How To Customize Production of Monoclonal and Polyclonal Antibodies

Posted 2 years ago in LIFE STYLE.

The technological advancements have allowed modern-day researchers to customize their approach towards antibody production, be it monoclonal or polyclonal. Speaking of which, antibodies tend to proffer potent tools for diagnostic and therapeutic purposes. The antibody specificity makes the

How To Customize Production of Monoclonal and Polyclonal Antibodies

In addition, when it comes to in vitro assays, researchers utilize antibodies to precipitate agglutinate (clump) cells, soluble antigens, kill and opsonize bacteria with complement, and neutralize toxins, drugs, and viruses. 

 

Custom Polyclonal Antibody Production

Antibodies utilized for research purposes are often acquired by injecting lab animals like goats or rabbits with specific antigens. After a few weeks, their immune system produces antibodies specific to their antigens. Furthermore, since most antigens are complicated structures with several epitopes, they lead to antibody production in lab animals. Such a so-called polyclonal response is also common to infection response by human immune systems. The antiserum extracted from animals can thus comprise antibodies through several B cell clones, with every B cell responding to a particular antigen’s epitopes.  

 

Protocols - Antibody Production 

 

As already discussed, antibodies are referred to as serum immunoglobulins with binding specificity for specific antigens. Therefore, antibodies are a significant utility element in experimental medicine, biology, diagnostic testing, biomedical research, and therapy applications. Polyclonal antibodies and monoclonal antibodies can be utilized for such purposes. However, the production of these specific antibodies needs considerable animal numbers with enormous animal welfare consequences.  

 

Regarding PAbs, researchers provide animal injections or adjuvant mixtures to collect blood and then monitor their antibody response through the experiment. This experiment also contributes towards acquiring the antibodies. Meanwhile, with MAbs, animals are given injections of adjuvant mixtures or antigens to induce B cells obtained from the lymph nodes or spleen to establish hybridomas.  
 

Animal Species Selection 

While choosing certain animal species for custom antibody production, one should consider the following factors: 
 

  • PAb amount required 
  • Obtaining blood samples 
  • Phylogenetic relationship between animal species and the antigen 
  • PAb use 

 

Researchers often choose rabbits to produce custom polyclonal antibodies because of the ease of handling, size and bleeding, production of high-titer, long life span, high-affinity, and precipitating antiserum.  

Antigen Preparation 

 

When you produce antibodies, it is essential to consider features of an antigen for custom antibody production, which comprise the quantity and quality of the antigen and its preparation. Moreover, its specificity depends on the applied antigen’s purity. It can be time-consuming but is quite worthwhile when it comes to antigen purification. Researchers recommend carefully preparing the specific antigen and maintaining the antigen’s quality control.  

 

Adjuvant Selection and Preparation 

 

When the specific antigen against which antibodies get evoked is poorly immunogenic, the entire immune system needs the stimulus to administer a practical immune response. For this purpose, adjuvants can be utilized, directing an immune response against a more humoral or cellular response. Meanwhile, when it concerns adjuvants utilized for custom polyclonal antibody production, it comprises Freund's complete adjuvant, aluminum salts, Freund's incomplete adjuvant (FIA 1 ), Iscoms, Quil A, TiterMax™, Montanide, and RIBI™. Researchers recommend the following preparations: 
 

  • Prepare mixtures aseptically. 
  • Monitor the emulsion quality and stability. 
  • Select the injection volume and route. 

 

Injection Protocol 

It is essential to know that the immunization protocol is prepared post-animal species selection and adjuvant. Such a protocol comprises various necessary steps that rely on specific preferences mentioned in a rundown: 

 

  • Route of Injection 
  • Volume of Injection 
  • Number of Injection Sites
  • Booster Injections 

 

Postinjection Observation 

 

Post immunization, researchers monitor animals daily and examine them for specific side effects thrice a week. Moreover, palpation and examination are crucial to determine the injected mixture’s side effects. Various investigators have stated adverse pathological transformations despite the absence of behavioral and clinical changes.  

 

Custom Monoclonal Antibody Production 
 

MAbs are antibodies produced through B cells’ single clones. Milstein and Kohler discovered and observed that such cells could undergo immortalization through myeloma cells, leading to hybridoma cells producing an unlimited number of monoclonal antibodies. Furthermore, the MAb development process comprises the following phases: 
 

  • Antigen-specific B cell generation  
  • Fusion of such cells with myeloma cells  
  • Selection and cloning of hybridoma clones through limiting dilution 
  • MAb production upscaling 

 

Generate Antigen-specific B Cells 

 

The process of specific B cell production needs animal immunization with particular antigens under study. In addition, crucial aspects that researchers should consider: 
 

  • Aseptic antigen processing 
  • Antigen selection 
  • Preparation of the antigen-adjuvant mixture 
  • Adjuvant selection 
  • Injection volume 
  • Choice of immunization route 
  • Aseptic inoculation to produce custom monoclonal antibodies  

 

When it comes to immunization, investigators use BALB/c mice since they have many myeloma cells available.  

 

MAb Production Through Ascites Induction 

 

Researchers generally prefer BALB/c mice for ascites production since the animal strain is syngeneic for myeloma cells. Initially, they provide the animals with the injection carrying a priming agent. Moreover, the primer effect is three-fold. 
 

 


 

During the ascites development, researchers then observe the animals thrice daily for the initial weeks and recurrently after that to monitor the abdominal distention degree and illness signs. 

The Bottom Line 

 

The custom production of polyclonal and monoclonal antibodies depends on several factors, from selecting a specific antigen to determining a host animal species. As a researcher, you must establish an application with the custom production process for a seamless yet advantageous output. 

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