Dear Ahmad,

You can go for Mouse hybridoma technology for production of MAb against specific antigen. If you are ready with purified antigen then you can make the MAb either in-house (on your own) or get it custom developed from external parties on payment basis.

In-house development:

You should have experience and thorough knowledge for generation of MAb. The steps involved in MAb production are:

1. Immunization of mice with antigen and sera titration by ELISA

2. Fusion of splenocytes with Myeloma and screening for Positive Supernatants

3. Subcloning and Isotyping

4. Production of MAb in a roller bottle/ multiwell plates or mouse abdominal culture to yield antibody-containing ascites fluid and finally affinity purification with Protein A/G resins.

This is the best possible way to produce MAb against purified antigens. If you don't want to handle this laborious work then simply hire outside party for custome development. It may cost approximately 3-4 Lakhs for a single antigen for a quantity of 5 mg of purified MAb starting from immunization till selection, production and purification.

Dear Ahmad,

You can go for Mouse hybridoma technology for production of MAb against specific antigen. If you are ready with purified antigen then you can make the MAb either in-house (on your own) or get it custom developed from external parties on payment basis.

In-house development:

You should have experience and thorough knowledge for generation of MAb. The steps involved in MAb production are:

1. Immunization of mice with antigen and sera titration by ELISA

2. Fusion of splenocytes with Myeloma and screening for Positive Supernatants

3. Subcloning and Isotyping

4. Production of MAb in a roller bottle/ multiwell plates or mouse abdominal culture to yield antibody-containing ascites fluid and finally affinity purification with Protein A/G resins.

This is the best possible way to produce MAb against purified antigens. If you don't want to handle this laborious work then simply hire outside party for custome development. It may cost approximately 3-4 Lakhs for a single antigen for a quantity of 5 mg of purified MAb starting from immunization till selection, production and purification.

Dear Dr. Nikonov: I am pretty much convinced with your idea of developing mAb if the the sequence is known. Unfortunately, I am working on the project for which we are going to make mAb using hybridoma technology because the sequences are not known. I really appreciate your approach. Thanks for you answer.

What are the best expression vectors used for cloning our gene of interest for mAb?

SA

Dear Dr. Ten Bokum: I am really novice in this field as am shifting my work from biochemistry and biophysics to molecular immunology. Could you please send me out how to make and select phage antibodies? A basic protocol for the same is required. Also please help me out, how much it will cost for the production of phage antibodies? my e-mail id: [email protected]

Mr. Shiv: It is pretty much tedious and time taking as it seems to be. I have done some work on immunization work, but that was for the polyclonal antibodies prodcution in rabbits. I am having some queries-

1. If I wanna make some mAb against some antigen X. So, how would we characterize these antigens..

2. Secondly since this is a pure research work so we can't get it manufactured. So, could you please send me out the basic protocol for mAb production in stepwise manner at [email protected].

The monoclonal antibody production is time consuming process until you make the precise clones for your antigen. but making MABs using mouse is the best of choice. selection of protein is the first choice. so please mention your protein/antigen.

I worked for three years in production of MABs against snake venom. snake venom has thousands of protein, making thousand of MABs is not easy instead we can use the conventional poly clonal antibodies. so we separated the lethal proteins by chromatography and immunising them in mice.

hybridoma using PEG is best of choice and you have the best probability of getting clones. in my team i produced more than 1000 MAB clones in which out of them nearly 150 clones only positive.

so again its like a blind calling so in other words its almost not cheap. but proper planning can minimize the cost of the experiments and maximizing the positive clones.

once you got the clones selection of clones. and confirming the clones to specific antingen every single step is vital.

but once you made i am sure you will be having a golden egg laying duck. you can get back the money by supplying your clones to some good companies.

Dear Dr. Suresh kumar.. Basically I am trying to find out an adduct of protein glycation which is majorly formed during glycation and I want to make monoclonal antibodies against them. Some of them are known and characterized and used in for detection of glycation both in human clinical samples and as well as in vitro. My primary goal is identify the protein adduct via HPLC and LCMS. Then purify them to produce mAB. It has commercial value in diagnostic field.. So could you please let me know what standard protocol should I follow. Plz write to me basic protocol right from the scratch. Your valuable help is appreciated..

Dear Susmita,

Thank you for your attention to our previous research on antigen-driven TARC production/its responsiveness to immunosuppressants in PBMCs from AD patients. Although I looked for the requested article, unfortunately it has not been stored as a PDF file properly. So, I am sorry but it would be better if you could ask the chief research supervisor, Prof. Koichiro Nakamura, who is currently working in the Department of Dermatology, Saitama Medical University of Medicine, Saitama, Japan. You may find him on the ReserchGate system. Again, I am sorry for this inconvenience.

Best regards,

Noritaka Oyama, MD and PhD

Hello Ahmad,

Following are the answers against your queries.

1. If I wanna make some mAb against some antigen X. So, how would we characterize these antigens.

ANS: You have to characterize your antigen before using it for MAb production. Following are the requisites for your antigen.

a) Purity: Determine the purity of your protein by using appropriate methods such as Electrophoresis or Chromatography. Protein purity of >95% is preferred.

b) Concentration: Determine the concentration of your protein by using appropriate methods such as wet chemistry (Lowry/BSA) or Bio-physical techniques (HPLC) or Immunochemical methods (ELISA).

c) Molecular weight: Determine the M.W. of your protein by using appropriate methods such as Electrophoresis or Chromatography. M.W. plays an imp. role in immunogenicity of the molecule.

d) Glycosylation: Determine the extent of glycosylation of your protein molecule.

e) Sequencing of protein molecule: AA sequencing.

f) Immunogenicity: Determine the immunogenicity of your protein in appropriate animal model such as mice or rabbit. Parameters such as animal species, the adjuvant used, and the route and frequency of injection are essential.

g) Toxicity: Determine the toxicity of your protein molecule. This may arise due to presence of pyrogens, chemical residues, an extreme pH level or sterility issues.

h) Vehicle: Put your protein in native buffers such as physiological salts (such as NaCl/PBS).

2. Secondly since this is a pure research work so we can't get it manufactured. So, could you please send me out the basic protocol for mAb production in stepwise manner at [email protected].

ANS: I strongly oppose the idea of making MAb in-house without having prior experience. Ultimately this will end up in selection of wrong clones instead of right ones. You will lose precious time. If you still want I can send you a basic protocol for your reference purpose.

Good Luck.

Dear Shiv: I have one research guy who is expertise in monoclonal antibody production. He has worked for a year in mAb production only at Serum Institute of India. Therefore, as far as expertise is concerned one is there to work day in and day out. The only thing which I mentioned in my post, the amount and time duration for the production. Yes sure you may send me out the protocol. I shall be highly thankful to you.

Hi Ahmad,

I strongly agree with Shiv as he correctly mentioned about starting the work only if you have expertise.

Two important point that I think is required for the successful generation of the mAbs depends of the antigen used for immunization.

1) First of all whether your antigen is conserved among various species, like in you case mouse. If you antigen X is conserved it will be tricky to deal with it.

2) Is your protein highly immunogenic.

So as Shiv has again mentioned, characterization is a must.

But since you have said that you have a experienced person it will not be a problem .

The time duration for completion of the mAb depends on the antigen used for immunization. Sometime the mice will not respond, you need to immunize with high dose, change immunization strategy etc,

Anyways good luck with our work

Dear Nishant: Convinced. Thanks for your valuable suggestions. Looking forward any help if desired.

Saheem