ELISA Kit to Detect the Antibody against Japanese encephalitis Reagents                                                                            

                                                                                      Volume
- Test plate, coat with JE                                          2 plate
- Positive control                                                        1 ml
- Negative control                                                      1 ml
- Goat anti swine IgG, labeled with HRP              10 ml
- 20× Wash concentrate                                           30 ml          
- Substrate solution A                                               10 ml          
- Substrate solution B                                               10 ml                                                                                                                          - Sample diluent                                                        100 ml    
- Stop solution                                                            10 ml          
- Serological Plate                                                      2 plate

Preparation
Wash Solution
   Determine the amount of 20× Wash concentrate needed for washing the test plates. Dilute the 20× Wash concentrate 1:20 with water (1 part concentrate with 19 parts water. This solution can be used during 7 days when stored between 2℃ and 8℃.

Sample Treatment
Serum to be tested: Diluted each serum to be tested 1:40 in the serological plate using sample diluent. (Example: 5μl serum + 195μl sample diluent)
Control: Diluted each control 1:40 in the serological plate using sample diluent. (Example: 6μl control + 234μl sample diluent)

Test Protocol
1. Dispense 100μl of treated samples and controls into each wells of the test plate. If only a  portion of a test plate is required , it is possible to use necessary amount. Incubate the plate for 30 minutes at 37℃.
2. Wash appropriate wells of test plate with 200μl of wash solution five times. Following each wash, aspirate liquid contents of all wells and tap residual wash fluid from each plate onto absorbent material.
3. Dispense 100μl of Goat anti swine IgG into each wells. Incubate the plate for 30 minutes at 37℃.
4. Repeat Step 2.
5. Dispense 50μl substrate solution A and substrate solution B into each well. Incubate the plate at room temperature (18~25℃) for 10 minutes.
6. Stop the color reaction by adding 50μl stop solution into each well.
7. Read the results using a photometer at a wavelength of 630nm within 10 minutes after the addition of the stop solution.

Results
  To validate the assay the optical density (OD) of the positive control must be≥0.6. The OD of the negative control should not exceed 0.3.

Interpretation of Results
 
OD ＞0.45 0.40-0.45 ＜0.40
interpretation positive suspect negative
 
Precautions and warning for users
? All reagents must be allowed to come to room temperature (18~25℃) before use.
? The reagents from different lots cannot mix use, and care should be taken to prevent contamination of kit components.
? Do not pipette by mouth.
? Do not expose substrate solution to strong light or any oxidizing agents.
? Do not open the sealing film on the antigen-coating plate that has not been used.
? When there are a lot of sera samples to be tested, first use a sera-diluting plate to dilute all samples and then transfer the diluted sera to the detection plate to make a unified reaction time.
? Use only distilled or deionized water for the reagents used in the test.
? An optimal result can be obtained when the operation strictly follows the instruction.

Pack Size   192 wells each kit

Storage    Tests must be storaged at 2~8℃.