Since the inception of antibody testing, several elements have transformed significantly. From valuable tools to clinics and laboratories, scientists and bioscience departments are more pronounced and equipped now. Not only have these allowed researchers to produce antibodies synthetically to develop vaccines, but they also enhanced research work. Speaking of antibodies, they are valuable elements in several laboratories. These comprise the secreted antibodies released by single clones of B lymphocytes, reckoned as monoclonal antibodies and generated by various B lymphocyte clone mixtures, known as polyclonal antibodies.

Both these antibody types have become integral instruments in immunohistochemistry, vaccine quality control, and diagnostic testing. In addition, antibody production needs a considerable number of animals as testing subjects. This is because they are entitled to several invasive procedures like blood collection and antigen injection. However, by designing immunization protocols and enhancing immunization responses, one can control an animal’s distress and pain while acquiring the most favorable immune reactions. The blog states vital steps included in the production of polyclonal antibodies. Moreover, you may also learn more about the animal species, age, ascites tapping, and injection protocol.

Protocols for Antibody Production

They are referred to as serum immunoglobulins with binding specificity for specific antigens when it comes to antibodies. As a result, antibodies have a significant utility in mediums like medicine, experimental biology, diagnostic testing, biomedical research, and therapy. Besides, researchers can use polyclonal and monoclonal antibodies for such purposes, although producing such antibodies needs several animals with pivotal animal welfare consequences.

Under polyclonal antibodies, researchers with prior experience on ELISA kits give antigen injections or adjuvant mixtures to animals to induce antibody responses. This is typically essential to obtain blood for monitoring purposes. However, researchers offer injections and combinations for monoclonal antibodies to induce particular B cells obtained from lymph nodes or spleen to establish and determine hybridomas. When in vitro production procedures are not viable, it is essential to utilize animals for producing antibodies through the abdomen cavity’s hybridomas.

Critical Steps

Some of the steps required in polyclonal antibody production include:

• Preparation of the antigen
• Animal species selection
• Preparation and selection of the adjuvant
• Injection protocol
• Post injection observation
• Antibody collection

Preparation of the Antigen

When producing antibodies, it is essential to determine every antibody feature, including the quantity and quality of the antigen preparation and antigen alone. Also, the immune response’s specificity acquired relies on an applied antigen’s purity. Consequently, minute impurities can also prove to be immunodominant and can lead to antibodies with more activity against the impurity if compared to the antigen of interest. Antigen purification is one of the laborious and time-consuming steps but is worthwhile. Before moving further with the immunization, a researcher should determine and consider the preparation of antigens through contaminating endotoxins like chemical residues or lipopolysaccharide utilized to inactivate the microorganism.

The diluents must be free from endotoxins, and the pH must be regulated within the physiological limitations. Post administration, such factors are essential since they can adversely affect the animal’s welfare and the ultimate immunological standings. Meanwhile, the quality of specific antigens depends on the antigen’s inherent properties, whether it is purified or an antigen mixture’s compound, an immunized animal species, or subjected to the injection frequency or sound.

If we go by the reports of Hanly and Coworkers, the typical dose of a single soluble protein for rabbits should be 10 to 200 μg; for goats, 50 to 1000 μg; for mice, and 250 to 5000 μg for sheep.

Animal species selection

While choosing animal species for polyclonal antibody production, one should consider the following:

• The amount of PAb needed
• The ease of acquiring blood samples
• The phylogenetic relationship between animal species and antigens
• The intended use of polyclonal antibodies

Researchers utilize a mouse, rat, rabbit, guinea pig, sheep, goat, hamster, and chicken for most laboratory settings to induce polyclonal antibodies. However, when it concerns the PAbs, rabbits are utilized more frequently since they comprise ease of bleeding and handling, convenient size, have a relatively long lifespan, high-affinity, adequate production of high-titer, precipitating antiserum. Meanwhile, when you need a more significant number of polyclonal antibodies, consider using goats, sheep, and horses. Chickens are also ideal animals for PAbs. Post-chicken immunization, antibodies make their way through the blood to the yolk.

After following such a process, the antibodies from chicken eggs can undergo extraction from the egg to concentrations of around 100-250mg of chicken egg antibodies per egg. From a modern yet practical point of animal welfare, the ease and comfort of acquiring blood samples can influence animal species selection. In addition, when one does not require identifying specific animals, the entire process of obtaining the blood samples becomes seamless and straightforward. In simpler terms, rabbits are ideal selections as compared to guinea pigs. This is because laying hens don’t need blood samples since antibodies reside in their yolks. Nevertheless, several disadvantages of using chickens exist.

Despite the integration of animal species, animal sex is also pivotal. Traditionally, female animals are utilized for polyclonal antibody production. This is because a researcher can group house such animals more successfully than the male ones. Females are less aggressive and more docile in social interaction. However, there are no overriding reasons for not utilizing male animals for polyclonal antibody production. Besides, you can also group-house castrated male rabbits.

To conclude, it is integral to consider an animal’s health status as well. Infectious agents may exist, suppressing, stimulating, and modulating the entire immune system. Having said that, using infection-free animals tends to minimize cross-reactivity to other antigens that the immune systems might have experienced.

Preparation & Selection of the Adjuvant

When antigens to which specific antibodies are evoked are immunogenic, the entire system needs a unique stimulus to instigate an immune response. One can utilize adjuvants for this reaction and simply redirect an immune response against more humoral and cellular responses. From a study point of view, it is essential to know that one can utilize only some adjuvants for PAb production. Meanwhile, several researchers use FCA to produce polyclonal antibodies since more significant antibody titers get induced to every antigen type. However, several investigators have stated adverse reactions and side-effects after the FCA injection.

Due to this, the available recommendations and guidelines on polyclonal antibody production focus on FCA. Besides, some universities in the US suggested that some undesirable FCA effects can be prevented and reduced through careful control of site selection and injection. Even though FCA is less problematic might be because of the improper former utilization. In addition, many pieces of literature prove that FCA use was more toxic and less pure, inducing more adverse inflammatory reactions. Moreover, the severity and extent of pathological developments and transformations depend on the adjuvant and the utilized antigen type.

Based on the elements and factors mentioned above, picking an alternative adjuvant for polyclonal antibody production that integrates high antibody titers and minimal side effects isn’t easy. While an infirm immunogenic antigen is utilized for PAb production, a researcher can perform comparative studies using various adjuvants blended with a specific antigen of interest to select an ideal adjuvant. Moreover, it is essential to include pathological examinations and results in such studies since many investigators have reported that behavioral and clinical changes do not occur after the FCA injection. However, they observed adverse pathological changes.

Refer to some antigen-adjuvant preparation recommendations:

• Prepare combinations and mixtures aseptically.
• Monitor the emulsion’s quality and stability carefully.
• Select the injection volume and route, primarily when the oil adjuvants are utilized.

Injection Protocol

The injection protocol is one of the most crucial selections of the adjuvants and animal species. Such a protocol comprises various steps that rely on every selection described below:

Route of Injection

The injection route’s choice is modeled extensively by animal species, adjuvant options, and through an antigen’s quantity, character, and volume. Moreover, several researchers use injection routes such as intradermal (i.d.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), and intravenous (i.v.) to produce polyclonal antibodies. Footpad, intra-lymph node, and intrasplenic injection are typically not essential for routine polyclonal antibody production and should undergo justification through case-to-case situations.

In addition to the discussion, the CCAC discourages the utilization of injection routes. Since injection into any isolated space is severe and painful, the Council rather opts for i.m. and i.d. routes to be examined. Furthermore, another set of participants participated in a workshop where they expressed their refusal of food pad injections and, i.p., intrasplenic, regardless of the adjuvant. Consequently, oil adjuvants are ideally administered to use the depot effect.

Several bioscience departments operating on peptide synthesis, ELISA kits, and various methods of antibody productions have eliminated the consideration of a handful of injection routes by the adjuvant choice. Instead, the i.v. routes for the water-in-oil emulsions (FIA and FCA) can be detrimental, whereas the i.v. injection route is not advised by experienced researchers since it can pose an embolism risk for more significant viscous or particulate gel adjuvants. However, for FCA injections, many do not recommend the i.p. route for polyclonal antibody production since it can integrate and administer peritonitis, inflammation, and behavioral changes.

Volume of Injection

Irrespective of the injected mixture, utilizing the minor most volume is pivotal to induce an adequate antibody response. Besides, the meager volume relies on the antigen’s specific characteristics. So, when you dissolve the antigen in a considerable volume that cannot be concentrated, you must inject a large volume.

Number of Injection Sites

A researcher tends to administer the adjuvant/antigen volume in single or multiple low volume injections. Looking at the situation from an animal welfare perspective, there can be potentially problematic opinions and positions between the two. Inducing the effects of injection at one of the sites can be severe and painful, especially if there’s limited space available. However, numerous injections can lead to additional pain and suffering because of the inoculation frequency and the number of painful sites. It is challenging to offer clear-cut recommendations since the present data is limited.

In many European nations, researchers recommend a maximum of four route sites while utilizing emulsions. However, it is ideal for integrating adjuvant or antigen blends at one of the sites only.

Booster Injections

It is essential to understand that the boosting protocol can lead to a determining effect on the immunization’s result. The specific time recorded between a couple of immunization steps can impact the class switch of B cells and the induction of B memory cells. In this case, you can rest the animals for long periods, preferably for months between boosting. In simple terms, consider the booster after the selected antibody titer declines or has reached a plateau.

Emulsion Use Recommendations:

• Utilize a maximum of 2-3 boosters.
• Administer it a minimum of 4 wk. post-prime.

Post injection Observation

Post immunization, you should thoroughly observe animals daily and examine them for particular side effects a minimum of three times every week. Besides, injection site palpation and examination are integral to evaluate the side effects of the entire injected combination. This is because clinical assessments and observations don’t predict adverse pathology at the injection site.

Monitoring Recommendation:

• Palpate the site of injection if you want to determine the injection’s side effects.

Monitoring of the Antibody Response

In mammals, one can monitor and examine the antibody responses while evaluating various blood samples for antibodies found in the serum. Furthermore, a researcher can study the titers in chickens without any aggressive action or activity because antibodies get excreted in the egg. Moreover, the blood collection frequency and the significant blood volume available can undergo removal from the animals as and when it’s safe or limited.

The Bottom Line

As already discussed, polyclonal antibody production occurs by injecting antigens into selected lab animals, like goats, rabbits, and many more. Even though these steps are integral to the entire approach, a researcher needs to understand the thorough process from immunizing an animal to antibody purification and quality control. And, after a few weeks, PAbs can be collected and harvested from the antiserum. To conclude, producing polyclonal antibodies is more cost-effective and seamless than producing monoclonal antibodies.