MACS Handbook

Antibodies

1 Introduction

Antibodies are arguably one of the most important components of a molecular biologist’s toolbox. They have revolutionized biological research and are now making their impact felt in the clinic as effective therapeutic agents. Following an introduction to antibody structure and types as well as recombinant antibody technology, this chapter details the properties of REAfinity™ Recombinant Antibodies and their use in flow cytometry.

1.1 Antibody structure

Antibodies are characterized by their unique Y shape. Each antibody consists of four polypeptides – two heavy chains and two light chains. According to their functional characteristics, antibodies can be subdivided into variable and constant regions. The variable region, made up of the N-terminal ends of the heavy and light chains, contains the antigen binding site. The constant region, which consists of the rest of the molecules, determines the immune response or the mechanism used to destroy antigens (fig. 1).

Each antibody can be proteolytically cleaved into three fragments – two Fab regions and an Fc region. Fab, short for fragment antigen binding, includes the variable ends of an antibody. The Fc fragment, short for fragment crystallizable, determines the antibody biological effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcγRs) on the surface of leukocytes, resulting in phagocytosis or lysis of the targeted cells. In CDC, the antibodies trigger the complement cascade at the cell surface leading to cell death. 

1.2 Antibody isotype classifications

Antibodies are divided into five major isotypes, based on their structure, biological properties and ability to deal with antigens. Table 1 outlines the isotypes and their properties, distribution, and immune functions. (Janeway, E. A. et al. (2001) The distribution and functions of immunoglobulin isotypes. Immunobiol. Immune Syst. Health Dis. 5th Ed.) 

Most assay antibodies belong to the IgG subclass which is subdivided into four isotypes: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. IgG isoforms exert different levels of effector functions – the level of ADCC effector function is high for human IgG1 and IgG3, low for IgG2 and IgG4, whereas the level of CDC effector function is high for human IgG1 and IgG3, low for IgG2, and null for IgG4.  

Table 1: Antibody isotypes

IsotypeSubclassesStructureMajor immune functionDistribution
IgAIgA1 and IgA2Monomer, dimerNeutralizationExtracellular fluid (monomeric IgA), secretions across epithelia like breast milk (dimeric IgA)
IgD MonomerFunction not clearMostly bound to B cells
IgE MonomerSensitization of mast cellsMast cells beneath epithelial surfaces (specifically of the respiratory tract, gastro-intestinal tract and skin)
IgGIgG 1–4 (in humans)MonomerNeutralization, opsonization, complement system activation, sensitization for killing by NK cellsPlasma, extracellular fluid
IgM PentamerComplement system activationPlasma, extravascular spaces

1.3 Primary-secondary antibody classification

Antibodies that bind to specific antigens of interest help biologists visualize a variety of biological entities – from proteins on a Western blot membrane to cellular and subcellular structures.  
Primary antibodies bind directly to the antigen. Depending on the type of experiment, primary antibodies are used alone or in combination with a secondary antibody. For example, in flow cytometry, primary antibodies conjugated to fluorochromes are almost always used alone, whereas in microscopy, a primary-secondary antibody combination is used to increase the signal.  

Secondary antibodies help detect, sort, or purify antigens of interest by binding to the primary antibody. They are raised against the species and isotype of the primary antibody. For example, for a primary antibody raised in rabbit, an anti-rabbit secondary antibody raised in a host other than rabbit would suffice. Primary antibodies are often IgG isotypes and therefore will need an anti-IgG secondary antibody. Secondary antibodies are often conjugated to enzymes, dyes, or fluorescent proteins. The choice here depends on the type of experiment; for instance, in immunofluorescence experiments, secondary antibodies conjugated to fluorescent proteins or dyes are the right choice.  

A two-antibody system – the primary-secondary system – is often employed for i) stronger dye localization to an antigen, ii) multicolor labeling within a specimen and across experiments using one primary antibody and multiple fluorescent secondary antibodies, and iii) economical reasons.  

1.4 Polyclonal vs. monoclonal antibodies

Polyclonal antibodies (pAbs) are a heterogeneous mixture of antibodies, produced by various B lymphocyte clones. They mostly consist of a mixture of IgG subtypes and recognize multiple epitopes of an antigen. Polyclonal antibody serum can be obtained within 4–8 weeks by injecting the host animal with the antigen of interest followed by observation of antibody response and collection of antisera. Their production is inexpensive compared to monoclonal antibodies. (PMID:15953834

Monoclonal antibodies (mAbs) are produced by a single clone of B lymphocytes and recognize only a particular epitope on the antigen. They consist of only one antibody subtype. The mAbs are produced by hybridomas, i.e., B cells immortalized by fusion with myeloma cells (fig. 2). (PMID: 15728446)

 Compared to pAbs, mAb production is more complex, expensive and time-consuming (3–6 months). (PMID: 15953834

Hybridoma-derived mAbs are often contaminated with antibody impurities from two sources: myeloma derived–Ig light chains and serum-derived IgGs. (PMID: 29485921) These impurities lower the specificity of the antibody resulting in irreproducible results (fig. 3). 

1.5 The reproducibility crisis in life science research: Antibodies are a major culprit

Irreproducibility of results is a major challenge faced by the scientific community today. Several factors contribute to this complex problem. According to the Second Annual State of Translational Research 2014 Survey Report conducted by the Sigma-Aldrich Corporation on the issue of reproducibility, antibodies were one of the major product classes contributing to the issue (around 36%) and second only to animal models (around 41%). In 2014 another survey conducted by the American Society for Cell Biology, the ASCB member survey on reproducibility, 50% of respondents named reagents including antibodies, sera, and plasmids to be the reason for the failure to replicate experiments.  

Antibody-related reproducibility can be improved by adhering to the following guidelines by all parties involved – vendors, users, and journals: 

  • Application-specific validation of antibodies 
  • Transparent and clear reporting of reagent use 
  • Selling and usage of consistent-quality antibodies

One solution is to use antibodies produced by recombinant antibody technology. (PMID: 25652980) Recombinant antibodies exhibit higher purity, lot-to-lot consistency, and definability because of standardized cell culture systems and access of DNA sequence, when compared to hybridoma-derived monoclonal antibodies. 

2 Recombinant antibody technology

Recombinant antibodies (rAb) are monoclonal antibodies generated in vitro and animal-free using rDNA technology. Recombinant antibodies overcome many of the limitations of conventional mAbs (see table 2). (PMID: 26482034)

2.1 Recombinant antibodies: production methods

The production workflow of recombinant antibodies consists of the following steps:

  1. Determination of the sequence of the desired antibody genes 
  2. Gene synthesis and cloning into expression vectors 
  3. Expression of antibody fragments in mammalian or bacterial cells 
  4. Purification and analysis of the antibody

For recombinant antibody production, stable cell lines such as CHO and HEK293 are often used.

Antibody fragments for recombinant antibody production can be obtained from existing monoclonal antibodies or can be selected from libraries of genes (encoding slightly different antibody proteins) according to the affinity of the antibodies for binding to target antigens. The library-based recombinant antibody production methods are divided into in vitro display platforms, such as phage display and ribosome display, and in vivo display platforms, such as bacterial, yeast, and mammalian cell-surface display. Figure 4 below outlines the early milestones in the production of recombinant antibodies. (PMID: 15953834, 4001944, 3140379, 3045807, 3210233, 2247164, 8248129, 9181578, 16763048)

Phage display is the most common method which involves the following steps:

  1. Library construction: The gene fragments encoding antibody heavy and light chains are cloned into phage vectors resulting in a phage library displaying antibodies with varied antigen binding sites.  
  2. Antibody selection by panning: Panning is a process by which antibodies are selected by incubating the phage library with the target antigen immobilized on a solid phase. Unbound phage particles are washed away, and the bound phages are eluted and amplified in E. coli.  
  3. Cloning and selection: The resulting antibody DNAs are cloned into expression vectors and expressed in E. coli.  
  4. Screening and affinity maturation: To improve antibody affinity, sequence diversity is introduced into the selected antibody molecules by methods like error-prone PCR or site-directed mutagenesis. Antibodies are evaluated for improved affinity and the selected clones are expressed and purified.  

3 REAfinity™ Antibodies: recombinant antibodies for flow cytometry

REAfinity™ Recombinant Antibodies are a new generation of recombinantly generated antibodies specifically designed for flow cytometry applications. They provide superior lot-to-lot consistency and purity, as compared to mouse or rat monoclonal antibodies. The following structural features of the REAfinity Antibodies makes them the superior antibody choice for flow cytometry experiments (fig. 5).

  • Universal human IgG1 isotype reduces the complexity of experiment planning by eliminating the need to include multiple isotype controls during flow cytometry analysis. 
  • Mutated Fc region eliminates background signals by abolishing any binding of the recombinantly engineered antibodies to FcγRs.
Figure 5: Structural features of REAfinity Antibodies.
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Figure 5: Structural features of REAfinity Antibodies.

3.1 REAfinity Recombinant Antibodies: production workflow

REAfinity Antibodies are generated by cloning the antigen binding region from a traditional hybridoma-derived monoclonal antibody (mouse IgG) with the human IgG1 Fc region. The Fc region is mutated to abolish binding of the recombinant antibodies to Fcγ receptors. This chimeric DNA is expressed in a mammalian cell line cultured under standardized, serum-free conditions. To ensure lot-to-lot consistency, the same cell line is used to produce all REAfinity Antibodies (fig. 6).

Advantages of this production method include:

  • No light chain impurities because of a highly defined chimeric IgG sequence 
  • No cell-derived impurities as the cells are programmed to solely express the defined antibody 
  • No serum-derived impurities as the mammalian cells are cultivated in serum-free media 

3.2 REAfinity Recombinant Antibodies: unique features and advantages

3.2.1 High lot-to-lot consistency – reproducible results

Hybridoma-derived monoclonal antibodies often contain contaminating light chains and serum-derived antibodies. The presence of these impurities and a non-standardized production method result in lot-to-lot variability and the lack of reproducibility of experiments.  
REAfinity Antibodies are derived from a defined DNA sequence, which encodes only one type of heavy and light chain, ensuring high antibody purity (fig. 7). They are produced in mammalian cells under biologically and chemically defined, standardized culture conditions, resulting in high lot-to-lot consistency and purity as compared to mouse or rat monoclonal antibodies. Moreover, every manufactured lot undergoes thorough analytical, biochemical, and cell-based analysis (fig. 8). 

Figure 7: Mass spectrometry analysis  of REAfinity Antibodies   and hybridoma-derived antibodies.
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Figure 7: Mass spectrometry analysis of purified antibodies shows that REAfinity Antibodies are defined products, while hybridoma generated antibodies can be a mixture.

(A) Mass spectrometry analysis of two examples of hybridoma-generated monoclonal antibodies shows that both contain a second light chain with a molecular weight of approx. 23,635 Da. In one example (bottom left), the amount of the contaminating light chain was by far exceeding the productive light chain of approx. 24,142 Da. (B) Two examples of recombinant REAfinity Antibodies show pure light chain populations.

Figure 8: Staining performance of REAfinity Antibodies. 
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Figure 8: Staining performance of REAfinity Antibodies is nearly identical between different lots. 

Two manufacturing lots of five different CD56 REAfinity Antibody conjugates were compared by flow cytometry using the MACSQuant® Analyzer. The two histogram curves (black and red) represent the two lots. Lot-to-lot staining performance was nearly identical when using human peripheral blood mononuclear cells (PBMCs) from a single donor.

3.2.2 No Fcγ receptor binding – reduced background signal

High background signals from antibodies often compromise the quality of flow cytometry results. One of the reasons for high background signals is non-specific binding of antibodies to Fcγ receptors on leukocytes. To avoid this type of non-specific binding, Fc blocking reagents are used during antibody staining. However, the blocking procedure is time-consuming and costly.  

REAfinity Antibodies feature a mutated human IgG1 Fc region which abolishes their binding to Fcγ receptors (fig. 9). This allows for background-free analysis and eliminates the need for additional blocking steps such as using an FcR blocking reagent (fig. 10). Therefore, staining protocols are much faster with REAfinity Antibodies (table 3).  

Figure 9: Recombinantly engineered REAfinity Antibodies do not bind to Fcγ receptors. 
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Figure 9: Recombinantly engineered REAfinity Antibodies do not bind to Fcγ receptors. 

CD144-specific hybridoma-derived mouse monoclonal antibodies (dark purple bars) bind to high-affinity FcγRI (CD64) as well as low-affinity FcγRIII (CD16) and FcγRII (CD32) receptors. In contrast, REAfinity Antibodies (light purple bars) show virtually no interaction with Fcγ receptors. Binding to  five cellular Fcγ receptors was compared using enzyme-linked immunosorbent assays.

Figure 10: Staining with REAfinity Antibodies shows no background signal, even without FcR blocking. 
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Figure 10: Staining with REAfinity Antibodies shows no background signal, even without FcR blocking. 

Human PBMCs were stained with either a mouse monoclonal CD158a-PE antibody or the REAfinity Antibody, CD158a-PE (clone: REA284). Staining was performed without (top) and with (bottom) FcR blocking. The mouse monoclonal antibody binds non-specifically to CD56-negative cells (A). Staining with the REAfinity Antibody shows no background signal. Cells were analyzed by flow cytometry on the MACSQuant® Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence.

Table 3: Staining protocols for immunophenotyping of human and mouse surface markers 

Steps involvedDuration of each step
Hybridoma-derived antibodiesREAfinity Antibodies
Prepare 106 cells--
Add blocking reagent to cells and incubate at 4° C or RT10–15 min-
Add primary antibodies and incubate at 4° C or RT10–30 min10 min
Wash cells1–2× 10 min10 min
Resuspend and analyze cells--
Total staining procedure30–65 min20 min
3.2.3 One universal isotype – easy experiment planning

All REAfinity Antibodies, i.e., REA clones, have the same human IgG1 isotype, eliminating any need to include multiple isotype controls during flow cytometry analysis (fig. 11). This offers the possibility of working with only one type of isotype control, reducing the complexity of experiment planning and saving time. 

In contrast, hybridoma-derived monoclonal antibodies are composed of antibody isotypes derived from different species and therefore require multiple isotype controls. This complicates not only the management of reagent inventory but also panel design when setting up experiments. 

Figure 11: Straightforward experiment planning with only one isotype control for all REAfinity Antibodies.
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Figure 11: Straightforward experiment planning with only one isotype control for all REAfinity Antibodies.

Besides these unique features, all REAfinity Antibodies undergo strict production and quality control processes. For each specificity, premium antibody screening technology is applied to identify the clone with the highest binding affinity and specificity. Only the top candidate is selected to become a recombinantly engineered antibody (REA) clone. 

3.3 REAfinity Recombinant Antibodies: optimized for flow cytometry applications

REAfinity Antibodies are designed with flow cytometry in mind. Their high purity, lack of background signal, and a standardized fluorochrome conjugation process ensure a superior performance in flow cytometry experiments in comparison to hybridoma-derived monoclonal antibodies (figs. 12–14). 

Figure 12: Specific detection of human CD116+ cells with clone REA 211. 
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Figure 12: Specific detection of human CD116+ cells with clone REA 211. 

Human PBMCs were stained with either a PE-conjugated mouse monoclonal antibody (A) or PE-conjugated REAfinity Antibody (B) recognizing CD116. Cells were also stained with CD14-FITC. Flow cytometry analysis was performed on a MACSQuant Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence.

Figure 13: Specific detection of human CD158b2+ cells with clone REA147. 
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Figure 13: Specific detection of human CD158b2+ cells with clone REA147. 

Human PBMCs were stained with either a PE-conjugated mouse monoclonal antibody (A) or PE-conjugated REAfinity Antibody (B) recognizing CD158b2. Cells were also stained with CD56-APC, human (clone: REA196). Flow cytometry analysis was performed on a MACSQuant Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence.

Figure 14: Specific detection of mouse dectin-1+ cells with clone REA154. 
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Figure 14: Specific detection of mouse 
dectin-1+ cells with clone REA154. 

Bone marrow cells from BALB/c mice were stained with either a PE-conjugated mouse monoclonal (A) or a PE-conjugated REAfinity Antibody recognizing dectin-1 (B). Cells were also stained with CD11b-APC. Flow cytometry analysis was performed on a MACSQuant Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide (PI) fluorescence.

3.4 Multicolor flow cytometry with REAfinity Recombinant Antibodies

REAfinity Antibody clones are available for hundreds of specificities and are conjugated to a variety of fluorophores to address multicolor flow cytometry needs. Current options include traditional dyes, like FITC, PE, APC, and newer versions like Vio® and VioBright™ Dyes (fig. 15). REAfinity Antibodies conjugated to Vio Dyes are powerful and reliable tools for multicolor cytometry analysis, resulting in highly sensitive, reproducible, and rapid cell profiling.