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Polyclonal antibody production
If you are aiming to increase the sensitivity and broaden epitope-specificity in your immunoassays, our polyclonal antibody production platform is your best choice:
Monoclonal antibody production
If you are aiming to generate highly specific antibodies or improve their properties, our monoclonal antibody production services are your best choice:
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Our access to state-of-the-art technologies and immense flexibility helps us to create unique solutions tailored to your specific goals and needs. All our resources and expertise are placed at your disposal to empower your groundbreaking discoveries in antibody development.
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Tailor-made solutions
Your antibody development projects by accessing our broad portfolio of state-of-the-art equipment and extensive expertise.
Vast choice of antigens
Don’t waste time and resources by using the wrong antigen design approach. Our experienced team will help you select the best antigen synthesis strategy for superior antibody production results.
Antibody format & species diversity
Produce your antibodies in any species (including human) and format (scFv, Fab, VHH, full-length, or antibody-fusion proteins).
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Your satisfaction is our highest priority and we instill that principle into everything we do. As a result, we offer the market’s best guarantees (yield, reactivity, etc.). “At ProteoGenix, your satisfaction is our obsession. We incorporate this principle in all aspects of the customer experience to ensure you are completely satisfied with your custom antibody and our customer service. That is why we offer the best guarantees in the industry including the highest antibody yields and reactivity.”
Holistic approach to antibody development
We learn your end goal objectives are to create the perfect antibody. Whether you are developing antibodies for therapy, research, or diagnostics, we put our extensive capabilities at your disposal.
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We put at your disposal highly skilled account managers who guide you along your decision-making process.
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ProteoGenix applies the highest ethical animal use standards to every custom antibody production project.
Antibody production refers to the entire process of producing an antibody with specificity to a distinct biological target. The antibody production process involves these general steps:
The engineered antibody can then be used in various medical or experimental applications.
Successful production of high-quality antibodies requires the careful preparation of antigen samples that are methodically designed to stimulate the production of high-affinity antibodies for a specific application. The engineered antigen samples are then carefully injected into a laboratory or agricultural animal to stimulate the secretion of antigen-specific antibodies by B cells. The secreted antibodies are retrieved from the animal by collecting the liquid portion of the blood called the serum. The antibodies located in the serum are termed polyclonal antibodies because they are secreted from different B cell clones, each producing an antibody that binds a unique epitope, the antigenic region recognized by the antibody.
Unlike polyclonal antibodies, monoclonal antibodies are produced from a single B cell clone (i.e. mono-clonal), which produces antibodies that bind a single epitope on an antigen. Thus, manufacturing monoclonal antibodies requires either the isolation of individual B cell clones or molecular techniques to identify the DNA sequence encoding single antigen-specific monoclonal antibodies. These production methods generally include single B cell screening, hybridoma development, and antibody phage display.
Single B cell screening is a monoclonal antibody production method involving the high-throughput isolation and characterization of individual B cells from immunized animal hosts. Specifically, B cells are isolated from the polymorphonuclear blood cell (PBMC) fraction after several rounds of immunization. Next, the B cells are isolated from the PBMC compartment using one fluorescence-activated cell sorting.
Individual B-cells can be isolated by fluorescence-activated cell sorting (FACS). This cell sorting mechanism is a way to purify B cells by detecting the presence or absence of molecular B cell markers expressed (or not expressed) among cells in a heterogeneous cell mixture (i.e. PBMCs).
First, the PBMCs are incubated with primary antibodies that specifically bind B cell antigens on the surface of the cell. The PBMC cell mixture is then incubated with a fluorophore-conjugated secondary antibody that binds to the primary antibody. This effectively labels each B-cell with a fluorescent tag.
Next, the cell solution is passed through a narrow nozzle under flow conditions, compartmentalizing the cells inside fluid droplets. The single-cell-containing droplets pass one by one through the light path of a laser, exciting the antibody-conjugated fluorophore attached to the cell. The presence of an excited fluorophore is processed by the detector, which initiates a process that electrically charges the B-cell-containing droplet. These steps occur in milliseconds before the cell droplets exit the nozzle.
Lastly, the electrostatically charged droplet exits the nozzle and is attracted to the oppositely charged electromagnetic plate. This electrostatic interaction redirects the vertical path of the droplet directly above a collection tube. Next, the isolated B-cells are seeded into individual wells of a tissue culture plate and cultured in cell culture media. The media is collected and the secreted antibodies are assessed for their ability to bind the target antigen by ELISA.
Hybridoma cell line technology produces large quantities of identical antibodies (also called monoclonal antibodies). The process of generating a hybridoma cell line starts by immunizing a mouse (or other mammals) with the purified target antigen to initiate the adaptive immune response. The B cell, a type of white blood cell, then produces antibodies that adhere to the immunized antigen. These antibody-producing B-cells are isolated from the animal and fused with immortal B-cell cancer cells, or myeloma, to create a hybrid cell line known as a hybridoma. The hybridoma cell line possesses the longevity of a myeloma cell and the antibody-producing capacity of a B-cell.
Antibody phage display is a laboratory method used to identify antigen-specific antibodies. Specifically, this method establishes a link between genotype and phenotype by inserting the antibody gene into a phage coat protein gene, forcing the phage to “display” the protein on its exterior while containing the gene for the antibody inside the phage. The interaction between the displayed antibody and antigen can be discovered by screening the phage display libraries using specific antigens. These libraries can be reused indefinitely to identify new and exciting antibodies that bind yet-to-discovered antigens.
Antibodies are known for their ability to bind specific targets and thus can be used for detecting, quantifying, delivering cargos (e.g. small drugs), blocking, or activating specific protein interactions. These properties make them useful for a wide range of experimental and therapeutic applications.
The first and most important choice is into choosing between polyclonal or monoclonal antibodies. Several critical parameters must be considered when selecting the proper antibody clonality for your project.
The table below compares the strengths and weaknesses of producing monoclonal antibodies vs producing polyclonal antibodies.
Explore our Monoclonal Antibody Production Services and Polyclonal Antibody Production Services
Overall, monoclonal antibodies can present several advantages over polyclonal antibodies:
Due to their high specificity, monoclonal antibodies are more sensitive to an epitope’s conformational changes. This property makes them less effective in applications that require higher sensitivity (low-abundance targets) but it also makes them advantageous for applications requiring highly selective applications such as differential diagnostics (detection of mutations or post-translational modifications) or immunotherapies to treat complex diseases.
The process of purifying antigen-specific antibodies varies depending on antibody clonality (monoclonal or polyclonal) and the method used to custom-make the antibody. Monoclonal antibodies generally do not require extensive antibody purification approaches. However, purification of polyclonal antibodies is required since the antigen-specific antibodies are isolated from the serum containing a heterogenous mixture of antibodies.
Purifying polyclonal antibodies usually involves protein A, G, or L purification to isolate antibodies from animal serum. Protein A, G, or L purified polyclonal antibodies might be sufficient for some research purposes, but they may not be appropriate in many other circumstances, either because of poor detection sensitivity or an unacceptably high background. Increasing detection sensitivity and decreasing unwanted background can frequently be achieved by further purifying and separating the antigen-specific antibodies from the total IgG antibodies. Further purification using antigen-conjugated resin can further affinity purify selective antigen-specific antibodies.
The methods ProteoGenix uses to make custom monoclonal antibodies (i.e. B-cell isolation, hybridoma generation, and antibody phage display) do not require sophisticated antibody purification methods. However, decades of experience are needed to successfully identify and characterize antigen-specific monoclonal antibodies. Thus, the following sections will focus on understanding the advantages of characterizing monoclonal antibodies.
Identifying the proper custom antibody that will perform well in your experimental, therapeutic, or diagnostic applications is a critically important process that should not be overlooked. However, the term antibody characterization can be confusing to most.
Antibody characterization consists of five different processes performed at different stages of the custom antibody production process:
Antibody Classes, Subclasses, and Heavy Chain and Light Chain Types.
Note: κ and λ are two types of light chains that can be associated with all of the heavy chain types
Of the five different antibody classes, only IgD, IgG, and IgA contain different antibody subclasses (see table above). Each antibody class and subclass has unique antigen binding characteristics and effector functions inside the body. For example, IgG antibodies are monomers known for their strong binding affinity to antigens, their ability to activate complement, and their potential to promote antibody-dependent cell-mediated cytotoxicity (ADCC) of infected cells or cancer cells. In contrast, IgM antibodies are pentamers and have a low antigen-binding affinity to individual antigens, but broad reactivity to a variety of antigens.
Antibodies belonging to individual immunoglobulin subclasses are highly conserved but differ in the constant region of the heavy chain, particularly at the hinge region. The variable regions among the immunoglobulins in an antibody subclass influence variability in Fc receptor binding.
An Fc receptor is a protein expressed on the surface of immune cells that binds to the Fc region of an antibody. Different Fc receptors are expressed on different types of immune cells and have different affinities for the various antibody subclasses. For example, IgG antibodies bind to Fcγ receptors on the surface of natural killer (NK) cells, macrophages, and neutrophils, promoting ADCC and phagocytosis. In contrast, IgE antibodies bind to Fcε receptors on mast cells and basophils, triggering the release of histamine and other inflammatory mediators in response to allergens.
Identifying antibody classes and subclasses is important when engineering antibodies because it allows for the selection of the most appropriate antibody for the intended application. For instance, monoclonal IgG antibodies would be the most effective antibody class for custom antibodies used clinically because they are monomeric immunoglobulins with high affinity against a target antigen and have the ability to facilitate target cell destruction via antibody-dependent cell-mediated cytotoxicity (ADCC).
Scientists making custom monoclonal antibodies can identify the immunoglobulin class of a specific antibody using an assay that involves designing DNA primers that hybridize to the complementary DNA sequence of a specific immunoglobulin heavy chain.
First, the mRNA from the clonal B cell population is collected and converted into copy DNA (cDNA). The cDNA is then mixed with Ig class-specific primers and thermostable DNA polymerase to generate numerous DNA fragments. These fragments can be quantified in real-time using a technique called quantitative real-time polymerase chain reaction (qRT-PCR). By amplifying the immunoglobulin cDNA isolated from the B cell population using primers against a specific heavy chain, but not other heavy chains, the immunoglobulin class can be identified.
For instance, if scientists can amplify the antibody encoding cDNA using primers against the γ heavy chain, but not the μ, δ, ɑ, and ε heavy chains, then the immunoglobulin class is IgG, not IgM, IgD, IgA, or IgE, respectively.
Other methods used to characterize the immunoglobulin class of a custom antibody include sequencing the cDNA that encodes the antibody heavy chain or performing mass spectroscopy on purified antibodies collected from clonal populations of B cells. Regardless of the method, characterizing the antibody class and subclass can vary in importance depending on the custom antibody’s intended purpose.
A reputable antibody production company understands the value of identifying the subclass of your therapeutic or diagnostic antibody. These antibody subclasses will likely be experimentally identified using methods such as ELISA, Western blot, or mass spectrometry.
ELISA and Western blot involve incubating the antibody sample with subclass-specific antibodies and visualizing any bound antibody using a detection system.
Mass spectrometry involves analyzing the protein sequence of the antibody of interest to identify unique peptides that are specific to each subclass. Collectively, the experimental identification of antibody subclasses requires the use of specific subclass-specific antibodies or methods for isolating and analyzing the antibody of interest.
Following immunization of the mammalian or avian host, polyclonal antibodies are collected from the serum (of mammals) or the egg yolks. At this stage, the isolated antibodies are heterogeneous. Some antibodies bind the target antigen at unique locations (epitopes) while others do not bind at all.
One common method for identifying the class and subclass of polyclonal antibodies is by using immunoassays, such as ELISA or Western blotting, that utilize specific antibodies against different heavy and light chains. Another approach involves using isoelectric focusing or HPLC to separate the antibodies by their charge or size, respectively, and then analyzing them using immunoassays or mass spectrometry.
Most polyclonal antibodies are used in biomedical research techniques, not as immunotherapeutics. Therefore rigorous evaluation of polyclonal antibody classes and subclasses is not necessary. However, there are some exceptions. For instance, anti-thymocyte globulin (ATG) is a polyclonal antibody used in the prevention of acute organ transplant rejection in humans.
ATG is produced by immunizing rabbits with human T cells, resulting in the production of antibodies that bind to multiple T cell epitopes. When administered to transplant patients, ATG binds to and eliminates T cells that are responsible for attacking the transplanted organ. Therefore, it is important to ensure that ATG antibodies contain the correct classes and subclasses to maximize effector functions via antibody-dependent cell-mediated cytotoxicity (ADCC).
In addition to identifying antigen-specific antibodies, measuring antibody titers, and identifying immunoglobulin classes and subclasses, epitope mapping is a monoclonal antibody characterization service that can reveal how a monoclonal antibody exerts its functional effects.
Specifically, epitope mapping is the experimental process of identifying an epitope, or binding site, of an antibody to its target antigen. The discovery and development of new therapeutics, vaccines, and diagnostics are assisted by the experimental characterization of antibody binding sites to target antigens. This is because epitope mapping can also help identify an antibody’s binding mechanism and strengthen intellectual property (patent) protection. In silico prediction of B-cell epitopes based on sequence and/or structural data can also be facilitated by incorporating experimental epitope mapping data into robust algorithms.
The expertise involved in engineering custom monoclonal antibodies using B-cell sorting involves screening the clonal B-cell populations for the highest performing antibody candidates. Screening B-cell clones starts with engineering an antigen and immobilizing it to the bottom of a well for use in enzyme-linked immunosorbent assays (ELISA).
B-cell clones are identified by collecting the antibody-enriched cell culture media and incubating it in the well to allow antigen binding. The unbound antibodies are washed out while the antigen-bound antibodies are detected using an enzyme-linked secondary antibody. The amount of secondary antibody bound to the antigen-specific primary antibody is detected by adding the enzyme substrate to a colorless solution. The enzymatic reaction produces a color change that is directly proportional to the amount of antigen-specific antibodies in the well.
The B-cell candidates that secrete antibodies with the highest antigen binding affinity are isolated so antibody-encoding genes can be sequenced. Next, ProteoGenix transiently expresses the antibody DNA sequences using our proprietary XtenCHOTM high-performance cell line, producing abundant antibodies with the correct post-translational modifications. Thus engineering a custom monoclonal antibody using single B cell isolation is the optimal approach if you need a high-affinity antibody with conserved native properties
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Hybridoma cell lines involve characterization methods similar to that of the B-cell isolation approach (described above). After B cells are fused with myeloma cells, they are cultured in a special medium containing a compound that kills any unfused myeloma cells. While the chemical does not kill unfused B cells, they eventually die in culture due to their short life span. The remaining cells are verified as hybridomas.
Hybridomas are screened by plating one cell per well in a tissue culture plate. The antibody-enriched cell culture media is collected and incubated with antigen-coated wells to allow the identification of antigen-specific antibodies by ELISA. The most reactive monoclonal antibodies are identified and delivered to our clients.
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Identifying an antibody from an existing antibody phage display library starts with making and purifying the target antigen. Next, the antigen is immobilized on a solid surface such as a nitrocellulose membrane, polystyrene tubes, or magnetic beads. The phage library is exposed to the immobilized antigen and any unbound phage is washed away. This process is called biopanning and is usually repeated several times to identify antibody-bound phages that consistently bind antigens with high affinity. The bound phages are isolated and sequenced to identify the monoclonal antibody candidates.
ProteoGenix has an array of existing antibody phage display libraries that are perfect for making humanized antibodies for clinical applications such as bispecific antibody production and antibody drug conjugation in various antibody formats such as scFv, Fab, and VHH.
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Antigen design can make or break a custom antibody production project. To maximize your chances of success industry-leading antibody production companies must always consider the native conformation of your antigen and the final intended application of your antibody. Great antibody production companies invest in the infrastructure and talent required to perfect the design, synthesis, modifications, and purification of the most biologically relevant target antigens. These include:
Proteins antigens remain the most important approach to antibody generation. Immunizing an animal host during polyclonal or monoclonal antibody generation using recombinant proteins ensures your antibodies recognize a single antigen with high specificity. While some antibodies produced by B-cells are high-affinity others could bind domains that are conserved among many proteins. Thus, polyclonal antibodies produced from mammals immunized using whole proteins might contain a fraction of antibodies that target conserved domains resulting in crossreactivity.
Immunizing animal hosts using peptides limits antibody crossreactivity when the synthesized peptides are unique and thus not conserved among other protein domains or motifs. Thus, peptide antigens favor the production of antibodies with minimal cross-reactivity and high specificity. However, they are often less immunogenic due to their small size. Designing a proper peptide antigen for antibody production requires considering several factors:
Immunizing mammalian hosts with DNA inside lipid-rich vesicles, allows the cells at the injection site to collect the genetic material, transport it into the nucleus, and produce the antigen. This method has several benefits over traditional protein immunization methods:
Overall, DNA immunization is a powerful tool that industry leaders incorporate into their antibody-production toolbox.
Learn how ProteoGenix’s DNA immunization strategies can produce high-performing antibodies that target your difficult-to-purify protein of interest.
Small molecules typically lack the complex three-dimensional structure that proteins and other macromolecules have. This makes it difficult to produce antibodies that recognize small molecules with high specificity. However, there are several strategies to overcome this challenge. One approach is to use an antibody phage display library to find an antibody that binds the small molecule. This is achieved by immobilizing the small molecule to a surface with or without a carrier protein followed by identifying the antibody-displayed bacteriophages that stick to the immobilized small molecule. Another approach is the use of synthetic haptens, which are small molecules that can be designed to mimic the structure of the target molecule. These haptens can be conjugated to a carrier protein and used to immunize animals to generate antibodies that recognize the target small molecule. To create antibodies that can specifically bind to small molecules, computational modeling, and machine learning algorithms are used. This method entails screening large antibody libraries and selecting those with the highest affinity and specificity for the target molecule. Discover how ProteoGenix can help you build the next big small molecule-targeting antibody for immunotherapy or diagnostic needs.
Building custom antibodies that recognize whole cells has numerous applications in research, diagnostics, and therapeutics. Some examples include:
Several different protein expression systems can be used to express recombinant proteins used to produce custom polyclonal and monoclonal antibodies. Each expression system has unique advantages and disadvantages so it is important to understand the limitations of each when engineering your target antigen.
Mammalian Expression Systems are often used for the production of therapeutic proteins that require correct folding and post-translational modifications. Mammalian expression systems such as CHO or HEK cell lines produce high-quality recombinant proteins in abundance.
Bacterial Expression Systems are often used as expression systems because they are easy to culture, rapidly proliferate, and are cost-effective. However, some proteins may not fold properly in a bacterial expression system.
Yeast Expression Systems such as Saccharomyces cerevisiae or Pichia pastoris are eukaryotic cells that can produce recombinant proteins with proper folding and post-translational modifications. These expression systems are often used for proteins that require translocation through the golgi apparatus to achieve (close to) native glycosylation. Yeast however maintain reducing conditions in the cytoplasm inhibiting disulfide bond formation.
Insect Expression Systems such as the Spodoptera frugiperda (Sf9) Baculovirus expression system can be used to produce large quantities of recombinant proteins that are membrane proteins and/or require post-translational modifications.
Understanding the basic steps of antibody production in vivo allows getting a better overview of the advantages of both in vivo and in vitro antibody production methods. Also, the particular advantages of each method may dictate the best choice for your projects.
In vivo antibody production occurs when the immune system encounters a foreign substance, the immunogen. Once an antigen binds to the surface of a B lymphocyte, these cells can be activated in 2 different ways: polysaccharides, lipopolysaccharides, and other non-protein antigens activate B cells directly in a T cell-independent manner (the activation signal comes from another source than T cells such as factors from the complement system) whereas protein antigens activate them in a T cell-dependent manner occurring in several steps:
Once activated by linked recognition, type 2 helper T cells activate B cells thanks to cytokine release leading to the proliferation of daughter cells. This process culminates in somatic hypermutation which results in random mutations of variable heavy and light chains. This step is followed by another selection step in which only the positive mutation (leading to an increased affinity against the antigen) will be conserved. Thus, somatic hypermutation allows the generation of antibodies and memory cells with higher affinity against the antigen.
Further cytokine secretion results in the differentiation of activated B cells into memory B cells and plasma cells, each having a defined function:
Stimulation of plasma cells by the cytokines released by type 2 helper T cells allows switching from IgM production to another class such as IgG, IgA, or IgE. This switch does not affect the affinity of the antibodies for the antigen as it only consists in a genetic modification a modification of the constant region (accomplished by genetic rearrangement).
The release of antibodies into the bloodstream occurs only once antibodies with sufficient affinities are generated. This antibody response occurs in two steps:
Adapted from: Abbas et al. Cellular and Molecular Immunology. Elsevier.
In the context of custom antibody generation, eliciting an immune response can be achieved by immunizing with various substances such as large molecules, viruses, or bacteria. However, some substances do not induce an immune response (or only a weak immune response) mostly due to their small size.
For this reason, antigen design remains a major step when producing a custom antibody. While the antigen itself can be modified to elicit an immune response, it is also possible to co-administer it with an adjuvant to enhance its immunogenicity. If chemical adjuvants are insufficient, in vivo antibody production might not be adapted to your project and in vitro solutions should be considered. In this case, naive library screening with antibody phage display remains the best solution.
Not sure about the best antibody generation technology to use for your project? Contact our antibody production experts who will elaborate a personalized solution adapted to your needs, budget, and time.
Understanding what stimulates antibody production is important to improve the efficiency of both active and passive immunization strategies. Due to the complexity of the human immune response, it is not only important to understand what types of molecules activate the immune system, but also, what types of cells and molecules coordinate and regulate antibody expression.
In our organism, antibodies are produced in response to foreign molecules, called antigens. The natural adaptive immune response can generate antibodies capable of recognizing a multitude of different molecules including proteins, carbohydrates, and lipids.
The adaptive response can happen through two different processes: T cell-dependent or T cell-independent process (TD or TI, respectively). The activation of each of these processes depends on the nature of the antigen. For instance, most molecules and pathogens need to be digested and processed before being able to cause an effective immune response. But large polymeric molecules are often sufficient to activate the quick TI response in adults.
The inherent limitation of the TI response arises from its inability to produce an immunogenic memory. This primary response requires only the interaction between a soluble antigen and a B cell with compatible receptors (BCR) and can quickly produce antigen-specific antibodies. However, most protective and active immunization strategies (i.e. vaccines) tend to rely on the more complex and stronger TD immune response.
In this case, the diversity of antigens able to stimulate antibody production in our organism increases exponentially. This happens because, unlike B cells, T cells are not activated by soluble antigens. Instead, T cells need to interact with professional antigen-presenting cells (APC), such as dendritic cells, before the immune response can be initiated.
Before naïve T cell activation can occur, these cells need to be activated by specialized APC. Notoriously, APC are responsible for degrading foreign molecules and microorganisms and complexing their fragments with MHC (major histocompatibility complex) molecules.
T cells responsible for interacting with antibody-producing B cells, express the CD4 receptor and are named helper T cells or CD4+ T cells. Via their T-cell receptor (TCR), these cells recognize only the foreign fragments that are bound to class II MHC molecules on the surface of APC.
One limitation of this mechanism occurs due to the natural bias of T cells. The proliferation and recombination of the genetic machinery of these cells are heavily regulated to avoid immunopathology. For this reason, the efficiency of the TD response also depends on the organism’s previous infections with the same or similar pathogens. This complicates the TD response to a completely new pathogen. In the case the organism has never encountered a similar antigen, it needs to produce large variations of precursor cells before obtaining compatible TCRs.
TCRs recombination is an extremely and tightly regulated process, however, it is also crucial for increasing the diversity of CDRs (complementary determining regions). CDRs, both in TCRs and B cell receptors (BCRs), are responsible for antigen interaction. For this reason, a high diversity of CDRs ensures the organism better and more effective protection against pathogens. In other words, the more a single organism is exposed to different pathogens and foreign molecules, the better equipped it becomes to deal with new threats.
This knowledge challenges the assumption that healthy individuals have unlimited antigen-recognition repertoires. Moreover, this constraint of naïve repertoires may limit the efficiency of conventional vaccination strategies.
This limitation has prompted many researchers to investigate personalized vaccination as a suitable alternative to conventional strategies. According to experts on the topic, vaccines may be tailored to specific groups of individuals (defined by common characteristics such as sex, age, ethnicity, Fc-gamma-receptor polymorphism, immune status, etc.) to specifically engage with their unique immune repertoires.
However, the personalization of vaccines comprises challenges with many different levels of complexity. Moreover, in many cases, the increase in efficiency may not compensate for the additional cost of developing different vaccine formulations according to each group’s specific needs. The main reason for this significant increase in cost is the lack of extensive studies on the immune repertoire of individuals from specific groups. Thus, as stated by Katja Fink, a scientist from the Singapore Immunology Network, there is an urgent need to increase our repertoire databases before the field can move forward.
Low molecular weight molecules (<1,000 DA) are often not naturally immunogenic. Hence, they cannot be directly used to stimulate antibody production in our organism. In these cases, effective active immunization strategies can be created by conjugating the small molecule with a larger protein. It is the complex, not the hapten itself, that stimulates antibody production. This process is called the anti-hapten response. Haptens are thus considered molecules that only elicit an immune response when linked to a macromolecule, also known as carrier. Nevertheless, haptens are a structurally and chemically diverse group, thus making the process of hapten conjugate design very complex from a chemical point-of-view. When a hapten is used for immunization, special consideration must be given to the coupling strategy and the hapten:carrier molar ratio. These are the two factors that directly influence the strength of the immune response. Many proteins have been successfully employed as carriers, the most frequent include globulins and albumins, gelatin, casein, and tetanus, cholera or diphtheria toxoids. The only requirements of a good carrier molecule include natural immunogenicity and the presence of sufficient reactive side chains that can be leveraged for conjugation with the hapten. Conjugation strategies are also diverse. For instance, when the hapten is chemically reactive, the conjugation can be spontaneous. However, when natural reactivity is weak, haptens may be conjugated by crosslinking with intermediary molecules such as carbodiimides or glutaraldehyde.
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