Download INTENDED USE:

Enzyme Linked Immunosorbent Assay
Goose Insulin-like growth factor-1
Collect Sample –Serum or blood plasma
Storage: 2 - 8 °C
Package size: 96 determinations
PRINCIPLE OF THE METHOD：
The IGF-1 kit is a solid phase phase sandwich
enzyme linked immuno sorbent assay(ELISA). Samples ,
including standards of known IGF-1 concentrations and
unknowns are pipetted into these wells. During the first
incubation, the IGF-1 antigen and a biotinylated
monoclonal antibody specific for IGF-1 are simultaneously
incubated.Afterwashing,
the
enzyme(streptavidin-peroxydase)is added. After incubation
and washing to remove all the unbound enzyme, a substrate
solution which is acting on the bound enzyme is added to
induce a coloured reaction product. The intensity of this
coloured product is directly proportional to the
concentration of IGF-1 present in the samples.
REAGENTS PROVIDED AND RECONSTTTUTION：
REAGENTS（Store
at 2-8℃）
96/48-wells
microtiter plates
Plastiv cover
Standard: 240pg/ml
Blank control
Standard Diluent
Biotinylated
anti-IGF-1
Streptavidin-HRP
Washing Buffer
Substrate A
Substrate B
Stopping Solution
1
×
WELLS
1
2
1Vials
(1.0ml)
1Vials
(1.0ml)
1Vials
(8.0ml)
1Vials
(8.0ml)
1Vials
(12ml)
1Vials
(20ml)
1Vials
(6.0ml)
1Vials
（6.0ml)
1Vials
(6.0ml)
96
0.5
×
WELLS
0.5
1
0.5Vials
(0.5ml)
1Vials
(0.5ml)
1Vials
(4.0ml)
1Vials
(4.0ml)
1Vials
(6.0ml)
1Vials
(10ml)
1Vials
(3.0ml)
1Vials
(3.0ml)
1Vials
(3.0ml)
96
MATERIAL REQUIRED BUT NOT PROVIDED：



Distilled water
Pipettes:10ul、50ul、100ul、200ul、1000ul。
Vortex mixer and magnetic stirrer.
SAFETY：
 For research use only
 Avoid any skin contact with H2SO4 and TMB. In
case of contact, wash thoroughly water.
 Do not eat, drink, smoke or apply cosmetics where
kit reagents are used.
 Do not pipette by mouth.
PROCEDURAL NOTES. QUALITY ONTROL：
 When not in use, kit components should be stored
refrigerated or frozen as indicated on vials or
bottles. All reagents should be warmed to room
temperature before use. Lyophilized standards
should be discarded after use.
 Once the desired number of strips has been
removed, immediately reseal the bag to protect the
remaining strips from edterioration.
 Cover or cap all reagents when not in use.
 Do not mis or interchange reagents between
different lots.
 Do not use reagents beyond the expiration date of
the kit .
 Use a clean disposable plastic pipette tip for each
reagent, standard, or specimen addition in order to
avoid cross-contamination, for the dispensing of
H2SO4 and substrate solution, avoid pipettes with
metal parts.
 Use a clean plastic container to prepare the
washing solution.
 Thoroughly mix the reagents and samples before
use by agitation or swirling.
 All residual washing liquid must be drained from
the wells by efficient aspiration or by decantation
followed by tapping the plate forcefully on
absorbent paper. Never insert absorbent paper
directly into the wells.
 The TMB solution is light sensitive. Avoid
prolonged exposure to light, also, avoid contact of
the TMB solution with metal to prevent colour
development. Warning TMB is toxic avoid direct
contact with hands. Dispose off properly. If a dark
blue colour develops within a few minutes after
preparation, this indicates that the TMB solution
has been contaminated and must be discarede.
Read absorbances within 1 hour after completion
of the assay.
 When pipetting reagents, maintain a consistent
order of addition from well-to-well. This will
ensure equal incubation times for all wells.
 Respect incubation times described in the assay
procedure.
SPECIMEN COLLECTION\ PROCESSING AND
STORAGE：






Serum---Avoid any inintentional stimulation of the
cells by the procedure. Use pyrogen\endotoxin free
collecting tubes. Serum should be removed rapidly
and carefully from the red cells after clothing. For
that, after clothing, centrifuge at approximately 1000
×g for 10 min and remove serum.
Plasma---EDTA\ citrate and heparin plasma can be
assayed. Spin samples at 1000×g for 30 min remove
particulates. Harvest plasma.
Cell culture supernatants---Remove particulates and
aggregates by spinning at approximately 1000×g for
10 min.
tissue homogenate-----Add normal saline to tissue
homogenate, 1000×g for 10 min, test supernatants
Storage---If not analyzed shortly after collection,
samples should be aliquoted(250-500ul) to avoid
freeze-thaw cycles and stored frozen at -70℃. Avoid
multiple freeze-thaw cycles of frozen specimens.
When possible, avoid use of badly hemolyzed or
lipemic sera. If large amounts of particles are present,
this should be removed prior to assay by
centrifugation or filtration.
Recommendation---Do not thaw by heating at 37℃
or 56℃. Thaw at room temperature and make sure
that sample is completely thawed and homogenous
before assaying.
PREPARATION OF REAGENTS：

Standards: Standard have to be reconstituled with the
volume of standard buffer diluent indicated on the
vial. This reconstitution produces a stock solution of
240pg/ml IGF-1. Allow standard to stand for 5
minutes with gentle swirling prior to making
dilutions. Serial dilutions of standard must be made
before each assys and cannot be stored.
Manual Technique：
STEP 1 ： Determine the number of microwell strips
required to test the desired number of samples,plus
appropriate number of wells needed for running blanks
standards. Each sample, standard and blank should be
assayed in duplicate. Remove sufficient microwell strips
from the pouch ， Add 50ul of standard diluent to
standard wells B1,B2, C1,C2, D1,D2, E1,E2,
F1,F2. Reconstitute standard vial with the appropriate
volume as described in the chapter reagents preparation.
Preparation. Pipet 100ul of standard into wells A1 and
A2 (see plate scheme below). Transfer 50ul from A1 and
A2 to B1 and B2 wells. Mix the contents by repeated
aspirations and ejections. Take care not to scratch the
inner surface of microwells. Repat this procedure from
the wells B1,B2 to wells C1,C2 and from wells C1,C2
to D1,D2 and so on creating two parallel rows of IGF-1
standard dilutions ranging，Add 50ul of standard diluent
to the bland wells. Add 50ul of sample to sample wells.
↓
STEP2：Add 50ul of diluted biotinylated anti-IGF-1 to
all wells.
↓
Incubate for 45 min.at 37℃Wash 5 times
↓
STEP3：Distribute 100ul of streptavidin-HRP solution to
all wells, including blank wells.
↓
Incubate for 30 min.at 37℃Wash 5 times
↓
STEP4： Add 50ul Substrate A and Substrate B to each
well。Incubate for 5 min at 37℃
↓
STEP 5 ：
Add 50μL of Stop Solution
↓
Read absorbance at 450nm within 30 min
CALCULATION OF RESULTS：

240pg/ml
120pg/ml
60pg/ml
30pg/ml
15pg/ml
7.5 pg/ml
0 pg/ml
6
Standard
5
Standard
4
Standard
3
Standard
2
Standard
1
Standard
Blank
Control
Original density 50ul。
100ul 6 Standard
+100ul diludent
100ul 5 Standard
+100ul diludent
100ul 4 Standard
+100ul diludent
100ul 3 Standard
+100ul diludent
100ul 2 Standard
+100ul diludent
50ul
Washing buffer 50 × concentrate:
distilled water.
Dilute 50 times in



Calculate the average absorbance values (A450) for
each set of reference standards, control, and
samples. Construct a standard curve by plotting the
mean absorbance obtained for each reference
standard against its concentration on linear graph
paper, with absorbance on the vertical (y) axis and
concentration on the horizontal (x) axis.Using the
mean absorbance value for each sample, determine
the corresponding concentration of in from the
standard curve.
The minimum detectable concentration in this
assay is estimated to be 0.1pg/ml.
Do not extrapolate the standard curve beyond the
max standard curve point. The dose-response is
non-linear in this region and good accruacy is
difficult to obtain.
This procedure will produce an adequate but less
precise fit of the data. If samples have been diluted,
the concentration read from the standard curve
must be multiplied by the dilution factor.