By Michelle Cheever and Alyssa Master, PhD
We recently encountered a customer who had purchased their fetal bovine serum (FBS) at a very low price. When we find FBS selling at or below the cost of the raw source material, we get a bit curious. We inquired about the IgG level listed on the product certificate of analysis; the customer asked “why?” We told them that it was quite possible that they had not received FBS but a product that included Newborn Calf Serum (NBCS). We explained that NBCS is significantly cheaper than FBS and contains different protein concentrations which can alter certain cell culture systems. If this was the case, not only did this customer overpay for their FBS by many thousands of dollars, but they also potentially affect their research.
We believe it is time to educate FBS customers. We want to explain the differences between FBS and NBCS and provide two simple tests that can be used to distinguish the two products.
All animal serum is complex and is comprised of water, protein, salts, sugar, amino acids, vitamins, enzymes, growth factors, and other components. All components will vary based on the age of the animal at collection, breed, sex, different geographic origin, feed, water, seasonal climatic variation, and many other factors that are yet to be determined. This is the reason every lot is tested for each customer’s suitability.
FBS is defined as the fluid fraction of naturally clotted blood (depleted of cell, fibrin, and clotting factors) derived from normal fetuses (which were not delivered by the birth process) from healthy dams deemed fit for human consumption. It is collected in government inspected, registered slaughter houses. FBS is the most commonly used serum type and is used as a supplement to provide a nutrient-rich environment for in-vitro cell growth and maintenance.
FBS, sometimes known as fetal calf serum (FCS), should never be mistaken for calf serum. NBCS is defined as naturally clotted blood from live animals collected from birth to 20 days of age who have been deemed fit for human consumption. Bovine Calf Serum (BCS) is derived from animals collected from 20 days to 12 months of age who have been deemed fit for human consumption. All these types of serums are commercially available and can serve a specific purpose.
The use of serum has grown considerably during the past decade. Because of high demand, a strong market, and other forces limiting availability, the price of FBS will continue to rise dramatically. FBS is a multimillion dollar global industry which has been historically under regulated. This has provided conditions that allow for intentional deception and mischaracterization. When a customer is evaluating FBS for cell line use, one must consider many factors including geographical origin of material, the product specifications, and the potential for adulteration.
The processes for manufacture, documentation, and traceability have improved over time, leading to greater control of FBS. Manufacturers of FBS should be willing to participate in audits and follow industry guidelines. Supplier transparency should include active participation in quality systems and traceability programs. International Serum Industry Association (ISIA) was formed to promote and provide guidelines on compliance for standards and ethics in the business practices of global animal-derived products including bovine serum. “ISIA traceability certified” facilities have been audited for and comply with traceability requirements.
Collection and source variations result in NBCS being approximately 90% less than the cost of FBS. This wide cost differential has sometimes led to products being mislabeled and, in some instances, bargain pricing of products labelled “FBS” which, in fact, are 100% NBCS or a mixture of both products. In other words, if you got an amazing price on your FBS, it may be because it wasn’t 100% FBS. Because of the biochemical differences between NBCS and FBS, this could lead to inconsistent reproducibility or worse, incorrect results.
Some scientists might say: “Hey, I don’t really care what it is. It’s cheap serum. I tested it and it worked for me. What’s the big deal?” We understand that budgets can be restrained, which puts pressure on organizations to find ways to be additionally cost-efficient. However, is buying cheap serum really saving you money? Is the cost of replicating an experiment because of lack of consistent data worth the savings? Is the potential reputational impact of having missed deadlines and/or failed experiments worth the savings? Some companies may say yes, but we suspect many will say no. In an effort to inform and educate end users, the serum industry has identified two biochemical markers that will distinguish FBS from NBCS. Immunoglobulin G (IgG) and Gamma-Glutamyltransferase (GGT), can be used to determine if a serum product is FBS, NBCS, or something in-between.
Serum samples were obtained from ISIA traceability certified suppliers, in association with ISIA, and given to a third party to be blinded and aliquoted or directly submitted for analysis. Biochemical properties were obtained from pooled lots of filtered and unfiltered serum, and individual fetuses.
The Biochemical Fingerprint of Fetal Bovine Serum (FBS)
Immunoglobulin G (IgG)
IgG is the most common antibody found in the blood. It is present in FBS and NBCS, though significantly higher levels are seen in NBCS. Anyone working in antibody production and purification can find varying levels of IgG in media incredibly problematic.
All healthy calves are agammaglobulinemic at birth because, in cows, the placental barrier prevents the transmission of IgG from the dam (mother) to the fetus. This antibody is largely acquired via colostrum from the maternal mammary gland causing the IgG levels of the calf to increase dramatically in the first 24-36 hours postpartum. The levels of IgG in the blood can determine if the serum is of fetal origin or calf origin. Although an individual fetus will have IgG levels that are not higher than 300 μg/mL, because of batch processing methods, the final expected levels of IgG in FBS have an overall value of less than 300 μg/mL. This difference in IgG presents a clear marker to demarcate between fetal serum and calf serum.
It is important to note that the end user should be cautious when comparing unit values of IgG in Certificate of Analyses. It can often appear that the values are within normal range but the units are expressed differently. For example, 1000 μg/mL can be also expressed as 100 mg/dL.
Gamma-Glutamyltransferase (GGT)
GGT, sometimes called gamma-glutamyltranspeptidase, is a transferase enzyme found in the cell membranes of tissues throughout the body. This enzyme is important to the transmembrane movement of amino acids as well as the metabolism of molecules such as glutathione. Inconsistent levels of GGT can have a profound effect on the reproducibility of cell-based assays, particularly in in-vitro cancer models due its key role in redox regulation. Since GGT is found in colostrum, the serum levels of this enzyme are vastly different prepartum and postpartum, like IgG. In mammals, the GGT levels in fetal origin serum change as gestation progresses but remain low as compared to calf origin blood. This enzyme serves as a distinct biomarker for determining if serum is fetal bovine or calf, based on the disparate differences in the levels seen within a fetus (~0-5 IU/L) and those seen in calves (~160-1000 IU/L). Variations are expected to be seen based on country of origin and type of analyzer. However, typical GGT levels for FBS in US origin product usually fall below 10 IU/L, while levels for Australia/NZ usually fall below 12 IU/L. 2
#FakeFBS
Experimental reproducibility continues to be an issue in the cell culture space; it is important to know the variables being introduced to in-vitro systems. By testing for GGT (gamma-glutamyl transferase) and IgG (Immunoglobulin G), scientists can know what they are really feeding their cells. This information will help select for product that will lead to better reproducible results. We are also closer to being able to determine the age of the animal, which is crucial to identifying NBCS or older material from FBS. The ISIA standardization team will be working to set expectations for levels of IgG and Gamma Glutamyl Transferase (GGT) to provide detailed differentiation of FBS.
We know what you’re thinking. Why should you believe us? We don’t want you to take our word for it. Recommendations for testing can be found throughout the industry, including the ISIA (www.serumindustry.org), United States Pharmacopeia (www.usp.org), EDQM (www.edqm.eu), and others. FBS suppliers should be publishing robust certificates of analyses that include, as a standard, the IgG and GGT values of their products.
We believe in asking basic questions about FBS before you purchase:
- Do the results match with other serum suppliers?
- Are IgG and GGT values within values that I have seen with other FBS?
- Are the values within industry standard guidelines?
- Does independently performed serum analysis match with the manufacturer’s certificate of analysis?
- Is your price quote for FBS significantly lower than what you see on the market?
- Is that price “too good to be true”?
If you have any doubt, test it. A simple, inexpensive test can give you peace of mind about the products you’re using. For example, GGT results can be produced in approximately a day and for less than 30.00 USD.
In the end you have a lot more to lose by using #FakeFBS than a supplier has to lose selling you overpriced NBCS.
Footnotes:
1 Standardization of Fetal Bovine Serum quality assessment and reporting definitions and sample certificates included retrieved from www.serumindustry.org/definitions.htm
2 Based on analysis of historic data
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