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Sincere Biotech supply ELISA KIT Type I , Type II and Type v(BS+)any customer 

can select the Kit according his /her research, Sincere Biotech promise to satisfy your 

all needs in higher level.

                 


Type I 

 

All the components in the kit should be stored up to 1 year at -20℃ and 2 months at 2-8℃,and should be kept according to the labels on vials. If the kit can not be used within 2weeks,
please keep in -20℃

 

ASSAY PROCEDURE

 Aliquot 100µl per well of the grades standard solutions into the  pre-coated 96 well plate. Add

    100µl of the Standard diluent buffer into the control wells. Add 100µl of each properly diluted

    sample of Serum, Plasma, Tissue lysates, Cell culture supernatants and other biological fluids to 

    each empty well. 

 Seal the plate with the Cover and incubate at 37°C for 90 min.

 Remove the cover, discard plate content, and blot the plate onto paper towels or other absorbent material. 

    Washing twice with 350 µl TBS each well, and each time let washing buffer stay in the wells around 1 min.

 Add 100µl of Biotinylated antibody working solution into each well and incubate the plate at 37°C for 60 min.

 Washing 3 times with 350 µl TBS each well,and each time let washing buffer stay in the wells around 1 min.

 Add 100µl of prepared ABC working solution into each well except the control well and incubate the plate 

    at 37°C for 30 min.

 Washing 5 times with 350 µl TBS each well ,and each time let washing buffer stay in the wells for around 1 min.

 Add 100µl of prepared TMB working solution into each well (include the control well)and incubate plate at 

   37°C for 30 min away from light. 

 Observe the color at all times, when shades of blue can be seen in the wells with the three - four maximum

    concentration of standard solutions,and the other wells show no obvious color,add 100µl of prepared TMB Stop

    solution into each well to stop the reaction. The color changes into yellow immediately.

 Read the O.D. absorbance at 450nm in the Microplate reader within 10 min after adding the TMB Stop solution 

    or even 620-630nm Secondary wavelength.

 


Type II

 

All the components in the kit should be stored up to months at 2-8℃,and should be kept according to

the labels on vials.  (Storage:2-8℃ Validity: 6 months)

 

ASSAY PROCEDURE

1) Preparation of the Standard: Set 10 Standard wells on the ELISA plates coated,label.

    Add Standard 100μl to , then add Standard diluent 50μl to , mix ; take out 100μl from,and then 

    add it to  separatelythen add Standard diluent 50μl to  ,mix ; then take out 50μl from,

    and discard, then take out 50μl fromand add to , then add Standard diluent 50μl to the, mix ;

    take out 50μl from  and add to, then add Standard diluent 50μl to the  ,mix ; take out 50μl from

    the  and add to , add Standard diluent 50μl to, mix , take out 50μl fromdiscard

2) Set wells separately: Set blank well ( Please do not add Sample and HRP- Conjugate reagent to the

    blank comparison wells,other each step operation is same), and Testing sample well. 

3) Add Sample: Add Sample Diluent 40μl to Testing sample well, then add testing sample 10μl (Sample final dilution

    is 5-fold) .Please add Sample to the bottom of pre-coated well , do not touch the well wall as far as possible, 

    and mix gently.

4) Incubate: After closing the plate with Closure plate membrane ,incubate for 30 min at 37.

5) Prepare the Washing Buffer: 30-fold Wash Solution, diluted 30-fold with Distilled water until 600ml,and reserve.

6) WashingUncover Closure plate membrane, discard Liquid, dry by swing, add Washing Buffer to each well, 

    still for 30s then drain, repeat 5 times, dry by pat.
7) Add enzymeAdd HRP-Conjugate Reagent 50μl to each well, except the Blank well.
8) IncubateOperation with 4).
9) WashingOperation with 6).
10) ColorAdd TMB Chromogen Solution A 50ul and then add TMB Chromogen Solution B 50ul to each well, mix gently,

    evade the light preservation for 15 min at 37
11) Stop the ReactionAdd Stop Solution 50μl to each well, to stop the reaction(the blue color change to yellow 

    color immediately).
12) AssaySet the OD of Blank well as zero , read absorbance at 450nm after adding Stop Solution within 15min.

Ø The judgment of result must take the OD of Microplate reader as a standard, when use the dual-wavelength to assay,

    reference wavelength is 630nm.

 


 A helpful guide to help you optimize your ELISA. View below  troubleshooting tips including:

  

Poor standard curve

                Cause

     Solution

Improper standard dilution

Confirm dilutions are made correctly

Standard improperly reconstituted

Make sure Standard all at bottom before opening; thoroughly resuspend powder

Standard degraded

Store/handle standard as the manual and label

Curve doesn't fit scale

Try plotting using different scale

 

  

No color present

                Cause

     Solution

Incubation time of reagent incorrect

Confirm the use time of Detection antibodies and ABC correct

Different kits or mix reagents from different lots.

Re-check and Not mix different kits or different batches of reagents.

Enzyme leakage

Inspection operation process

Curve doesn't fit scale

Try plotting using different scale

Buffer preparation of contamination

Please use new Buffer

Too low protein concentration beyond the Range

Concentrated specimens, re-test

Too high protein concentration beyond the Range

Diluted specimens, re-test

Using incompatible sample type

Detection may be reduced or absent in untested sample types

Sample prepared incorrectly

Ensure proper sample preparation/dilution. Samples may be incompatible with microtiter plate assay format.

 

  

Poor color developing

                Cause

     Solution

Over validity of the product

Checking the validity

Reagent and the sample can not balance prior to use

Re-check and Not mix different kits or different batches of reagents.

Shorter the incubation time

Check the incubation time

Incubation Temperature Unstable

Check and stable,avoid to vary

Reagent preparation of contamination

Please use new Buffer

Instrument settings are incorrect, filters do not match

Instrument is set correctly .

 

  

Standard curve well, but did not produce any positive signals

                Cause

     Solution

No target protein in the samples

Use nal control, repeat the experiment, the corresponding parameters reconsider

Stromal of specimens cover the detection

Re-check and Not mix different kits or different batches of reagents.

Drift

                Cause

     Solution

Inconsistent experiment procedure

Confirm the consistent procedure

The reagent not be balance prior to use

Balance the reagent

 

  

Large coefficient of variation (CV)

                Cause

     Solution

Sample loss bioactivity

Replace the sample and re-test

Buffer preparation of contamination

Please use new Buffer

Bubbles in wells

Ensure no bubbles present prior to reading plate

Wells not washed equally/thouroughly

Check that all ports of plate washer are unobstructed/wash wells as recommended

Edge effects

Ensure plate and all reagents are at proper temperature before beginning assay. Don't put plate near hot or cold air source that could alter temperature of wells on periphery but not interior wells

Well dry

DO NOT let the wells completely dry at any time

Incomplete reagent mixing

Ensure all reagents/master mixes are mixed thouroughly

Inconsistent pipetting

Use calibrated pipettes and proper technique to ensure accurate pipetting

Sample centrifuged insufficiency, coagulation reaction occurred or residualcellular components

Centrifuged  sufficiency

If you have any other questions ,please Contact us directly or send the FEEDBACK to us!


Here is some Abbreviation for your reference.

ChIP        Chromatin Immunoprecipitation

ELISA      Enzyme-linked Immunosorbent Assay (EIA)

FACS        Fluorescence-Activated Cell Sorting (FC, Flow cytometry)

HAI           Hemagglutination Inhibition Assay (HI)

HPLC        High Pressure Liquid Chromatography

IEP           Immunoelectrophoresis

IF/ICC     Immunofluorescence/Immunocytochemistry (IFA)

IP             Immunoprecipitation

IRMA        Immunoradiometric Assay

NEPH        Nephelometry

PCR          Polymerase Chain Reaction

P-ELISA    Peptide ELISA (assess sensitivity of antibody using synthetic peptide used as immunogen)

RIA           Radio Immuno Assay

RID           Radial Immunodiffusion

SDS-PAGE   Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis

WB           Western Blot (IB, immunoblot)

Cy3           Indocarbocyanine

Cy5           Indodicarbocyanine

FITC          Fluorescein Isothiocyanate

GFP           Green Fluorescent Protein

PerCP      Peridinin-Chlorophyll-Protein Complex

IHC           Immunohistochemistry

CLIA         Chemiluminescence immunoassay

 

 

 

 

 

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