What are your methods?
Our standard media for growing established hybridoma cells is DMEM containing 8-10% fetal bovine serum supplemented with glutamine (2 mM final concentration) and 50 U/ml penicillin/streptomycin. Cell densities are between 2 x 105 to 1 x 106 cells/ml in 8% CO2 at 37⁰. Doubling times are approximately 20-22 hrs.
Will this serum work for my project?
Please contact us if you need help growing your cells. Not all fetal bovine serums are identical, and we can send you small amounts of our media to help you get your cultures going. We can also share with you our approach to growing cells under serum-free conditions.
Cell Culture Services
How will you ship my cells?
Frozen and shipped on dry ice. We HIGHLY RECOMMEND thawing and culturing a vial immediately upon receipt.
How should I produce the antibody?
There are a variety of different flasks, vessels, and bioreactors for growing hybridoma cells as well as different media and additives. We recommend gradually weaning the cells from the media we grew them in before freezing. We are happy to send you small amounts of our media to help with this.
Two cautions:
- All hybridoma cells lines have an inherent instability that leads to cells that grow but do not express antibody or express antibody at low levels. These so-called “non-producers” will tend to overpopulate the culture as time goes on. The solution is to routinely subclone the line and to freeze cells at different stages of the process; for example, before the line is subcloned, after subclones have been selected and expanded, before seeding a bioreactor, after culture in a bioreactor, etc. Having these frozen stocks make it easier to go back and determine where in the process the problem began if you see loss of antibody production.
- Always archive the line by freezing multiple vials, at different times, and checking to ensure that the cells will thaw successfully and produce the expected amount of antibody. It is also critically important, along with having multiple batches of frozen cells, to have them stored in multiple liquid nitrogen freezers. We can’t stress this enough: store cells in multiple freezers!
And of course, we are here to help produce the antibody so that you don’t have to do this.
What kind of antibody yields can I expect?
Production levels typically run from 5-200 μg/ml in spent culture media.
Can we produce the monoclonal antibody in ascites?
You can, but our recommendation is to grow it in culture. We believe it is more humane and there are no longer overriding technical reasons to make ascites. Also, antibodies purified from ascites typically contain varying amounts of normal mouse immunoglobulin.
Production & Purification
How is the antibody produced?
By in vitro production: cells are cultured in roller bottles or flasks in serum free media or the media of your choice. Antibody is purified on protein G with low pH elution into Tris neutralizing buffer. We analyze the purified antibody using SDS-PAGE and dynamic light scattering.
How will I receive the antibody?
Purified antibody will be provided to you in a buffer of your choice. If you do not specify a preferred storage buffer, we recommend storing antibody in 50% glycerol/water at -20°C or in phosphate-buffered saline containing 0.02% sodium azide.
What information will I receive with the purified antibody?
A product data sheet is supplied outlining the process used for purification, estimated percent homogeneity, concentration, buffer, recommended storage conditions, and isotype.
Who owns the cell lines at the end of the project?
Once payment is received, you do.
How long does it take to produce and purify the antibody?
Starting with frozen cells, this process takes approximately 4-6 weeks, depending on the number of cell lines selected for production and the growth characteristics of the cell lines.
How is pricing determined for this stage?
Pricing for this stage is set in our hybridoma development package. Price may vary if an extra number of hybridomas are selected for antibody production or if larger amounts of antibody are required.
Fusions & Subclonings
What cells are used in the fusion?
Splenocytes, and if requested, lymphocytes from mice with acceptable sera titers are fused with a murine myeloma cell line.
How are positive hybridomas selected?
Hybridoma supernatants are screened using a solid-phase ELISA; generally, with the same assay used to determine serum titers. Our criterion for a positive clone is signal twice the negative control.
Can I also screen at my lab?
Yes! We will send you the culture supernatants. The more data we have on antibody binding in different assays, the better the decision we can make selecting positive hybridomas.
Are positive hybridoma parent wells preserved, even if not selected for subcloning and production?
Cryopreservation of positive hybridomas is possible and should be discussed with your Project Manager. There is additional cost for freezing additional hybridomas.
Why do the positive hybridomas have to be subcloned?
Parent wells are mixtures of hybridoma clones. To isolate stable monoclonal lines, we clone the parent well by limiting dilution. A second round of screening identifies positive, stable, cloned hybridomas. Two rounds of subcloning are the minimum to ensure the cells are monoclonal.
What happens to positive subclones?
You and your Project Manager will decide which clones to expand, freeze, and put into the production phase. The selection is made on the basis of screening assay data. The more data from different assays that is available, the more ideal the selection of clones for expansion and/or freezing.
How long does it take to prepare clonal hybridoma cell lines?
Typically, 4-6 weeks on cells from a single fusion. Some hybridomas are unstable and require more time.
How is pricing determined for these phases?
Pricing is set in our hybridoma development package but may vary if more clones are selected for cryopreservation or cloning, or if different assays are required. You receive a custom quote at the beginning of a project so your special needs can be addressed from the beginning, at a price you can afford.
Which Antigen Works Best?
The ideal antigen: large, pure, and removed in evolution.
Although a wide range of substances are antigenic (cells, proteins, organelles, small molecules, conjugated peptides, carbohydrates, lipids), not all antigens elicit an immune response. In addition, individual animals in a given species have different humoral immune responses. We find the best approach is to use large (greater than 20kD) proteins or peptides conjugated to carrier proteins. For proteins with high homology to rat or mouse, we suggest varying the immunization strategy, typically by varying the adjuvant, route of administration, or length of the protocol. It may also be necessary to increase the number of animals to increase the likelihood of getting a rare unique idiotype.
With the recent explosion in gene sequences, many scientists and researchers are requesting antibodies to protein antigens that have never been isolated. Two methods are useful in preparing antigens from gene sequence data: 1) chemical peptide synthesis of the expressed protein, and 2) expression of recombinant proteins. Because there are pros and cons to both approaches, please contact us to discuss your options in the early stages of your project.
Chemical peptide synthesis
Making monoclonal antibodies to synthetic peptides that also bind the protein can be challenging. If this is your chosen method, call us before you synthesize a peptide so we can recommend ways to increase our chances of success. For example, one approach is to immunize with a peptide conjugate and screen hybridoma supernatants using native protein. Since some anti-peptide antibodies fail to bind native protein, this approach can be used to obtain antibodies that blot or bind antigen using immunofluorescence following methanol or glutaraldehyde fixation.
Peptides and other small molecules are weakly immunogenic and must be coupled to a carrier, such as keyhole limpet hemocyanin or ovalbumin. We recommend that you quantify the amount of peptide conjugated to your carrier (not all conjugation reactions occur with high efficiency) and consider preparing a second conjugate using a different carrier for use in screening.
Expression of recombinant proteins
Recombinant proteins should be 20,000 Daltons or larger and may contain their expression tags (GST, FLAG, 6 x His, etc.) during the immunization stage. Ideally, purified antigen with the tag removed should be available for screening. A screening strategy using recombinant protein with and without the tag is one reasonable approach since the tag itself is often immunogenic.
Highly conserved antigens
Highly conserved antigens are a challenge to prepare antibodies against since most species are tolerant to self. The greater the degree of sequence differences between your antigen and the host sequence, the greater the likelihood of producing antibodies. In general, the more homologous the antigen, the greater the need to try alternative immunization strategies including using higher numbers of animals of different strains.
Other antigen properties
The more you know about your antigen the more straightforward it is to make antibodies. Important properties include purity, biological activity, solubility, and sequence homology to the host animal. All of these factors come into play when there is a low or marginal immune response. One common suggestion is to prepare a rabbit antiserum before attempting the production of a monoclonal antibody. This oftentimes identifies difficulties in making antibodies to a particular antigen. The serum is useful as a positive control in other immunoassays.
Immunoassay Selection
Which Assay Should I Use?
Immunoassay Selection
We are committed to getting antibodies fit for your intended purpose, so please share with us your end use for the antibodies we will be producing. There are many different immunoassays and not all antibodies bind their antigens in all assays. For example, if your goal is to routinely assay vast numbers of clinical blood or plasma samples, you might require antibodies that bind native antigen in the presence of blood components, some of which could interfere with antigen-antibody binding. Likewise, a monoclonal antibody useful in Western blots will need to bind antigen that has been denatured with sodium dodecyl sulfate and heat. By knowing your end use for the antibody, you need made, we can consider alternative sample and assay conditions during the hybridoma selection phase of antibody production.
Below are several routine assays we use to select hybridoma antibodies:
Solid-phase ELISA (with antigen coated plates)
Our standard solid-phase ELISA uses antigen-coated microtiter plates, a secondary antibody (for example, goat anti-mouse IgG conjugated with horseradish peroxidase), and a chromogenic substrate. The output signal (absorbance of the chromogenic substrate following oxidation) depends on the formation of primary antibody-antigen complexes. Under certain conditions, the assay can be linear with respect to antibody concentration and, therefore, useful for comparing the relative concentrations of antibodies in different samples. One factor to consider is that some epitopes are highly dependent on the solution versus the surface properties of the antigen. This is an important consideration for small linear peptides which often require conjugation to a larger carrier molecule.
Antigen Capture ELISA
This solid-phase assay uses antibody-coated microtiter plates (with antibody either directly bound or bound via another protein, such as protein A). One format uses wells coated with rabbit or goat anti-murine immunoglobulin to capture mouse mAbs. Addition of biotin-labeled antigen followed by avidin peroxidase identifies murine antibodies that bind to the antigen. As with any solid-phase assay, critical parameters include the type of plate, the method of blocking nonspecific protein binding sites on the plate, incubation times and temperatures, the relative concentration of assay components, and the number of washes and composition of the wash buffer. This assay is often used for projects having less than 1 mg total antigen.
Competitive ELISA for antigen in solution
This assay measures the amount of free antibody remaining in an equilibrium mixture of antibody and antigen in solution. One use for this assay is the identification of anti-idiotypic mAbs. Binding of the mouse mAb in solution to the target antibody prevents binding of the target to its ligand on the surface of a well.
Immunoblotting (Western Blot)
Antibodies recognize both linear epitopes and nonlinear epitopes determined by the conformation of the antigen. Immunoblotting assays often detect a different set of antigenic determinants than other immunoassays because the antigen is denatured before electrophoresis. This assay is useful for detecting antigens in complex mixtures and for estimating antigen molecular weight.
Blocking Options
Most immunoassays require the addition of protein, detergent, or polymer to decrease non-specific binding. Although not common, it is possible that these substances can influence antigen-antibody binding. Another consideration is the requirement for some level of fetal bovine serum in the hybridoma screening assay. If your antigen is found in serum, or affected by serum components, it can be more difficult to obtain useful antibodies.
If you know or suspect that serum components bind or modify your antigen, or if serum contains homologous antigen, we will need to discuss alternative approaches before starting your project. Please contact us to discuss your project.
Octet / Gator Operations
The Octet and Gator systems uses Bio-Layer Interferometry (BLI), an optical analytical technique that allows for label-free, real-time monitoring of bimolecular interactions. As the biosensor tip is exposed to different analytes the number of molecules bound to the biosensor tip will change. This causes a change in the optical layer thickness and results in a shift in the interference pattern that can be measured in real time.
EXAMPLE:
Our murine antibodies (GMA-092 and GMA-095) show specific binding to human prothrombin on ELISA. We used the Octet to determine if they bind the same epitope on prothrombin. The results below are with sensors loaded with GMA-095 and show that GMA-092 binds a distinct epitope from GMA-095:
Each new step in the method is denoted by a vertical dashed red line (9 steps total):
Step 1: Baseline – kinetics buffer to establish a baseline for the next step.
Step 2: Capture antibody (GMA-095) binds to the sensor.
Step 3: Baseline.
Step 4: Analyte of interest (prothrombin) binds to the capture antibody.
Step 5: Baseline. This step can also be used to access the dissociation curve of the analyte from the capture antibody.
Step 6: Quench. This step is used to fill any empty antibody binding sites on the sensor tip so that all subsequent binding should be only to the analyte of interest. In this example Ms IgG is used.
Step 7: Baseline.
Step 8: Binding of second antibody to the analyte of interest (prothrombin). The blue line represents GMA-092 and shows binding to prothrombin. The red line represents additional GMA-095 and shows the expected lack of additional binding, consistent with its prothrombin binding site already being occupied.
Step 9: Dissociation. The stability of the sandwich protein complex is monitored in kinetics buffer. Minimal dissociation is seen here.
Antigens & Immunization
How much antigen is required for immunizations and screening?
In general, 2-5 mg of antigen for immunizations and 1-3 mg for screening. We can adjust project strategies if the antigen is difficult to isolate or not readily available.
Why is adjuvant used?
Adjuvants enhance the cellular immune response against the injected antigen. When mixed with antigen, they help deposit or sequester the injected material. The effect of adjuvants on antigen immunogenicity is uncertain.
How long does it take to produce an adequate immune response?
Mice typically receive 3-6 injections over 2 to 3 months. The first injection exposes the cells of the immune system to the antigen. Subsequent booster injections are needed to increase the number of Ig-secreting plasma cells.
What happens if no immune response is produced?
The immune response of any given animal is dependent on several variables in addition to the immunization time frame. Important variables include: the size of the antigen; the genetics of the mouse; the evolutionary distance between the species of the antigen and the immunized animal; how the antigen is processed by the immune system; the amount of antigen given in each immunization. Because of these variables, there may be little or no response to a given antigen. If this occurs, we will work with you to outline more aggressive immunization protocols or suggest a change in your antigen. Alternative immunization protocols will be recommended if serum titers are unacceptable for fusion.
How do you determine immune response?
We define titer as the dilution of serum at 50% maximum signal using a solid-phase ELISA. If you require antibodies that function in other immunoassays, we will send you the sample sera for testing.
Should my antigen be coupled to a carrier?
Haptens, peptides, and weak or nonimmunogenic antigens may require conjugation to carrier proteins, such as keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin.
What protein sequence should I select if I want to make an antibody to a peptide?
Longer is better (greater than 12 residues) and lots of hydrophilic residues (Glu, Asp, Arg, Lys). Consider putting a Cys at one of the ends for coupling to a carrier. Your peptide should be soluble in aqueous solvents. Although there are a number of predictive algorithms for picking sequences, we know of no data saying that one is better than any other for all proteins. Our history of making antibodies to synthetic peptides goes back to 1983 (Church, et al. (1983) PNAS, 80, 255.
Do you make peptides?
We don’t. But we can suggest some excellent companies who do. If you have a favorite synthesis company, ask if they will determine coupling efficiency. Not all peptides couple to carrier with high efficiency and this is a project stopper if peptide is not conjugated. Our recommendation is to use amino acid composition to determine coupling efficiency.
How pure does my antigen need to be?
As pure as you can make it. Remember, contaminants are usually as good at producing antibodies as your antigen of interest. There are strategies for using non-homogenous preparations of antigen but please talk to us first.
My expressed protein has GST (or His, or Flag). Will it matter if I don’t remove it?
Yes, it will make a difference. We’ve made lots of antibodies through the years to GST. Some even have specificity for the non-GST part but require GST for binding. Our recommendation is to remove the expression tags for screening. If this is a problem, call and we can discuss alternative strategies.
My antigen is hard to make (or purify). What if I only have 0.5 mg?
At the present time, our personal best is generating two specific monoclonal antibodies using 120 ug total protein. Talk to us but remember: if it doesn’t look like we can do it or that you won’t be happy with the length of time it will take us, it’s probably best to go back to the drawing board and redesign how and where the antigen will come from. Our procedures, processes, and technical staff are only as good as your antigen.
The Antibody Development Process
STEP 1: ANTIGEN DESIGN AND HYBRIDOMA DEVELOPMENT
Once we have agreed on an approach, mice are injected with antigen to produce an immune response. The specific strain of mice and the design of the antigen are chosen based on our many years of experience and your end-use for the antibody. Once the mice have an acceptable serum titer of specific antibodies, antibody-producing cells are fused to myeloma cells to produce “hybridoma” cells.
STEP 2: HYBRIDOMA SELECTION, VARIABLE REGION SEQUENCING, AND CRYOSTORAGE
Our standard hybridoma selection assay is a high throughput assay using hybridoma cell supernatants to select hybridoma cells producing antigen-specific antibody. Additional alternative assay methods narrow the selection of antibody-producing clones to a manageable number. After several rounds of subcloning and assaying, the light and heavy chain variable regions can be sequenced to ensure antibodies from different clones have unique variable regions. Cell archival and storage under liquid nitrogen conditions assures the integrity and availability of selected clones for future use.
STEP 3: ANTIBODY PRODUCTION
Our antibody production services can prepare purified antibody in amounts ranging from milligrams to grams. If necessary, cells are adapted to serum-free conditions to eliminate contamination of bovine immunoglobulin from fetal calf serum. We believe this method of production is superior to antibody produced from ascites. Purified antibody can be prepared to customer specifications, including purification under low-endotoxin conditions, labeling of the antibody with biotin or fluorescent dyes, construction and expression of recombinant antibody in HEK cells, fragmentation of antibody to produce Fab or F(ab)’2 or attachment of purified antibody to Sepharose for antigen purification.
STEP 4: ANTIBODY CHARACTERIZATION AND ASSAY DEVELOPMENT
Once the antibodies are purified, they can be further characterized and modified to assure a proper match to your specific diagnostic or therapeutic end-use. We offer a number of services to meet customer needs including development of sandwich ELISA, determination of antibody-antigen association rate, effect of solution conditions (pH, ionic strength, divalent metal ions, etc.) on antigen binding, competitive inhibition by similar analytes for antibody binding, and pharmacokinetic experiments in mice.
Blocking Options
Most immunoassays require the addition of protein, detergent, or polymer to decrease non-specific binding. Although not common, it is possible that these substances can influence antigen-antibody binding. Another consideration is the requirement for some level of fetal bovine serum in the hybridoma screening assay. If your antigen is found in serum, or affected by serum components, it can be more difficult to obtain useful antibodies.
If you know or suspect that serum components bind or modify your antigen, or if serum contains homologous antigen, we will need to discuss alternative approaches before starting your project. Please contact us to discuss your project.
Octet / Gator Operations
Both the Octet and Gator system uses Bio-Layer Interferometry (BLI), an optical analytical technique that allows for label-free, real-time monitoring of bimolecular interactions. As the biosensor tip is exposed to different analytes the number of molecules bound to the biosensor tip will change. This causes a change in the optical layer thickness and results in a shift in the interference pattern that can be measured in real time.
Example:
Our murine antibodies (GMA-092 and GMA-095) show specific binding to human prothrombin on ELISA. We used the Octet to determine if they bind the same epitope on prothrombin. The results below are with sensors loaded with GMA-095 and show that GMA-092 binds a distinct epitope from GMA-095:
Each new step in the method is denoted by a vertical dashed red line (9 steps total):
Step 1: Baseline – kinetics buffer to establish a baseline for the next step.
Step 2: Capture antibody (GMA-095) binds to the sensor.
Step 3: Baseline.
Step 4: Analyte of interest (prothrombin) binds to the capture antibody.
Step 5: Baseline. This step can also be used to access the dissociation curve of the analyte from the capture antibody.
Step 6: Quench. This step is used to fill any empty antibody binding sites on the sensor tip so that all subsequent binding should be only to the analyte of interest. In this example Ms IgG is used.
Step 7: Baseline.
Step 8: Binding of second antibody to the analyte of interest (prothrombin). The blue line represents GMA-092 and shows binding to prothrombin. The red line represents additional GMA-095 and shows the expected lack of additional binding, consistent with its prothrombin binding site already being occupied.
Step 9: Dissociation. The stability of the sandwich protein complex is monitored in kinetics buffer. Minimal dissociation is seen here.
Protein Concentration Correction
What is your method for determining protein concentration?
We use a corrected Absorbance at 280 nm, and an extinction coefficient (0.1% w/v) of 1.4 to determine antibody concentration.
The scatter correction derivation is shown below:
A280 = A°280 + Isc,280
Where:
A280 = Observed absorbance.
A°280 = Actual intrinsic absorbance of the protein.
Isc, 280 = Absorbance due to scatter or dispersion.
A280 = A°280 + Isc,280
A°280 = A280 – Isc,280
Isc,280 is proportional to 1/λ4 and equal to K (1/λ4) = K (1/ (280)4
Where K = proportionality constant.
Likewise:
A320 = A°320 + Isc,320
For proteins, A°320 = 0. (Not valid for proteins absorbing at 320 nm or for proteins larger than λ (e.g., von Willebrand factor).
Therefore:
A320 = Isc,320 = K (1/ (320)4)
K = A320 x (320)4 = Isc,280 x (280)4
Isc,280 = A320 ((320)4/ (2804) = 1.7 x A320
And:
A°280 = A280 – 1.7(A320)