Question

1. You are the head of a bioanalytical laboratory in a hospital. Doctors asked you to check the presence of Hepatitis virus (the dsDNA-based virus) in new patients arriving in the emergency room. Please suggest a set of appropriate bioanalytical techniques to solve the problem

2. Your colleague recently discovered a new mouse antibody. This antibody binds to known bacteria and eliminates it from blood. He asked you to isolate 100 micrograms of the active antibody from 1 mL. of mouse serum. Please, develop a protocol involving serval preparative and analytical techniques for the efficient isolation of the antibody from the blood serum, its purity check and antigen binding activity validation

Answer 1

1. The presence of Hepatitis virusn( HBV) can be confirmed by the presence of either HVB DNA with PCR method or an ELISA test can carried out to verify the presence of hepatitis virus specific surface antigen HBsAg in pateint serum or plasma sample. So when pateints are coming in emergency room a hepatitis B specific PCR analsyis can be done to check the presence of viral DNA. Pateints blood samples will be taken upon arrival and checked for the existance of viral DNA.

2.To isolate the antibody from mouse serum following steps will be performd-

1. whole blood from the mouse will be drawn.

2. next blood serum will be separated by centrifugation.

3. blood serum will next passed through a sepharose column which will contain the antigen specific for the antibody to be isolated.

4. now, the desired antibody will get the specific antigen and will make a antigen-antibody complex and stay in the column and other non-related antibodies will flow out through the coloumn.

5. Next elution buffer can be used to elute out the desired antibody.

Now , to check the purity of the protein, obtained protein can be loaded on SDS-PAGE gel. Prefered band of desired length can be cut out and sequenced for the confirmation of the antibody. For validation of the antigen activity, ELISA test can be performed. In ELISA method the desired antibody will be coated onto the wells of ELISA plate.Next, the mouse serum will be added on the plate. If the desired antibody is there , antigen.antibody complex formation will occur which can be visualized by addition of substrate.