Service manual of PPA- ELISA Kit for japanese encephalitis virus (JEV IgG) antibody

【Products Introduction】The japanese encephalitis (JE) is a central nervous system lesions of acute infectious diseases and is a natural foci of zoonotic diseases, caused huge losses of the domestic pig industry. The pig vaccinated japanese encephalitis virus vaccine, duing to a variety of factors that could cause does not produce or produce low levels of japanese encephalitis virus antibodies. JEV E protein is the major structural protein of the particle surface, the surface epitopes possess the hemagglutination activity and neutralizing activity, can stimulate the body to produce hemagglutination inhibition antibody, complement binding antibodies, neutralizing antibodies, causing the host to produce a protective immune response.The ELISA test kit applies the indirect ELISA principle to test the anti-E antibodies of japanese encephalitis virus in the serum. The japanese encephalitis virus ELISA test kit is used for detection of japanese encephalitis virus antibodies in the serum; assessment of immunity conditions against japanese encephalitis virus in the pig farm; and investigation of the epidemiology of the japanese encephalitis virus.

【Products components】

【Component traits】

【Operation Steps】

1. The equipment need to be prepared：

1.1 Pipette： 10μl、100μl、200μl和1000μl range.

1.2 Tips、500ml graduated cylinder，1.5ml EP，Microplate Reader.

1.3 Distilled or deionized water.

2. Sample preparation：

Dilution of samples to be tested serum samples were diluted 100 times (2μl of serum samples diluted by adding 198μl sample diluent solution), the sample need tofully mix before joining antigen-coated plates, dilution of the sample to be seized attention in different replacement tips.

Note: ① Preparation of blood serum samples should not be used for hemolytic phenomenon; ② can not dilute the negative and positive control serum, long time do not set the positive and negative serum and IgG put in -20 ℃ preservation.

3. Steps(* kit need to restore to room temperature and reagents need to shock mix before using.)

3.1A1and A2 well were added to 100μl negative control serum

3.2 A3and A4 well were added to 100μl positive control serum

3.3Other wells were added to 100μl the diluted sample(1:100 dilution) and make a record

3.4 ELISA plates were Incubated 1 hour under room temperature or 37℃ conditions

3.5Removed the reaction solution in microplate, and add 300μl wash solution per well to wash. Washing time should be controlled in 2-3 minutes,andWashing times should be controlled in 3-5 times.(*20×concentrated washing solution has diluted with distilled water or deionized water before use. If crystals, you must first dissolve before they are allowed dilution).

3.6 Added 100μl diluted enzyme conjugates (SPA-HRP) per well

3.7 ELISA plates were Incubated 1 hour under room temperature or 37℃ conditions

3.8 Repeat steps 3.5

3.9 Added 100μl per well of substrate solution

3.10 ELISA plates were Incubated in room temperature and dark conditions

3.11 At last, end the reaction by adding stop solution.

3.12 Test the OD value in each reaction well with ELISA at 450nm wavelength.

4. Result determination

4.1 Negative control: Under normal circumstances, the OD450 value of the negative control well ≤ 0.2.

4.2 Positive control: Under normal circumstances, the OD450 value of the positive control well≥0.4.

4.3 Critical value (CO) calculation:critical value =0.18 + negative control mean. If more than 0.20,the negative control OD450 value should be discarded. If all the negative control OD450 values are greater than 0.20, the experiment need to be repeated. Negative control well below the 0.05 to 0.05 calculated.

4.4 Results found: test specimens OD450 value ≥critical value to determine the sample was positive; test specimens OD450 value <critical value to determine the test sample was negative

【Attention】      

(1) Substrate solution and stop solution are irritating to the skin, pay attention to protection.

(2)Substrate solution not exposed to strong light and any oxidant.

(3) All reagents should be stored at 2 ~ 8 ℃. Return to room temperature before use, after use back into the 2 ~ 8 ℃.

(4) Attention to prevent contamination of kit components

(5)Do not use expired reagents, the composition of different batches of kits do not mix.

【Specification】96 wellx 2 /kit

【Storage and Expiry date】: store 2-8℃, not frozen, 6 months

【Sales Contact】WANG Jinliang                       Telephone：+86-543-3403060

【Technical Support】DONG LIN                       Telephone：+86-543-3405361

Veterinary diagnostic use only