Rat IgE ELISA KIT
Research Reagent


For in-vitro laboratory use only


Please, read this instruction carefully before use.

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


Merit of the Kit

(1) Rat IgE can be measured rapidly (about 3.5hours).
(2) Small volume of sample (serum, plasma*:5ml) is enough
(3) Simple assay procedure without any pretreatment of samples.
(4) A wide assay range (1-100ng)
(5) Not influenced by hemolysis (hemoglobin concentration less than 40mg/dl).
(6) High specificity (Cross-reaction with IgG, IgA, IgM less than 0.01%).
*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 100ml x1
E:HRP-conjugated avidin 200ml x1
F:Chromogenic substrate reagent(TMB) 12ml x1
H:Reaction stopper (1M H2SO4) 12ml 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 1:100 with the buffer solution.
2 HRP-conjugated avidin
Prepare by diluting the concentrated solution to 1:100 with the buffer solution.
3 Chromogenic 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.


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 wells 3 times with the washing buffer by pipetting 250ml washing buffer and discarding. Thereafter, place the plate upside-down on paper towel for a while to remove the excess buffer.
(2)   Place 50ml of the standard IgE solution prepared above to the wells for standard curve.
(3)   Place 45ml of the buffer solution and 5ml of sample to sample wells.
(4)   Shake gently using preferably a microplate shaker.
(5)   Pipette 50ml of the biotin-conjugated antibody solution to each well, and shake gently using preferably a microplate shaker.
(6)   Stand the plate for 2 hr at room temperature(20-25C) for the reaction.
(7)   After the reaction, discard the reaction mixture, and rinse the wells 3 times with each 250ml washing buffer. And remove the excess buffer as stated in (1).
(8)   Pipette 100ml of HRP-avidin solution to each well, and shake gently on a microplate shaker.
(9)   Stand the plate for 1 hr at room temperature(20-25C) for the reaction.
(10) After the reaction, discard the solution, and rinse the wells 3 times with 250ml washing buffer, and remove the excess buffer.
(11) Pipette 100ml of the chromogenic substrate (TMB) reagent to each well, and shake gently on a microplate shaker.
(12) Stand the plate for 20 min at room temperature (20-25C) for the reaction.
(13) Pipette 100ml of the reaction stopper to each well to stop further color development, and shake gently.
(14) Measure absorbance of each well at 450 nm (sub-wave length, 620nm) within 30min.

Assay Range


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 absorbancies.
3. Multiply the IgE concentrations by dilution factors of the samples (in our assay procedure, dilution factor is 10) to obtain the IgE concentrations of the original samples.

    * If sample absorbancies are scale-out of the standard curve, further dilute the sample and measure again.
    * In computer calculation, use 3rd order regression.

Summary of Assay Procedure

Antibody-coated plate
      ¯
      Washing
      ¯
IgE standard solution or Buffer + sample; 50ml
      ¯
      Shaking
      ¯
Biotin-conjugated anti-IgE antibody; 50ml
      ¯
      Shaking , Reaction at room temp.(20 - 25C) for 2hr
      ¯
      Washing
      ¯
HRP-avidin; 100ml
      ¯
      Shaking , Reaction at room temp.(20 - 25C) for 1hr
      ¯
      Washing
      ¯
Chromogenic substrate solutin; 100ml
      ¯
      Shaking , Reaction at room temp.(20 - 25C) for 20min
      ¯
Reaction stopper; 100ml
      ¯
Shaking , Measurement of Absorbance(450nm,sub-wave length, 620nm)

Storage Conditions / Term of Validity

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

Reference

IgE in male SD rats of 5weeks old, n=7, serum sample (Shibayagi's data)
Mean: 45.5 ng/ml, (Standard deviation: 6.2ng/ml)
The value may change by keeping conditions.


      Former page Page top