The Developmental Studies Hybridoma Bank (DSHB) was created as a National Resource by the National Institute for Child Health and Human Development (NICHD) in 1986. It was originally housed at Johns Hopkins University and directed by Dr. Thomas August. It was funded through a contract to Dr. August, with a subcontract to Dr. Michael Solursh at the University of Iowa. As is the case for most NIH-contracted National Resources, there was the expectation by NIH that the resource would become self-sufficient (i.e., self-funded) in a reasonable amount of time, a rare outcome. The resource was established to bank hybridomas developed primarily with NIH funds, and to distribute them and the monoclonal antibodies (mAbs) they produced, at a price slightly above cost, leaving enough room for growth. It was conceived as a mechanism to relieve scientists who developed mAbs, from the responsibility of distributing them, to provide the reagents at a reasonable price to basic researchers and to make available mAbs with low demand. The original target group consisted of cell and developmental biologists. Its basic mission was, and still is, to facilitate basic research.

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Figure 1. Developmental Studies Hybridoma Bank (DSHB).

I entered this story in 1996, after Dr. Solursh passed away. I was at the time the director of a program project grant, “The Developmental Biology of Cell Motility”, funded for 23 years by the same institute that funded the DSHB. Probably for that reason more than any other, the Institute personnel in charge of the extramural DSHB contract selected me to direct the subcontract. The DSHB in 1996, 10 years after its inception, was far from fulfilling the expectation of achieving self-funding. Within a year after Dr. Solursh’s death, the NICHD personnel selected me to direct the entire DSHB, and it was moved to Iowa, with a new contract containing a four year exit strategy. Realizing the restrictions inherent in a government contract and the route that had to be taken to achieve self-funding, the DSHB terminated NIH funding three years ahead of contract and went solo. It was revamped as a nonprofit resource with a company-like structure and an aggressive plan for growth and self-sufficiency. The latter was achieved within a year. In the past 18 years, the DSHB collection has grown from approximately 280 to more than 2,000 different reagents, and distribution has increased from 4000 samples per year to over 65,000 samples per year. And because of two recent NIH initiatives and an in house initiative, we expect the DSHB reagent collection to grow in future years at an even faster pace, keeping quality and service high, and client costs at a fraction of commercial prices. In the following sections, I will attempt to describe the model we have developed to achieve our goals, new initiatives, what the DSHB can and can not do, the future of the DSHB and the problems we will have to overcome to remain successful.

The model originally developed by NIH and codified in the first contract, 28 years ago, for the DSHB was simple and unique, but did not include a rigorous-enough approach for growth and independence. Its key point, and one that remains a major by-law, was that the DSHB would not own donated hybridomas (i.e., it would not own intellectual property-IP). It simply would bank the hybridomas created and characterized by other scientists and institutions, mainly through grants from public sources (e.g., NIH, NSF, European public funding sources, of other countries). The fact that the DSHB was a nonexclusive distributor of IP owned by others, and that distribution was at cost, was spelled out in the depositor’s agreement. But this agreement or contract went further. The depositor’s contract contained, and still does, a number of key elements, both explicit and implicit.

• 1. The depositor, not the DSHB owns the IP of the deposited reagent and maintains the sole right to commercialize that intellectual property (IP). The DSHB is simply a bank and distributor.
• 2. Once a reagent is deposited, it can not be recalled and the distribution contract could not be terminated by the depositor, because the latter wishes to sell it to a company for commercialization or because of any other reason or change of mind. The reason for this is the expense shouldered by the DSHB to bank and distribute the reagent. The DSHB expands the deposited sample, retests the mAb by ELISA, produces supernatant to store for distribution, many times tests the reagent, and banks the deposited and expanded hybridoma. When a preparation stops producing high-enough titers, or becomes ineffective for other reasons, the DSHB reclones original substocks or recombinant constructs. The DSHB also builds, through costly publicity, reputation and quality control, a market for each reagent. The value added by the DSHB to each banked reagent is therefore substantial. The possibility that the DSHB would be used by depositors as a free storage facility, therefore, is excluded by DSHB bylaws.
• 3. The DSHB commits to distribute the IP to a second party, with the stipulations that that party can not commercialize the reagent or provide it to a third party. This protects the IP of the depositor. This is accomplished through a purchaser’s agreement signed by the client prior to distribution.
• 4. The DSHB will not return IP to a depositor for commercialization, since one of the components of the depositor’s agreement is that the DSHB will not provide IP to anyone or any entity (including the depositor) for commercialization. Therefore, depositors had to maintain their own cell lines, constructs or other basic reagents, for future commercialization.
• 5. The DSHB distributed to nonprofit as well as for-profit entities, but the latter were scrutinized more intensely to be sure they understood the non-commercialization clause. This many times entailed letters of intent by a commercial entity to the Director prior to purchase. The depositor can not exclude for-profits as clients, but is made aware of IP protection by DSHB licenses signed by every client receiving their IP.
• 6. The depositor does have the right to limit distribution to supernatants (i.e., solutions or protein capture reagents), thus excluding distribution of hybridomas and recombinant constructs.
• 7. Neither the depositor nor anyone else could obtain the names of DSHB customers and the content of their purchases. The depositor could only obtain the volume of distribution of each deposited reagent.

These by-laws have been rigorously maintained for the past 18 years, and expanded to protect protein capture reagent other than mAbs.

The DSHB provides to basic researchers at cost mAb supernatant ($35 per ml), hybridoma lines ($210 per vial of frozen cells) for most hybridoma reagents, the highest quality production controls, experts in hybridoma technology with Ph.D.s that will answer client questions within 24 hours by phone or email, and warranties on reagents. If a reagent is petering-out and the DSHB is notified of this by a client, it will reclone and have replacements within three weeks, or the return of the cost of the purchase. If a client has trouble getting a reagent to work, the DSHB will work with that client diligently on the technology. Because the DSHB was created to serve basic scientists, not to expand profit, it places on the shelf mAbs and other protein capture reagents with very low demand.

But what can’t we do? We can’t afford to provide particular services, given the near-zero profit margin on sales. We can not afford to characterize a reagent unless we believe a customer’s complaint is valid. Usually, customer queries and complaints revolve around application, and we readily work with the customer to solve their problem. Several recent articles demand greater quality control given the number of mAbs commercially sold that don’t perform [55-59]. We vehemently agree with the criticisms and solutions, but do not have the resources to comply with some of the rigid standards and characterizations proposed. For most of our reagents, there are literature histories (publications), which help in validation but, unfortunately, for many of the reagents we are distributing through new initiatives, validation and characterization is initially thin. However, we carefully monitor usage, and expand our data sheets as new characterization data appear in the literature or are communicated to us. The second thing we cannot afford to do is interact with companies that are producing capture reagents for a fee for prospective depositors. And finally, we can not afford to generate hybridomas in response to most requests by our clients. The generation of hybridomas targeting the green fluorescent protein was an exception. Given our price structure, however, these limitations appear to be a reasonable trade-off.

First, the depositor profits because the DSHB banks, maintains, publicizes and distributes the depositor’s reagent, with no cost to the depositor. The DSHB relieves the depositor financially and time wise of that responsibility. Because of our large customer base and our continual advertising efforts, a reagent may become popular and the depositor may be widely referenced in the literature for creating the reagent. Referencing is a requirement in the purchase agreement signed by the client. This strengthens the depositor’s reputation, and impacts funding. Second, our clients profit by buying at cost reagents created and characterized by others, for the most part through public sector funding. When a client buys and tests 10 different mAbs from the DSHB, it costs $350, compared to approximately $2000 to $3000 from for-profit companies. This represents a large stimulus given, first, that most purchased mAbs usually languish in the refrigerator, and, second, that it allows scientists to test, with greatly reduced cost, multiple mAbs.

In the 18 years since the entire DSHB moved to Iowa, proprietary methods have been developed in the production labs and the business office to keep costs low. As the DSHB grew, we worked with three initial software companies and spent a small fortune searching for customized software to handle the ever increasing paper work for domestic and foreign clients, with no success. Then we succeeded in finding a company that had developed software for several companies, including two large department store chains in the United States. They worked with us to customize software for our needs and graciously discounted their services because we were nonprofit. And the DSHB grew because it was unencumbered by a dependency on outside funding, since that source is unreliable and can disappear at the whim of a funding agency or a decrease in funding legislated by the U.S. Congress. One indeed gets used to outside funding, which can indeed become an impediment against searching for ways to achieve self-sufficiency. But most importantly, the DSHB proceeded because of the continuity of personnel. The DSHB depends on a dedicated staff, some of them with the DSHB since it moved to Iowa. The DSHB has been housed through the generosity provided over the last 26 years by the Deans of the Liberal Arts College, the Chairpersons of the Biology Department, the support of the Presidents and Provosts of the University, and help by the faculty of the biomedical science departments of the University of Iowa, which has been a major resource for characterizing and testing new mAbs. It benefits from the advice of an international board of advisors that meets in Iowa City once a year, and from me being a proactive director. In return, the DSHB provides over 15 full time salaries and sponsors an advanced, full semester course on monoclonal antibody technologies and disease. The DSHB provides directly to the Department two graduate student fellowships each year, contributes the interest of an interest-bearing account to the Biology Department graduate endowment, pays to the University fees for facility administration, pays for the graduate student retreats each year, funds meetings at Iowa and all over the world, and provides mAbs to University of Iowa researchers with no service or shipping fees. The University of Iowa and the Department also benefit from the reputation it has received for housing the DSHB, a reputation noted by major NIH and European initiatives, which have signed contracts with the DSHB to function as their reagent bank and distributor for protein capture reagents. And the reputation of the University of Iowa and the Biology Department have also benefited from the service the DSHB provides to over 100,000 customers worldwide, who use DSHB reagents in their basic research.

Most of our collection has been contributed over the past 28 years from individual scientists and institutions. We also receive full collections as they evolve from various initiatives. The hybridoma collection generated and banking by Dr. Glenn Morris at Keele University, United Kingdom, for the Muscular Dystrophy Association, now includes over 200 hybridomas, and is still growing. A rapidly growing collection of cancer-related hybridomas, now numbering 250, generated by the Clinical Proteomic Technologies for Cancer (CPTC) initiative funded by the National Cancer Institute of NIH, continues to grow. A collection of hybridomas and recombinants targeting primarily human transcription factors continues to be generated by the Protein Capture Reagent program funded by the Common Fund of NIH, and banked in the DSHB. And we are presently in negotiations for a collection of over 500 hybridomas from the European Cell Biology Consortium. Over 1700 mAbs are currently on the shelf for distribution.

Approximately 80% of DSHB distributions represent less than 10% of the DSHB collection. Many of these mAbs are highly characterized and have been validated for specific uses by a number of researchers through publication and, therefore, have provided data for different applications in peer reviewed journals. The distribution of a mAb however, depends on a variety of other factors as well. Two important ones are cost and quality. In response to continued pleas from our clients to generate effective and low cost mAbs against green fluorescent protein (GFP), especially for chromatin immunoprecipitation, we generated a stable of six hybridomas against native GFP. In the past year, two of these mAbs have entered our top 10 products list. Over 1,200 supernatant and frozen cell samples have been distributed to clients during this period. The DSHB characterization of these mAbs was recently published to provide a reference for clients [60]. The mAbs were characterized for interacting with native protein, live cell staining, fixed-embedded staining, immunoprecipitation and chromatin immunoprecipitation. Since they were generated against native protein, they do not work in western blots, which separate denatured protein. Both supernatant and cell lines are available at cost. The mAbs are now being epitope-mapped by a collaborator. And a new stable of mAbs targeting denatured GFP is now under development. The success of these anti-GFP mAbs is based on high demand and very high commercial prices. But not all mAb stories are as rosy. When a new mAb is developed against an antigen, for which mAbs are already commercially available, and when the latter mAbs have a publication history, distribution of the new mAbs is usually low for several years, increasing only when publications appear validating their efficiency. This may take years. Scientists are wary of new mAbs without a literature history no matter how good the characterization appears to be on a product data sheet. Scientists in the trenches really believe in literature-based validation. Perhaps the least expensive aspect of using a mAb for research is the price of the mAb. The cost of staff hours and other reagents far exceed the cost of the mAb.

Even though the DSHB doubled its collection over the past six years, its income remained relatively fixed for the first five years and fell approximately 5% in the last year. Because of decreased biomedical research funding in the US, the DSHB must continually find new customers at an ever increasing rate. And the costs to run the DSHB keep rising, forcing it to downsize staff. The volume of work has increased as the DSHB collection rapidly expands, primarily as a result of the initiatives by the NCI and Common Fund of NIH. The DSHB never has and does not at present receive funds from any agency for banking and distribution of reagents. Unfortunately, the voluminous number of new reagents from these new initiatives lack publication histories. Therefore, demand for them at present is extremely low. They will, therefore, not have an immediate effect on DSHB income to cover the costs of placing them on the shelf for distribution. Because the DSHB is completely self-funded, it functions as a partner rather than grant recipient in the aforementioned initiatives, and must, therefore, subsidize all costs for banking them. But the DSHB is committed to these initiatives and the role it plays. The task of the DSHB, therefore, is to increase distribution by increasing awareness of the reagents through newsletters and publicity. The DSHB is also committed to generate in house, reagents that our customers repeatedly request, such as the mAbs against GFP. We have begun to produce batteries of mAbs against protein tags other than GFP, including mCherry, HA(YPYDBPDYA) and DYKDDDK.

We have also created in the past year the Monoclonal Antibody Research Institute (MARI). Its objectives are the following: 1) to develop new ways of generating antibodies, for example through the use of less toxic adjuvants [60] ; 2) to generate complex antibody chips allow an individual to assess 100 of protein targets in a single sample, such as a Drosophila larva on a cancer cell preparation; 3) to produce recombinant rabbit antibodies that are partially humanized and that exhibit increased avidity to high profile antigens, and 4) to identify and generate mAbs against new, medically important targets on cancer stem cells, metastasis cells and cells in the process of tumorigenesis.

The use of protein capture reagents is not a transient technology, as have been so many other technologies that are extremely popular in a small time window and are then replaced by new technologies. Indeed, in the face of shrinking funds, as has been experienced in the past six years, the DSHB has remained relatively healthy. It is still financially sound, continues to expand its collection of reagents and has funds, albeit minimal, to experiment with emerging protein capture reagent technologies. The DSHB has developed new and unique methods for generating and maintaining conventional mouse hybridomas, and is in the process of developing new protocols for producing recombinant antibodies in CHO cells, bacteria and yeast. And it has remained self-funded for over 18 years, independent and not beholding to any commercial interest. Most importantly, it remains faithful to its original mission, to facilitate basic research. It is, therefore, surprising that so many scientists have trepidations purchasing DSHB mAbs and hybridomas, at prices less than an eighth of commercial price. The attitude may be that if something is too cheap, it can’t be good. In these days of shrinking funds, it seems like it should be the responsibility of researchers to get the “biggest punch for their buck”, and see if the hybridomas, mAbs and recombinants they need can be purchased from the DSHB at great savings. It should also be recognized that if a scientist has created a new protein capture reagent and has previously purchased hybridomas and/or mAbs from the DSHB, and especially if public funds have paid for the production of the new hybridoma, it seems reasonable to expect that the scientist and his or her institution to deposit it in the DSHB. It does not hamper future commercialization by the scientist’s institution and it allows the scientist to share immediately their reagent with other scientists.

While commercial antibody providers tend to focus on antibodies against human or rodent proteins, DSHB provides antibodies to proteins in other species. For example, JH Lee et al stained mouse brain sections with DSHB LAMP2 antibody ABL-93, which serves as a lysosomal marker [61]. AR Palla et al stained mouse myofibers with MHC2a (SC71) and MHC2b (BF-F3) antibodies from DSHB [62]. G Dixon et al labeled pancreatic progenitors with an anti-NKX6.1 antibody from DSHB ( F55A12) [63]. Y Lu et al stained wholemount mouse retinas with mouse anti-AP2 alpha (clone 3B5, 1:100) from Developmental Studies Hybridoma Bank [64]. HY Kim et al labeled frog intermediate filaments with anti-keratin antibody (clone 1h5) from DSHB [65]. H Seok et al labeled cardiomyocyte with the MF20 anti-myosin heavy chain antibody from DSHB [66]. A Marconi et al identified cartilage in paraffin sections from skate, Leucoraja erinacea, with the anti-COL2A1 antibody (clone II.II6B3) from Developmental Studies Hybridoma Bank [67]. Hu CK et al stained killifish embryos with a mouse anti-MYL1 antibody from DSHB ( F310) [68]. Z Wu et al stained mutant huntingtin in mouse brains with clone MW7 from DHSB [69].

The section "Application Examples of DSHB Antibodies" is compiled and updated by Materials and Methods editorial staff.