Instructions for use

IGFBP-1 ELISAME E-0400R

IGFBP-1 ELISAINTENDED USE
For the direct quantitative determination of Insulin-Like Growth Factor Binding Protein-1 by an enzyme
immunoassay in human serum.
For research use only.
PRINCIPLE OF THE TESTThe principle of the following enzyme immunoassay test follows a typical two-step capture or ‘sandwich’ typeassay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for
IGFBP-1 is immobilized onto the microplate and another monoclonal antibody specific for a different regionof IGFBP-1 is conjugated to horse radish peroxidase (HRP). IGFBP-1 from the sample and standards areallowed to bind to the plate, washed, and subsequently incubated with the HRP conjugate. After a second
washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of thestopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour
formed by the enzymatic reaction is directly proportional to the concentration of IGFBP-1 in the sample.
A set of standards is used to plot a standard curve from which the amount of IGFBP-1 in samples andcontrols can be directly read.
PROCEDURAL CAUTIONS AND WARNINGS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.

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.
Control materials or serum pools should be included in every run at a high and low level for assessing thereliability of results.
When the use of water is specified for dilution or reconstitution, use deionized or distilled water.
In order to reduce exposure to potentially harmful substances, gloves should be worn when handling kit
reagents and human specimens.
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.
A Standard curve must be established for every run.
The controls should be included in every run and fall within established confidence limits.
Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent
storage may be indicated when assay values for the controls do not reflect established ranges.
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.
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 be used.
When dispensing the substrate and stopping solution, do not use pipettes in which these liquids will comeinto contact with any metal parts.
To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent,
sample, standard and control.
Do not mix various lot numbers of kit components within a test and do not use any component beyond
the expiration date printed on the label.
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 IGFBP-1 in human serum. The
kit is not calibrated for the determination of IGFBP-1 in saliva, plasma or other specimens of human oranimal origin.
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.

Version: 7.0-r

Effective: 2020-02-13

2/8

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 foundto be nonreactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies toHCV and 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 reagentsshould be considered a potential biohazard and handled with the same precautions as applied to any bloodspecimen.
CHEMICAL HAZARDS
Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any ofthese reagents, 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
for up to 24 hours or at -10 °C or lower if the analyses are to be done at a later date. Consider all humanspecimens 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 20, 50, 80, 100 and 300 μl
2. Disposable pipette tips
3. Distilled or deionized water
4. Plate shaker
5. Microplate reader with a filter set at 450 nm and an upper OD limit of 3.0 or greater*(see assay procedure step 13).
REAGENTS PROVIDED
1. AA E-0030

Wash Buffer Concentrate – Requires Preparation X10

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.
Stopping Solution - Ready To Use.

3. AA E-0080
Contents:

One vial 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.
Version: 7.0-r

Effective: 2020-02-13

3/8

4. Standards and Controls- Ready To Use.
Listed below are approximate concentrations, please refer to vial labels for exact concentrations:
ME E-0401

Cat. no.

SymbolStandard
Standard AConcentration
0 μg/lME E-0402

Standard B

1 μg/l

0.5 mlME E-0403

Standard C

5 μg/l

0.5 mlME E-0404

Standard D

30 μg/l

0.5 mlME E-0405

Standard E

100 μg/l

0.5 mlME E-0406

250 μg/l

0.5 mlME E-0451

Standard F
Control 1

ME E-0452

Control 2

Refer to vial labels for expectedvalue and acceptable range!

Volume/Vial
2.0 ml

0.5 ml
0.5 mlContents:

IGFBP-1 in a protein-based buffer with a non-mercury preservative. Prepared by spiking
buffer with a defined quantity of IGFBP-1.

Storage:

Refrigerate at 2-8oC

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.

5. ME E-0413
Contents:

Assay Buffer - Ready To Use.
One vial containing a protein-based buffer with a non-mercury preservative.

Volume:26 ml/bottle

Storage:

Refrigerate at 2 - 8 oC

Stability:

12 months or as indicated on label.
Mouse Anti-IGFBP-1 Antibody-Coated Break-Apart Well Microplate Ready To Use.

6. ME E-0431
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-IGFBP-1 Antibody-Horseradish Peroxidase (HRP)
Conjugate Concentrate – Requires Preparation X100

7. ME E-0440
Contents:

Anti-IGFBP-1 monoclonal antibody-HRP conjugate in a protein-based buffer with a nonmercury preservative.

Volume:250 μl /vial

Storage:

Refrigerate at 2 - 8 oC

Stability: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 the wholeplate is to be used dilute 120 μl of HRP in 12ml of assay buffer. Discard any that is left over.

Version: 7.0-r

Effective: 2020-02-13

4/8

ASSAY PROCEDURE(Specimen Pretreatment: None)
All reagents must reach room temperature before use. Standards, controls and specimen samples should beassayed in duplicate. Once the procedure has been started, all steps should be completed withoutinterruption.
1.

Prepare working solutions of the anti-IGFBP-1 - 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 samples into correspondingly labelled wells induplicate.

4.

Pipette 100 μl of the assay buffer into each well.
(We recommend using a multichannel pipette).

5.

Incubate on a plate shaker (approximately 200 rpm) for 30 minutes 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 the conjugate working solution into each well.
(We recommend using a multichannel pipette).

8.

Incubate on a plate shaker (approximately 200 rpm) for 30 minutes at room temperature.

9.

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)

10.

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

11.

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

12.

Pipette 50 μl of stopping solution into each well at the same timed intervals as in step 10.

13.

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 415nm filter may
be substituted. The optical densities will be lower, however, this will not affect the results of 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 Standard A from the mean absorbance values of theStandards, 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 curveis recommended.
5. Read the values of the unknowns directly off the Standard curve.
6. If a sample reads more than 220 μg/l, 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.
StandardOD 1
A
0.077
B
0.086
C
0.120
D
0.459
E
1.404
F
2.591
Unknown
0.120
Version: 7.0-r

OD 2
0.075
0.088
0.125
0.452
1.356
2.639
0.117Effective: 2020-02-13

Mean OD
0.076
0.087
0.123
0.456
1.380
2.615
0.119

Value (μg/l)
01530100250
4.55/8TYPICAL CALIBRATOR CURVESample curve only. Do not use to calculate results.

OD 450nm

101

0.1

0.01

1530100 250

IGFBP-1 (ug/L)

PERFORMANCE CHARACTERISTICSSENSITIVITY
The lower detection limit is calculated from the standard curve by determining the resulting concentration ofthe mean OD of Standard A (based on 10 replicate analyses) plus 2 SD. Therefore, the sensitivity of the DirectIGFBP-1 ELISA kit is 0.5 μg/l.
SPECIFICITY (CROSS REACTIVITY)
The specificity of the Direct IGFBP-1 ELISA kit was determined by measuring the apparent IGFBP-1 value ofStandard A spiked with the following compounds:
Substance
IGFBP-2
IGFBP-3
IGFBP-4
IGFBP-5

Concentration Range
Up to 5000 μg/l
Up to10,000 μg/l
Up to 5000 μg/l
Up to 5000 μg/l

Apparent IGFBP-1 Value (μg/l)
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 μg/l) are tabulated
below:
Sample
123

Mean
5.5
22117

SD
0.14
0.75
2.8

CV %
2.5
3.4
2.4

INTER-ASSAY PRECISIONThree samples were assayed ten times over a period of four weeks. The results (in μg/l) are tabulated below:
Sample123Version: 7.0-r

Mean
4.8
21113

Effective: 2020-02-13

SD
0.31
1.6
5.6

CV %
6.4
7.4
4.96/8

RECOVERY
Spiked samples were prepared by adding defined amounts of IGFBP-1 to three serum samples (1:1). Theresults (in μg/l) are tabulated below:
Sample1 Unspiked+6.5+35+174
2 Unspiked+6.5+35+174
3 Unspiked+6.5+35+174

Obs. Result
5.0
5.8
2090
20
1429100
1106280
155

Exp. Result
5.75
20
89.5
13.3
24.5
97
58.3
72.5
133

Recovery %
100.9
100.0
100.6
105.3
118.4
103.1
106.3
110.3
116.5

LINEARITY
Three serum samples were diluted with Standard A. The results (in μg/l) 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
13.5
6.9
3.4
1.6
38
20.9
8.24.212058.2
22.1
11.5

Exp. Result
6.8
3.4
1.4
197.6
3.8
60
24
12

Recovery %
101.5
100.0
114.3
110.0
107.9
110.5
97.0
92.1
95.8

HIGH DOSE HOOK EFFECTThe Direct IGFBP-1 ELISA kit did not experience a high dose hook effect when it was tested up to an IGFBP-1
concentration of 200,000 μg/l.
EXPECTED NORMAL VALUESEach laboratory should collect data and establish their own range of expected normal values.
GroupAdults

N55

Mean (μg/l)
4.4Abs. Range (μg/l)
0.6 - 14.4

REFERENCES
1. Rutanen EM. Insulin-Like Growth Factor Binding Protein-1 (Review) Seminars in Reprod Endocrinol. 1992;
10:154–63.
2. Rutanen EM, et al. Aging is Associated with Decreased Suppression of Insulin-Like Growth Factor Binding
Protein-1 by Insulin. J Clin Endocrinol Metab. 1993; 77(5):1152–5.
3. Rutanen EM, et al. Relationship Between Carbohydrate Metabolism and Serum Insulin-Like Growth Factor
System in Postmenopausal Women: Comparison of Endometrical Cancer Patients with Healthy Controls. JClin Endocrinol Metab. 1993; 77(1):199–204.
4. Rutanen EM, et al. Measurement of Insulin-Like Growth Factor Binding Protein-1 in Cervical/Vaginal
Secretions: Comparison with the ROM-Check Membrane Immunoassay in the Diagnosis of Ruptured FetalMembranes. Clin Chim Acta. 1993; 214(1):73–81.
5. Lockwood CJ, et al. Fetal Membrane Rupture is Associated with the Presence of Insulin-Like Growth Factor
Binding Protein-1 in Vaginal Secretions. Am J Obstet Gynecol.1994; 171(1):146–50.

Version: 7.0-r

Effective: 2020-02-13

7/8

Symbols:

Version: 7.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2020-02-13

Contains sufficient for<n> tests

For research use
only!

8/8