/ml)

91.60

158.08170.47173.55190.48Average Recovery (%)

103.5

102.0

95.8

109.2

95.9

from

96.8

96.6

86.1

98.8

87.5

to

106.6

106.0

114.9

126.4

101.2

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Concentration (ng/ml)

141.55

199.55201.36250.29301.28Average Recovery (%)

94.4

94.3

97.7

95.1

97.4

from

88.2

85.4

87.5

89.8

91.4

to

101.3

107.3

112.7

101.8

105.6

Range of Recovery (%)
9.6 Linearity

Range of Recovery (%)
10 LIMITATIONS OF USE

Reliable and reproducible results will be obtained when the assay procedure is performed with a complete
understanding of the package insert instruction and with adherence to good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.
10.1 Interfering Substances
Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.25 mg/ml) and Triglyceride (up to 30 mg/ml) have noinfluence on the assay results.
A biotin concentration of up to 1200 ng/ml in a sample has no influence on the assay results.
10.2 Drug Interferences
Until today no substances (drugs) are known to us, which have an influence to the measurement of IGF-1 ina sample.
10.3 High-Dose-Hook EffectNo hook effect was observed in this test.

Version: 11.0b-r

Effective 2019-10-28

9/10

12 REFERENCES / LITERATURE
1.

2.

3.
4.

5.

6.
7.

8.

9.

10.

11.

12.

13.
14.
15.

Daughaday E, Rotwein P: Insulin like growth factors I and II. Peptide, messenger ribonucleic acid andgene structures, serum and tissue concentrations.
Endocrin Rev 10:68-91, 1989.
Baxter RC, Martin JL, Beniac VA: High molecular weight insulin-like growth factor binding protein
complex.
J Biol Chem 264:11843-11848, 1989.
Rechler M: Insulin-like growth factor binding proteins.
Vit Horm 47:1-114, 1993.
Zapf J, Hauri C, Waldvogel M, Froesch ER: Acute metabolic effects and half-lives of intravenously
administrated insulin-like growth factors I and II in normal and hypophysectomized rats.
J Clin Invest 77:1768-1775, 1986.
Guler HP, Zapf J, Froesch ER: Short-team meatbolic effects of recombinant human insulin-like growth
factor-I in healthy adults.
New Engl J Med 317:1237-140,1987.
Costigan DC, Guyda HJ, Posner BI: Free insulin-like growth factor I (IGF-1) and IGF-1I in human saliva.
J Clin Enocrinal Metab 66:1014-1018, 1988.
Lewitt MS, Denyer GS, Cooney GJ, Baxter RC: Insulin-like growth factor-binding protein-1 modulates
blood glucose levels.
Endocrinology 129:2254-2256, 1991.
Lewitt MS, Saunders H, Baxter RC: Bioavailability of insulin-like growth factors (IGFs) in rats
determined by the molecular distribution of human IGF-binding protein-3.
Endocrinology 133:1797-1802, 1993
Lieberman SA et al.: Effects of recombinant human insulin-like growth factor-I (rhIGF-1) on total andfree IGF-1 concentrations, IGF-binding proteins, and glycemic response in humans.
J Clin Endocrinol Metab 75:30-36, 1992.
Schneiderman R, Maroudas A, Lee PDK: Concentrations of IGF-1 and its complexes in normal andosteoarthritic human cartilage: in situ values.
Orthopedic Res Soc, submitted, 1994
Brabant G et al.: German KIMS Board. Serum insulin-like growth factor I reference values for an
automated chemiluminescence immunoassay system: results from a multicenter study.
Horm Res. 2003;60(2):53-60.
Elmlinger MW et al.: Reference ranges for two automated chemiluminescent assays for serum insulinlike growth factor I (IGF-1) and IGF-binding protein 3 (IGFBP-3).
Clin Chem Lab Med. 2004;42(6):654-64.
Bonefeld K, Møller S: Insulin-like growth factor-I and the liver. Liver Int. 2011; 31(7):911-9.
Ameri P et al.: Interactions between vitamin D and IGF-I: from physiology to clinical practice.
Clin Endocrinol (Oxf). 2013; 79(4):457-63.
Bidlingmaier M et al.: Reference Intervals for Insulin-like Growth Factor-1 (IGF-I) From Birth toSenescence: Results From a Multicenter Study Using a New Automated Chemiluminescence IGF-I
Immunoassay Conforming to Recent International Recommendations
J Clin Endocrinol Metab, 2014, 99(5):1712–1721.

Symbols:

Version: 11.0b-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective 2019-10-28

Contains sufficient for<n> tests

For research use
only!

10/10