Antibody Production FAQs

Q1) How much antigen do you need for a custom project?
Ans: A standard two rabbit schedule uses 1mg of antigen (peptide, protein, etc.), but please provide 1.5-2mgs of protein are recommended, especially if you require ELISA or extensions. For affinity purification, 2-3mg of lyophilized peptide and 3-5mg of soluble protein are required. We prefer a concentration of 1mg/mL or greater.

 

Q2) What if I don't have enough peptide or protein?
Ans: This is not a problem, just send us what you have ready and we will start your project. You can send us more when it becomes available.

 

Q3) Can I send you a gel strip?
Ans: Sure, however gel strips can only be accepted for our package I. Gel strips cannot be used in conjunction with ELISA and affinity purification. Additionally, the protein should be reasonably concentrated prior to running on the gel (2-5 mg/ml). Just stain the whole strip with coomassie blue. Then de-stain, rinse, and cut the desired strip from the gel (Please don't chomp it up). After you cut the band from the gel, place it in a 15 or 50ml conical tube with an icepack- don't freeze it.

 

Q4) How do I de-stain my gel strip?
Ans: Use 5% methanol, 7.5% acetic acid, and fill the remainder with ddH2O. De-stain gel in 100mL solution overnight and replace with fresh de-stain if needed. The gel will remain blue even though the coomassie dissociates from the proteins.

 

Q5) What types of antigens do you accept?
Ans:

  • DNA plasmid: we can use a DNA plasmid to generate antibodies; however you need to provide an immunization protocol (bleed/boost schedule and quantity of plasmid to be immunized, etc.) because different plasmids require different schedules. Immunizing with a DNA plasmid is different from immunizing with a protein or peptide antigen. The plasmid can be shipped to us on blue ice.
  • Enzymes: enzymes can easily be used for antibody production; we need to know concentration in order to administer immunizations and to perform ELISAs.
  • Polysaccharide: at this time we do not accept polysaccharide samples for immunization; they are difficult to get to elicit immune response and we can make no guarantees.
  • Cells: cells (i.e.: isolated from an organ like the spleen) can be used as an antigen, but we cannot perform an ELISA to determine titer. Blood cells must be washed away before we can accept the immunogen cells.
  • Cell Lines/Lysates: cell lines and lysates can be used as polyclonal antigen; however they cannot be used for a monoclonal antigen as screening via ELISA is not available.

Note: We cannot use any living organisms for immunization. If the recombinant organisms are dead, and contain no toxic materials harmful to the animals or personnel, they could be used.

 

Q6) Do you offer immunogenic peptide sequence analysis?
Ans: Yes, and its FREE. Our scientists will help you select a sequence that has potential to raise a good antibody response. Just email your full length protein sequence(s) along with any supplemental information to antibody-sg@base-asia.com and we will have a response in 1 to 2 working days.

 

Q7) What kind of guarantee do you offer for your custom antibody production and services?
Ans: If you choose one of the sequences we recommend for peptide synthesis, we will guarantee an immune response to the antigen by ELISA titration. This is a great reason for taking advantage of our free antigen design assistance!

 

Q8) What is immunoaffinity purification?
Ans: The peptide/protein is bound covalently to CNBr activated Sepharose 4B or SL gel in a column. The antibodies within the polyclonal pool that are specific to this antigen are allowed to bind. The unbound antibodies and other serum proteins pass through the column, and the antigen bound antibodies are eluted. The resulting purified antibody is highly specific. We will provide you with the serum, flow through, purified antibody (a yield from 0.1 mg to 0.5 mg per ml of serum), re-usable column to the antigen of interest and ELISA results.

 

Q9) Do I need to ship my antigen on dry ice? How are you currently storing your protein?
Ans: You only need dry ice if the storage conditions are -80°C. If the protein is stable, ice packs are fine. Contact us to arrange for sample collection, or ship to our office using standard courier services eg. FedEx.

 

Q10) What do I need to send with my antigen?
Ans: Please include an order form within your package (preferably in a zip-lock bag) as well as the concentration of the antigen and the buffer used.

 

Q11) Do I have to cleave the tag from my recombinant protein before sending it to you?
Ans: Please cleave the larger tags (GST, MBP, etc.) as they can interfere in specific antibody production. Small tags such as His, Myc, and FLAG do not need to be cleaved prior to antibody production.

 

Q12) What is the smallest antigen that will produce an immune response?
Ans: Approximately 10-12kDa is necessary for an immune response. Anything smaller needs to be coupled with a carrier protein, typically KLH, so that the immune system can recognize it.

 

Q13) How many samples of hybridoma required for Ascites Production? 
Ans: We require two samples of hybridomas for the production of ascites. The first is used to screen the hybridomas for the presence of mycoplasma, the second is cultured to inject the production mice.

 

Q14) How do we prepare the hybridomas for the ascites production? 
Ans: Rapidly growing and dividing hybridomas (log phase) with more than 90% viability should be used for cryo-preservation (freezing). Hybridomas should be grown for a minimum of two passages in an antibiotic-free medium (DMEM or RPMI with 10% FBS). Under Sterile conditions, remove hybridoma cells from the flask and transfer to a centrifuge tube. Count the cells and determine the necessary volume to have 5 x 106 hybridomas per cryo-vial. Centrifuge cells at 200 g for 10 minutes. Carefully remove the media and re-suspend cells in freezing media (90% fetal bovine serum (FBS) and 10% DMSO) to have 5 x 106 hybridomas per ml in each cryo-vial. Cells should be frozen slowly. Place the cryo-vials in to one half of polystyrene tube racks and completely enclose the vials with the other half of the rack. Tape the halves together and leave them overnight at – 80° C freezer (alternatively special cryogenic freezing container can be used). Next day transfer the cryo-vials to liquid nitrogen tank or ship them overnight with dry ice. Wear gloves and face mask when working with liquid nitrogen tank and frozen cryo-vials. Duplicate samples should be prepared from the same hybridoma culture to be tested for mycoplasma and preparation of ascetic fluid.

To optimize mycoplasma detection, hybridomas should be grown for a minimum of two passages in an antibiotic-free medium as antibiotics generally suppress (but do not eliminate) mycoplasma growth. Hybridomas should be harvested when they are growing rapidly (log phase). Trypsin-EDTA should not be used for harvesting. For best results between 1,000 and 10,000 cells should be frozen under sterile conditions as described previously.

 

Q15) Which antibodies are better: polyclonal or monoclonal?
Ans: There are advantages and disadvantages to both. Polyclonal antibody production is less expensive. It is technically easy and fast, but it gives a limited amount of antibodies with multiple specificity. Monoclonal antibody production takes more time and is more complicated, but it gives a limitless amount of antibodies with single epitope specificity. These antibodies provide clear signal and less background in their usage.

 

Q16) What does KLH stand for? Why is conjugation to KLH necessary when using a peptide as an antigen?
Ans: KLH, Keyhole Limpet Hemacyanin, is the most commonly used carrier protein because species cross-reactivity is very minimal. It is conjugated to small molecules of haptens or short peptides for antibody production. Most peptides are not large enough in size to be immunogenic on their own. Conjugation to a carrier protein will make the peptide antigen adequately large to be immunogenic.

 

Q17) What does ELISA stand for?
Ans: Enzyme-Linked Immunosorbent Assay. ELISA is used to determine the specific antibody presence and titer.

 

Q18) Why are chickens chosen as host animals for antibody production?
Ans: Chickens are recommended for production of antibodies to mammalian antigens. Chicken antibodies usually provide good results because there is less cross reaction of those antibodies with mammalian proteins. In addition, chickens lay eggs regularly, providing a continual source of antibodies.

 

Q19) What are the preferred storage conditions for antibodies?
Ans: 100 µg in 200 µL phosphate buffered saline solution (pH 7.4) containing 0.02% Proclin300 or sodium azide can be stored at 2-8°C for up to one month. Antibodies should be stored at -20oC for long-term storage. Repeated freezing and thawing should be avoided.

 

Q20) What is sodium azide used for?
Ans: Sodium azide is a preservative. It prevents bacteria from growing in the serum. Please notify us if you are planning to use the serum directly in cell culture; for cell culture usage, sodium azide should not be added to the serum. If you already have sodium azide in the serum, it can be easily removed by dialysis. Alternatively, Proclin300 can be used in place of sodium azide.