Mouse IgE ELISA KIT
Research Reagent


For in-vitro laboratory use only


Please, this instruction carefully before use.

This is a highly sensitive ELISA(Enzyme Linked Immunosorbent Assay) kit
for mouse IgE using sandwich method


Merit of the kit

1. Mouse IgE can be measured rapidly (5.5 hours).
2. Small mount of sample (serum, plasma*: 5 ml) is enough.
3. Simple assay procedure without any pretreatment of samples.
    *Use heparin to obtain plasma.

Contents of the Kit

A:Antibody-coated plate 96well(8x12) x1
B:IgE standard solution (100ng/ml) 600ml x1
C:Buffer solution 60ml x1
D:Biotin-conjugated anti-IgE antibody 10ml x1
E:HRP-conjugated avidin 20ml x1
F:Chromogenic substrate reagent(TMB) 10ml x1
H:Reaction stopper (1M H2SO4) 10ml x1
I:Concentrated washing buffer (10x) 100ml x1


Apparatus Necessary But not Included

(1) Micropipette (1-1000ml)
(2) Microplate washing apparatus (microplate washer, shaker, wash bottles, etc.)Microplate reader.

Preparation of Reagent Solutions

Use reagent solutions immediately after preparation.
Use all the reagent solutions of the Kit after getting back to room temperature.
(1) Biotin-conjugated anti-IgE antibody:
Prepare by diluting the concentrated solution to 1:1000 with the buffer solution.
(2) HRP-conjugated avidin
Prepare by diluting the concentrated solution to 1:2000 with the buffer solution.
(3) Cromogenic substrate reagent (TMB)
Use the attached solution without dilution.
(4) Concentrated washing buffer
Prepare by diluting concentrated washing buffer to 1:10 with distilled water.
(5) IgE standard solution
Prepare by diluting the original solution as an example shown below.
An example of preparing standard IgE solutions
Concentration(ng/ml) 100 75 50 25 10 1 0
IgE originail solution(ml) Orig. 150 100 50 20 5 0
Buffer solution(ml) 0 50 100 150 180 495 Buffer


Assay Procedure

(1)   Wash the plate 3 times with the washing buffer by filling the wells with the buffer and discarding. Thereafter, place the plate upside-down on paper towel f or a while to remove the excess buffer.
(2)   Place 50ml of the standard IgE solution prepared above to the wells for standard caurve.
(3)   Place 45ml of the buffer solution and 5ml of sample to sample wells.
(4)   Shake gently using preferably a microplate shaker.
(5)   Stand the plate for 2 hr at room temperature (20-25C) for the reaction.
(6)   After the reaction, discard the solution, and rinse the plate 3 times with the washing buffer. Thereafter, remove the excess buffer as described above.
(7)   Pipette 50ml of the biotin-conjugated antibody solution to each well, and shake gently using preferably a microplate shaker.
(8)   Stand the plate for 2 hr at room temperature (20-25C) for the reaction
(9)   After the reaction, discard the solution, and rinse the plate 3 times with the washing buffer. And remove the excess buffer.
(10) Pipette 50ml of HRP-avidin solution to each well, and shake gently on a microplate shaker.
(11) Stand the plate for 1 hr at room temperature (20-25C) for the reaction
(12) After the reaction, discard the solution, and rinse the plate 3 times with the washing buffer, and remove the excess buffer.
(13) Pipette 50ml of the chromogenic substrate (TMB) reagent to each well, and shake gently on a microplate shaker.
(14) Stand the plate for 20 min at room temperature (20-25C) for the reaction.
(15) Pipette 50ml of the reaction stopper to each well to stop further color development, and shake gently.
(16) Measure absorbance of each well at 450 nm (sub-wave length, 620nm) within 30 min.

Assay Range

1 ~ 100 ng/ml (with 5 ml sample)

Calculation for IgE concentration

1. Prepare a standard curve by plotting absorbances obtained from standard wells against IgE standard concentrations.
2. Obtain IgE concentrations of the samples from their absorbances.
3. Multiply the IgE concentrations by dilution factors of the samples to obtain the IgE concentrations of the original samples.
* If sample absorbances are scale-out of the standard curve, further dilute the sample and measure again.
* In computer calculation, use 3rd order regression.

Storage Conditions/Term of Validity

2 ~ 8C(Shield the kit from the light. Do not freeze.)/Valid period is indicated on the container.

Summary of Assay Procedure

Antibody-coated plate
      ¯
      Washing
      ¯
IgE standard solution or Buffer + sample; 50ml
      ¯
      Shaking , Reaction at room temp.(20 - 25C) for 2hr
      ¯
      Washing
      ¯
Biotin-conjugated anti-IgE antibody; 50ml
      ¯
      Shaking , Reaction at room temp.(20 - 25C) for 2hr
      ¯
      Washing
      ¯
HRP-avidin; 50ml
      ¯
      Shaking , Reaction at room temp.(20 - 25C) for 1hr
      ¯
      Washing
      ¯
Chromogenic substrate solutin; 50ml
      ¯
      Shaking , Reaction at room temp.(20 - 25C) for 20min
      ¯
Reaction stopper; 50ml
      ¯
Shaking , Measurement of Absorbance(450nm) (sub-wave length, 620nm)

Statements and Precautions as to Our Kits

*  This assay kit should be used only for research works.
*  The reagent solutions of the kit should be used principally immediately after dilution. Otherwise, keep them in a dark place at 2-8C,and use them within 3 days.
*  The reagents were prepared to give accurate results by their combination within the kit. So, do not combine the reagents in the kit of other lot number. Even the lot number is the same, do not mix the reagents with those that are preserved for some period.
*  Pipetting and dilution of the reagent solutions should be made accurately because these steps influence the assay precision.
*  Do not dry the assay plate to avoid denaturation of the coated antibody or antigen.
*  The reaction time should be counted from the onset of reagent pipetting.
*  Prepare the standard curve in every assay.(For KIT with standard solution.)
*  Dilution of the assay sample must be carried out using the buffer solution attached to the kit.
*  Preservation condition for the kit should be strictly kept.
*  Be careful not to allow the reagent solutions of the kit to contact to skin and mucus. Especially treat the stopping solution very carefully because it contains sulfuric acid.
*  In treating assay samples of animal origin, be careful for possible biohazards.


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