Assays, Reagents, and Antibodies

ASSAYS, REAGENTS, AND ANTIBODIES RESOURCE GUIDE

? Questions to Ask When Buying Assays, Kits, and Reagents

? How to Make Your Antibodies Work for Your Lab

? Antibody Generation and Validation

? Efficiency, Reliability, and Possible Danger in Protein Quantification

? Automating Cell Culture for Therapeutic Monoclonal Antibody Development

Questions to Ask When Buying Assays, Kits, & Reagents 

It is very important to purchase assays, kits, and reagents from trusted sources, as the experiments conducted will only be as good as the components in these products

by Lab Manager

Assays, kits, and reagents are used in many different life science, environmental, and research labs. Assay kits have become very important in providing the materials needed to conduct specific tests that require complex or hard-to-develop components like enzymes, antibodies, genomics, and cells. It is very important to purchase assays, kits, and reagents from trusted sources, as the experiments conducted will only be as good as the components in these products.

7 Questions to Ask When Buying Assays, Kits, and Reagents:

• How pure do the components need to be for this application?

• Is the same lot needed for an extended experiment?

• How similar or dissimilar are the results from different lots?

• Does the assay, kit, or reagent work well with the existing lab equipment?

• Does the assay, kit, or reagent reduce lab work time to implement?

• How important are potential contaminants from the packaging?

• What is the unit price per test?

Quality Considerations

Purchasing Tip

The recent pandemic has created havoc in some lab supply chains. Due to issues with supply and delivery of some assays, kits, and reagents, it is important to establish the robustness of the supply of important items for the lab. It may be necessary to have multiple sources of the key assays, kits, and reagents to ensure that the lab can continue to be productive, even in uncertain times.

Given the wide range of assays, kits, and reagents available, as well as a large number of manufacturers, it is very important to understand the level of purity required, and the importance of trace components included. These can vary for items purchased from different vendors, and can even show important lot-to-lot variations from a single producer. Before starting a long-term set of experiments, it is worth understanding the precision required to obtain the desired results.

How to Make Your Antibodies Work for Your Lab 

Not all antibodies work. Learn how to optimize them

by Andy Tay, PhD

Antibodies are a common lab consumable used in diverse applications including flow cytometry, immunohistochemistry, western blot, and as clinical therapeutics. They are produced by B lymphocytes upon exposure to molecules known as antigens, such as proteins and carbohydrates, like glycans.

While antibodies are used prevalently, their use has been linked to false scientific results, causing a reproducibility crisis in science. A study in 2008 found that fewer than ~6,000 routinely used commercial antibodies are specific to their targets. Poorly characterized antibodies might have also led to poor replication of landmark preclinical studies, costing the biomedical research community an estimated $530 million per year in the United States.

When an antibody is not specific to its target, a cell type could be wrongly identified by its surface protein expression, causing a misreporting of its cellular functions. In addition, when an antibody binds to multiple targets in a non-specific manner, it can also lead to confusion on mechanisms that adversely affect downstream drug discovery. Here, we will first discuss some common applications of antibodies before sharing tips to optimize them for use in labs.

Application of antibodies

Antibodies can be used to label a variety of biomolecules. Western blot is a technique that is used to detect the presence and compare relative amounts of a target protein in a protein mixture. The protein mixture is first loaded into polyacrylamide gel and separated using electrophoresis by their molecular weights. Primary antibodies that bind to the target proteins are added before adding fluorescence molecule- or enzyme-conjugated secondary antibodies that bind to the primary antibodies. Fluorescence molecule- or enzyme-con- jugated secondary antibodies subsequently emit and amplify signals, such as through breaking down substrates for detection via methods like fluorescence, colorimetry, or radioactivity.

Antibodies are also useful for immunohistochemistry such as in situ tissue staining to localize the spatial distribution of specific cell types or proteins. For such application, tissues are first frozen and fixed before adding primary and secondary antibodies. To promote the diffusion of antibodies into densely packed tissues, electric fields, and physical swirling can be introduced to improve the migration and uniformity of antibody binding.

Antibodies are also widely used as therapeutics. For instance, monoclonal antibodies are used to inhibit actions of proteins such as programmed cell death 1 receptor (PD-1) that is implicated in cancer. PD-1 signaling negatively regulates T cell-mediated immune response and an antibody that blocks PD-1 has been shown to enhance anti-tumor immunity and prolong survival. There are already three US Food and Drug Administration-approved monoclonal antibodies against PD-1 including atezolizumab, durvalumab, and avelumab.

While incredibly valuable in scientific research, antibodies may not always work effectively due to reasons like poor specificity and denaturation. Below are some tips to optimize your antibodies.

Check for host species and clonality

Antibodies are typically used in pairs: the primary antibody binds to the antigen of interest, such as a protein, and the fluorescent molecule- or enzyme-conjugated secondary anti- body binds to the primary antibody for signal detection and amplification. The host species of the primary and secondary antibody should be different from the antigen species to prevent cross-reactivity and unwanted background staining or interference.

The antibody clonality is also an important factor. Monoclonal antibodies are more specific as they bind only to a single antigen, but they cannot be used across multiple species. On the other hand, polyclonal antibodies bind to multiple epitopes with greater tolerance of antigen changes (such as those unintentionally induced by sample treatment and preparation), but they suffer from greater batch-to-batch variability.

Talk to other users

Before attempting to perform antibody-related experiments, it pays to read scientific literature to check which antibodies are being used by researchers in the same field. While there is a possibility that everyone might be using a non-specific antibody, the pooled risk is lower. Users can also consider attending webinars by The Antibody Society to learn tips on choosing antibodies.

Purchase validated antibodies

Different manufacturers can adopt widely different standards for antibody manufacturing, which affect material quality. It is advisable to check databases of validated antibodies such as Antibodypedia, which contains data on 19,000 human protein targets (~95 percent of human genes). It is also recommended to read the guidelines by the International Working Group for Antibody Validation on techniques to validate antibodies and outline which applications each antibody is suitable for.

Scrutinize quality analysis documents from manufacturers

Reputable manufacturers will provide detailed information about their antibodies, such as the antigen sample, host species, and cross-species reactivity unless there are proprietary reasons. Bordeaux et al. reported three levels of antibody validation efforts from companies. The first is “low/no validation” where there is minimal information except for a brief description of the target and recommended applications, without examples of references that have successfully used the antibodies. The second is “moderate validation” where manufacturers include at least one example of the antibody being used to correctly identify targets through western blots. The third is “high validation” where companies include western blot data for multiple cell sources with high batch-to-batch consistency. Users should try to find suppliers that share antibody manufacturing and quality analysis data, which is useful in troubleshooting.

Adopt good techniques to handle antibodies

Being proteins, antibodies are vulnerable to extreme environmental conditions like heat and acidity that can cause denaturation and affect binding specificity to targets. Some malpractices include repeated freezing and thawing of the same antibody samples. Users should aliquot the antibodies into smaller quantities and thaw only when needed. Users can also read protocols from suppliers and publishers to know the ideal temperature to handle antibodies, duration for incubation with antibodies, and various troubleshooting tips.

Incorporate experimental controls

As much as possible, users should include positive and negative controls in their experiments to validate antibodies. Positive controls can be lysate or recombinant proteins that are known to bind to the specific antibody. Negative controls can include genetic knockout samples that no longer produce the antigens. Users can also omit a primary antibody to understand whether secondary antibody can cause unwanted background interference.

Antibodies are popular and valuable materials in labs and for use in clinics. Nevertheless, how they are manufactured and handled can lead to poor reproducibility. To fully optimize antibodies for use, tips such as sharing and learning from your peer community, choosing only reliable manufacturers, and incorporating good handling practices will greatly benefit the science done in your lab.

Antibody Generation and Validation 

Conjugated antibodies for flow cytometry is an essential tool that requires validation

by Andy Tay, PhD

Antibodies, also known as immunoglobins, are produced by immune B cells in response to foreign molecules called antigens to neutralize them. They are used for numerous applications in biology and diagnostics.

Shaped like a Y, an antibody consists of the antigen-binding (Fab) variable region at the top, and a constant (Fc) region at the base. The Fab region enables an antibody to bind to a specific segment (or epitope) of an antigen through interactions like electrostatic forces and van der Waals forces. Attributed to their molecular recognition properties, antibodies are a popular tool in biological experiments. They are widely used in experiments to identify, isolate, and quantify specific protein antigens to understand their roles in physiology and diseases. Some examples include western blot analysis, immunostaining, and flow cytometry.

There are two main types of antibodies—polyclonal or monoclonal—and they are used in different settings. To detect the presence of multiple antigens for a simple yes/ no diagnostic test, polyclonal antibodies have an advantage as they can bind to different epitopes. Hence, they are more tolerant of minor changes in antigen structure resulting from polymorphism and slight denaturation. Monoclonal antibodies are more widely used in cases where specific antigen-antibody binding is crucial and cross-reactivity may invalidate experimental interpretation.

This article is a discussion of how antibodies are generated and the challenges associated with validation. The use of conjugated antibodies for fluorescence flow cytometry and cytometry by time-of-flight (CyTOF) applications is also explored.

Generating and purifying antibodies

Polyclonal antibody production exploits animal immune systems (often in rabbits or donkeys). Typically, the antigen of interest is repeatedly injected into the animals to evoke high expression of antigen-specific antibodies in the serum. For weakly immunogenic antigens, an adjuvant is also used to release the antigens in a sustained manner to increase the probability that an immune response will be elicited.

Polyclonal antibodies can then be purified from blood serum using antigen affinity chromatography. The column matrix can either contain antigens against unwanted antibodies, allowing elution of the target antibody, or it can contain an antigen against the target antibody, allowing elution of unwanted antibodies.

Monoclonal antibodies can be produced using hybridomas. The antigen of interest is injected into a mouse to generate antibody-producing B cells. Using electro-fusion or chemicals like polyethylene glycol, the B cells will be fused with an immortal myeloma cell line to create hybridomas. Only myeloma cells that do not secrete antibodies are used, and these cells also lack the hypoxanthine-guanine phosphori- bosyltransferase (HGPRT) gene, making them sensitive

to hypoxanthine-aminopterin-thymine (HAT) media that contains aminopterin that blocks DNA synthesis, and thus cell division. After electrical or chemical treatments, the cells are cultured in HAT media for 10 to 14 days and only hybridomas survive, as they contain the HGPRT gene from B cells while B cells with a shorter life span perish. The cell population is then diluted to one cell per well plate and as the antibodies in a well are produced by the same B cell cone, they recognize the same epitope, and are monoclonal.

Besides this method, advances in genetic sequencing have also given rise to recombinant antibody technology. Rather than using animals, the gene responsible for producing the target antibody is identified and cloned into an appropriate vector before being introduced into a host cell like Chinese hamster ovarian cell. The genetically-engineered host cell produces the monoclonal antibody, which can then be purified using methods like affinity chromatography.

Validating antibodies

Reduced antibody specificity, due to batch-to-batch variability, or antibodies that bind the wrong targets lead to poor experimental reproducibility and can result in data misinterpretation and wasted resources. It is, therefore, important to check that the antibody is specific to its target antigen.

There are a few simple ways to validate antibodies. First, labs should request characterization information of the same antibody batch being purchased from the manufacturer. Second, seek assistance and peer review from the community. If you are using an antibody that has been reported in a publication, it may be valuable to reach out to authors in that paper about their experience using the antibody. Initiatives like Antibodypedia and The Antibody Registry also list user-validated antibody information for reference. Third, perform simple lab tests using positive and negative controls. Some examples include using cells with a knock-out gene expressing the antigen of interest or using siRNA to block the mRNA translation of the target antigen.

Antibodies in flow cytometry

Fluorescence flow cytometry is a technique in which fluid-suspended fluorescently labeled single cells flow through a laser beam and the properties of the cells can be determined by their light-scattering profiles. Fluorescence-tagged anti- bodies are used in flow cytometry to isolate and quantify cells expressing target antigens.

This technique relies on a primary antibody that targets specific antigens binding with a secondary antibody conjugated with fluorescence tags. The secondary antibody specifically binds to the heavy chain (Fc) region of the primary antibody. As an example, the primary antibody binds to antigen X and is produced in a rabbit. Then a suitable secondary antibody should bind to the Fc region of a rabbit immunoglobin and be tagged with a fluorescence label (antirabbit-fluorescence). It should also come from a non-rabbit source and preferably an animal that shares little homology.

An emerging flow cytometry technique is cytometry by time of flight (CyTOF). This technique overcomes spectral overlaps in fluorescence flow cytometry, and it enables the detection of more antigens at a single time (approximately 100 antigens, compared to 40 antigens using traditional methods). In CyTOF, antibodies targeting antigens of interest are conjugated with metals instead of fluorescence tags.

Antibody-bound cells are then sent through an argon plasma, which ionizes the metal-conjugated antibodies, and the metal signals are analyzed by a time-of-flight mass spectrometer.

CyTOF is becoming a popular way to label intracellular proteins such as cytokines involved in complex signaling pathways that involve many proteins.

Antibodies are an indispensable tool in biology. Although the technology to manufacture them is established, antibody validation remains poorly standardized. Before using antibodies in experiments like flow cytometry, users should always perform a few extra steps to ensure they are validated.

Product Spotlight

Incucyte® Live-Cell Analysis Systems 

Incucyte® Live-Cell Analysis Systems automatically acquire and analyze images around the clock, enabling you to derive deeper and more physiologically-relevant information about your cells, plus real-time kinetic data — without ever removing your cells from the incubator.

The Incucyte® instrument, proprietary assays, and reagents provide you with the ability to gain new insights into biological processes via real-time, quantitative analysis of live cells.

Furthermore, the Incucyte® system accommodates multiple users and applications seamlessly and combines information-rich, image-based analysis with the convenience and throughput of microplate assays. Incucyte® technology is featured in over 3,000 peer-reviewed publications in journals.

Efficiency, Reliability, and Possible Danger in Protein Quantification 

Biochemical assays are dependent on the fidelity with which one can quantify the relative concentrations of proteins in solution

by Brandoch Cook, PhD

Biochemical assays are powerful tools in laboratory biomedical science, and they can often answer questions clearly and digitally in ways other procedures cannot.

However, their accuracy is wholly dependent on the fidelity with which one can quantify the relative concentrations of proteins in solution.

To give an idea of the importance of protein quantification, the top three most-cited articles in the history of scientific publication describe techniques for measurement or preparation of protein samples. The all-time champion is the 1951 paper (with over 305,000 citations!) in which its lead author, Oliver Lowry, introduced an eponymous procedure for protein quantification.

Ease of use and reliability have been the keywords in subsequent variations on protein assays since the days of Lowry et al. Most modern assays rely on colorimetric evaluation of a solution via its linear change in absorbance at a given visible-spectrum wavelength when exposed to a protein-binding reagent. One way the Lowry method retains an advantage over contemporary procedures is that it is an “end-point” assay; in other words, the reaction between protein and detection reagent reaches a finite maximum so a single standard curve using a known protein can be used repeatedly to calibrate sample concentrations across multiple experiments.

In other commonly used assays, color continues to deepen indefinitely, and a new standard curve must be generated every time. This is achieved by measuring absorbance across a range of known concentrations in serial dilution of bovine serum albumin or immunoglobulin and calculating the concentrations of experimental samples from the equation generated by the standard curve. If it sounds tedious to repeat this procedure every time you run a protein gel, that’s because it is.

Improvements in reliability and throughput

Tedium aside, where Lowry is deficient compared with newer assays is in almost every other aspect relevant to modern biochemical techniques. Therefore, when the BCA assay re- placed the Folin-Ciocalteau reagent with bicinchoninic acid, the throughput and reliability of copper-chelate protein quantification improved dramatically. The BCA assay proceeds in one easy step, making it more amenable to simultaneous measurement of many samples, in terms of both time and re- agent usage. Moreover, the detection reagent is more stable in an alkaline solution, with less interference from components typical of many protein-containing cell lysates, including detergents, Tris, cations, EDTA, and reducing agents.

Similar in colorimetric principle, although quite different in chemistry, the Bradford assay employs the intrinsic property of Coomassie dye to change from red in an acidic solution to blue when bound to protein. This is another straightforward, one-step procedure that allows for high throughput, with the added benefit that it develops more quickly. However, Bradford is also sensitive to the presence of detergents in protein buffers and suffers from high variability. Although other assays use a similar procedure while offering more compatibility in buffer constituents, variability is still a problem compared with biuret-based methods. Consequently, BCA has unofficially become the gold standard among biomedical laboratories for protein measurement preparatory to standard procedures such as Western blotting. Because of differences in chemistry, BCA and Bradford are mutually incompatible in the affinity of one detection reagent over another for particular amino acids. Proteins with excess cysteine, tyrosine, and tryptophan residues will skew BCA assay absorbances because of their affinity for cuprous ions, while the affinity of Coomassie dye for arginine and lysine will do the same, providing inaccurate readings.

Specialty assays

Finally, for specialists who may be examining small peptides at low concentration or proteins associated with lipid bilayers that can interfere with standard assay reagents, there are several kits and protocols that allow reliable and reproducible measurement. One example is the CBQCA assay, designed for highly sensitive measurement of proteins in lipid solutions, which uses potassium cyanide (danger!) to stimulate reaction with amine groups, resulting in fluorescent excitation.

In addition to CBQCA, there is a seemingly limitless variety of specialty applications for protein quantification. The table below provides a summary of differences and compatibilities between the more standard assays. Although BCA is perhaps currently the most favored, there are rationales for choosing other assays based on factors such as buffers, time frames, detection limits, and wavelengths particular to spectrophotometer and microplate reader setups. What works best for the end user depends on considerations that are unique to every laboratory’s field of interest. This table uses kits provided by several large suppliers, and the more adventurous and thrifty among you can always make your own.

Assay Comparison Table for 'Efficiency, Reliability, and Possible Danger in Protein Quantification'

Automating Cell Culture for Therapeutic Monoclonal Antibody Development 

Advances in automation eliminate the tedious, time-consuming aspects of cell culture and cell line development

by Michelle Dotzert, PhD

An astonishing 38 percent of men and women are diagnosed with cancer at some point during their lives, according to the National Cancer Institute at the National Institutes of Health. While this statistic is disheartening, it is important to note that the fight against cancer is not a losing battle. Between 1991 and 2015, the overall cancer death rate in the United Sates was reduced by 26 percent. Early-detection strategies and cutting-edge treatments are continuously evolving as a result of ongoing research. Therapeutic monoclonal antibodies (mAbs), for example, have emerged as a promising treatment strategy. Numerous mAbs have been approved for clinical use, and dozens more are under investigation as potential cancer therapeutics. The use of mAbs for cancer treatment began in 1997, when rituximab was the first mAb approved by the U.S. Food and Drug Administration (FDA) for treatment of some forms of non-Hodgkin lymphoma. Since then, mAb therapeutics has grown into a multibillion-dollar market. Prior to commercialization, mAb development begins with cell culture for cell line generation. Technological advances in cell culture automation have dramatically accelerated the development process by increasing efficiency, consistency, and sterility.

Predefined Specificity

In 1975, Georges Köhler and César Milstein were the first to describe the creation of hybridomas for monoclonal antibody production. In an early study, they fused mouse myeloma and spleen cells from an immunized donor to produce cell lines that secreted anti-sheep red blood cell antibodies. The hybridoma technique is still used to develop cell lines for mAb production. Dr. David Fox is a professor of internal medicine in the Division of Rheumatology and the director of the Hybridoma Core at the University of Michigan. The Core offers antibody development services for preclinical antibody research and investigation, using the same process involved in therapeutic mAb development. Dr. Fox explains this process: “We receive the antigen from the person who wishes to develop a monoclonal antibody. We then immunize mice and test the serum to determine if the mouse is mounting an immune response. Once there is sufficient antibody titer in the serum, the spleen is removed to create hybridomas by fusing spleen cells with a cell line. Supernatant [fluid] is taken from the hybridomas, and the investigator screens for the monoclonal antibody of interest that will recognize a specific antigen and is appropriate for their application.”

Humanization techniques are required for antibodies destined for therapeutic use in humans. “Once they are considered a therapeutic lead, these mouse antibodies must be humanized (by genetic engineering) unless they are made in a humanized mouse, which allows more rapid progress to a potential drug stage,” explains Dr. Thomas Moran, a professor of microbiology at the Icahn School of Medicine at Mount Sinai and the di- rector of the Center for Therapeutic Antibody Development.

Targeting Cancer

Humanization techniques have moved mAbs into the clinic, and several are FDA-approved for use as cancer therapeutics. mAbs are used to target and bind tumor cells expressing cancer-specific antigens and induce cell death. Individual antibodies exhibit unique mechanisms of action. “Monoclonals can be used directly to bind to surface proteins on cancer cells to kill or inhibit their growth,” explains Dr. Moran. Trastu- zumab (Herceptin), for example, targets the HER2 protein on the surface of breast and stomach cells. “[They] can work by recruiting immune elements, delivering toxins, producing CAR-T cells, or as bispecific antibodies that recruit T or NK cells to kill the cancer cells.” Tositumomab is used in the treatment of some B-cell non-Hodgkin lymphomas and is an example of a monoclonal antibody used to deliver toxins, as some of the antibody delivers a linked radioactive substance to the B-cells. Bispecific antibodies consist of two different monoclonals capable of binding to separate proteins simultaneously. Blinatumomab is a bispecific T cell engager that binds to the CD19 protein on leukemia and lymphoma cells and CD3 proteins on T cells to direct an immune response toward the cancerous cells. The unique targets and mechanism of action of each mAb enable a highly specific approach to cancer treatment compared with other treatments such as chemotherapy. As a result, they are often associated with fewer side effects among patients.

The Automation Advantage

Antibody development on a small scale is often done without automated devices. “For the scale of culture we work with, we mainly use in vitro bioreactor flask systems,” says Elizabeth Smith, assistant director of the Core. Sterility is critical for all cell culture applications, and smaller-scale mAb production may not require highthroughput equipment. “I like the control that I have over the sterility of what I’m doing when I’m handling the liquids myself,” says Smith.

However, for large-scale production of therapeutic mAbs, automating cell culture can offer numerous advantages, including reducing time-consuming, repetitive tasks for laboratory staff and enhancing throughput. Automated liquid-handling systems eliminate hours spent pipetting and ensure highly accurate volumes. Systems that offer dedicated

pipette tips for each cell line and noncontact liquid dispensing reduce the risk of cross-contamination. Automation, by design, reduces the amount of human interaction with cell cultures, thereby eliminating various opportunities for contamination. Automated systems range in capability, with sophisticated systems enabling automated media changes, passage, harvesting, and monitoring when integrated with plate readers and imaging devices.

Working with an automated system has contributed to more than just increased productivity for Dr. Moran and his col- leagues. “The traditional method was to test and then identify the clones through a laborious replating and retesting step. An automated system is more accurate, automated, and less prone to contamination,” he notes.

Challenges Remain

While mAbs are a promising line of investigation in the search for cancer treatments, their development is not without challenges. Cell culture contamination from chemicals, bacteria, yeasts, viruses, and even cross-contamination with other cell lines can have severe consequences, including destruction of the culture and even the cell line. Practicing good aseptic techniques and implementing automated cell culture technologies can dramatically reduce the risk of contamination.

Another methodological challenge is to “make antibodies that bind to native molecules as they appear on the surface of cells,” says Dr. Moran. “Often, synthesized proteins produced to simulate the real protein are not folded the same way and antibodies made against them do not bind effectively to the ‘real protein.’” It is extremely difficult to predict if an anti- body developed with a specific protein will effectively recognize the protein on the cell surface. Immunogenicity also poses challenges. Genetically engineering the antibodies to render them more human has shown some success; however, even fully human antibodies can elicit a negative immune reaction. Scalability, yield, and cost are also considerations in the development of mAbs.

Given their highly specific nature, monoclonal antibodies are promising cancer therapeutics. Their development relies heavily on cell culture techniques, which can be laborious, time-consuming, and prone to contamination. Automated cell culture technologies ensure greater accuracy and precision, reduce the risk of contamination, and alleviate scientists of tedious and time-consuming tasks.

The Sartorius Group is a leading international partner of life sciences research and the bio-pharmaceutical industry. With innovative laboratory instruments and consumables, the group’s Lab Products & Services division concentrates on serving the needs of laboratories performing research and quality control at pharmaceutical and biopharmaceutical companies and those of academic research institutes. The Bioprocess Solutions division, with its broad product portfolio focusing on single-use solutions, helps customers manufacture biotech medications and vaccines safely and efficiently. The company is growing at double-digit rates on average per year and regularly expands its portfolio through the acquisition of complementary technologies. In fiscal 2021, the company generated sales revenues of around 3.45 billion euros.

www.sartorius.com

Featured Manufacturer