How to Develop an Antibody

You can develop an antibody in a laboratory.

Antibodies are molecules produced by white blood cells, called the B-cells, against different antigens. Antigens are molecules that are foreign to your own body. Your B-cells can respond to an antigen and produce a specific antibody against it. Antibodies are important molecules of your immune system and help eliminate disease-causing pathogens and toxins from your body. Antibodies can also be made in a laboratory and used in many medical and scientific applications. Most commonly, these antibodies are produced in a mouse or a rat against a specific antigen. You can develop an antibody against an antigen with few challenging steps.

Things You'll Need

• Antigen
• Mice
• Myeloma cells
• Culture medium and other chemicals
• Tissue culture plates and other laboratory equipment

Instructions

1. Prepare correct amount of the antigen you want to make the antibody against. Your antigen can be a whole cell, a protein or a carbohydrate molecule. According to an article published in "Molecular Pathology", 50 to 100 mcg of protein or ten million cells, should be enough to immunize a mouse. You might also need to use an adjuvant, especially if your antigen is poorly immunogenic.

2. Inject proteins subcutaneously -- sc -- and cells intraperitoneally -- IP -- into the mouse. Give a boost injection after few days or weeks and follow the antibody titers using tail bleeds. Once you can detect antibody titers, remove the B-cells from the mouse spleen using aseptic and humane methods.

3. Fuse the mouse splenic B-cells with HGPRT negative, histocompatible myeloma cells, by mixing the cells with a fusing agent, such as polyethylene glycol. The resulting myeloma-splenic B-cells are called hybridomas or hybrid cells.

4. Plate the hybrid cells into tissue culture wells and begin selecting against unfused myeloma cells. Use a selective medium that contains hypoxanthine, aminopterin and thymidine, also known as HAT. All unfused myeloma cells will die during this selection and only the hybridoma or antibody secreting cells will survive.

5. Examine the growing cells with a microscope and record poorly growing, newly emerging and established hybridomas. One you have established hybridoma cell lines, transfer them to bigger tissue culture plate. Within 30 days, switch the HAT medium to HT medium to end the selection process.

6. Screen the established hybridomas for antibody production by ELISA assay and continue with a cell line that shows the highest antibody production. Also cyropreserve your best producing hybridoma cell lines and store in liquid nitrogen.

7. Purify your antibody from the growth medium by using an affinity chromatography like protein A high-performance liquid chromatography -- HPLC.

Tips & Warnings

• Always use good laboratory practices and safe working techniques at the laboratory. Read the material safety data sheet for each chemical you will use during your antibody development.

References

• "Immunobiology"; Charles Janeway, et al.; 2001
• "MOlecular Pathology"; Demystified Monoclonal Antibodies; P.N. Nelson, et al.; 2000