Antibodies and ELISA Kits

AFSBio has access to different high quality protein manufacturers, each with unique areas of expertise

Along with standard high quality/purity antibodies, we can provide the following:

- For clients that require antibodies from mammalian cell lines, we have access to patented CHO-based QMCF technology

- For clients that need large volume of high quality antibodies with a quick turnaround time, we have access to antibodies from chickens

- Since our product range is quite wide, it is very likely if you require a rare protein we can source it for you

- We have quite a large range of CE-IVD approved ELISAs for therapeutic drug monitoring

- For autoimmune researchers, we have highly pure native antigens that outperform their recombinant counterparts both within the assay and in terms of LOT to LOT consistency and reproducibility


CHO-Based QMCF Technology

Chicken Polyclonal Antibodies

Proteins and Peptides

ELISAs

Associated Reagents and Tools

Isotype Controls

 

We have access to over 3 million products in this field! Please contact us with what you need and we will be happy to support you with product info and a quote.

We welcome custom projects

AFSBio is proud to partner with :

 

CHO-Based QMCF Technology

Icosagen provides recombinant protein production services using proprietary CHO-based QMCF Technology in serum-free conditions. Customer benefits are a short production time and cost-efficiency.

QMCF Technology comprises of genetically modified eukaryotic cell line and expression plasmids that stably replicate and maintain in these cells.

QMCF cell lines are stable suspension or adherent cells (for instance CHOEBNALT85) into which two genes have been introduced in order to provide replication initiation and effective maintenance functions to appropriate QMCF expression vectors. The replication initiation function is provided by mouse polyomavirus Large T antigen, and the stable maintenance function for the vector is provided by the Epstein-Barr virus EBNA-1 protein, which by acting on the FR-element provides chromatin attachment and segregation/partitioning function to the expression vector in mitosis and cell division.

QMCF expression plasmids consist of two viral-originated DNA elements for replication and maintenance of the plasmids in dividing cells. The replication of the plasmid is assured by Py minimal replication origin. For maintenance of the QMCF plasmids FR of EBV is used which consists of multiple binding sequences for EBV EBNA-1. DNA binding domains of EBV EBNA-1 interact specifically with appropriate binding sequences in plasmids and non-specifically with chromatin DNA using second DNA binding domains. These interactions assure stable maintenance and partitioning of the expression plasmid during mitosis. There is a large set of QMCF plasmids that in addition to the regulatory maintenance and replication elements contain different expression cassettes and allow to design protein production experiment in most proper manner.

Outstanding Chicken Polyclonal Antibodies - the IgEASY way

AffinityImmuno has expertise in rapidly producing high affinity, high quality antibodies to a diverse number of targets.

Our standard polyclonal chicken egg IgY antibody service is a 35-day immunization program, followed by 14 days of egg collection, with the freedom to continue IgY production thereafter.

We are flexible on the immunization schedule and will use serum and egg titers to guide the egg collection timing in order to ensure maximum quality and yield.

From antigen design and peptide synthesis to antibody purification and labeling, AffinityImmuno’s expert staff is available to assist you with every phase of antibody development. The success of your antibody project is very important to us. As such, we encourage you to take advantage of our free Antigen Design and Protocol Review. Tell us what you need, we’ll create a customized solution for you.

IgY Immunization:
White and Brown Leghorn hens are ideally suited to antibody production and will lay approximately 300 eggs per year. Each egg yolk will have approximately 100 mg of total IgY antibody and 0.5 mg of specific antibody. That is A LOT of antibody! These antibodies can be sent to you in the form of total IgY antibody or affinity purified IgY antibody.

This process is easily scaled, meaning that if you need more antibody, we can deliver.

The Benefits of Polyclonal Chicken Antibody:
Higher Affinity: Most mammalian proteins exhibit enhanced immunogenicity in chickens due to phylogenetic distance, and thus raise antibodies of higher affinity. This also makes production of antibodies against conserved mammalian proteins more successful in chickens than in mammals.

Higher Specificity: Compared with mammalian IgG, chicken IgY has less cross-reactivity with mammalian proteins other than the immunogen.

Lower background: IgY and IgG are structurally different in the Fc region; IgY does not bind to IgG Fc receptors and causes less false positive staining.

More flexibility: Chicken antibodies are not bound by protein A, protein G, or most anti-mammal secondary antibodies allowing greater flexibility in reagent selection in multiple labeling schemes.

High yield: Chickens lay eggs regularly, providing a continual source of IgY antibody. One hen can produce 10 times as much antibody as a rabbit in the same time period.

Services for Polyclonal Antibodies:
Antibody Purification: from salt fractionation to antigen-specific chromatography, our expert staff offers purification services to complement your antibody project.

ELISA testing: ELISA testing can be useful in measuring and tracking the concentration of antibody in culture supernatants, bleeds or purified materials.

Antibody Characterization: AffinityImmuno offers several services to help evaluated the purity, concentration and specificity of your antibody.

What should you consider when thinking about your source and type of antigen?

Native autoantigens – the benchmark
Native autoantigens have a proven track record in autoimmune disease diagnostics. They have been used extensively by benchmark manufacturers of autoimmune diagnostic kits for over 20 years and have proven their reliability many times over. At AROTEC Diagnostics, autoantigen production is a result of extensive process development and control combined with proven scale-up procedures. We are therefore able to produce large batches of quality native autoantigens that are most appropriate to the diagnostic industry, and remain excellent value for money. The use of recombinant versions of autoantigens where the authentic human sequence is required (Ro52 and tissue specific autoantigens) or where there is no viable native source available (CENP proteins) is however a valid strategy. Indeed both Ro52 and CENP B proteins are produced at AROTEC Diagnostics using recombinant technology. The continuing improvement in native autoantigen quality due to the application of advanced chromatographic process technology will ensure that these reagents retain their benchmark status for many years to come.

Recombinant autoantigens often do not have the correct conformation
It is now well documented that many autoantibodies react with their corresponding autoantigen in a conformationally-dependent manner. Although this is especially evident for tissue-specific autoantibodies (e.g. thyroid), it is also becoming apparent that tertiary and quaternary autoantigen structures contribute to the binding of many systemic (ENA or ANCA) autoantibodies. The scientific literature is littered with cases of recombinant autoantigens, even though they have the correct primary sequence and may have been expressed as “soluble” proteins, being unable to effectively bind their corresponding autoantibodies. Sm, Ro60 (SSA) antigen and Proteinase 3 (cANCA) antigen are important examples where native proteins are clearly superior to their recombinant versions because autoantibody binding is conformation-dependent.

Recombinant autoantigens have aggregation and solubility issues
Overexpression of a foreign protein is usually a destabilizing process for a host cell. The expressed protein will therefore often be ejected from the cell or be compartmentalized into inclusion bodies. Host cell efforts to neutralize the foreign protein will usually result in aggregation and reduced solubility. In our experience even recombinant proteins that are extractable using buffers appropriate for “soluble” proteins often display degrees of aggregation that can vary considerably between fermentation runs. Aggregation and solubility issues have marked effects on purified autoantigen application procedures (e.g. immunoassay plate coating) and can be a significant source of assay variability.

Association with non-protein entities may be different or lacking in recombinant autoantigens
Many native autoantigens associate with non-protein entities which in many cases are thought to be involved in autoantigen binding, e.g. the ENA autoantigens Sm, RNP, SSA and SSB all associate with specific RNA molecules. It is not known if recombinant autoantigens associate with such entities and if so, it could only be with those of the host cell. It is unlikely that an autoantigen associated with, say, bacterial RNA possesses the same autoantibody reactivity as the native mammalian complex

Post-translational processing may be deficient in recombinant autoantigens
Many expression systems, while being able to reproduce the primary sequence of the protein of interest, are unable to post-translationally process the expressed autoantigen as would be the case in the native human or mammalian cell (examples of post-translational processing include phosphorylation, glycosylation, acylation, dimethylation and citrullination). Sm antigen is a well known example where native autoantigen effectively binds patient autoantibodies even in a denatured stated (e.g. in Western blot) whereas the recombinant version is ineffective. In host cells which are capable of some post-translational modifications, the modification carried out may not be identical to what would be the case in human mammalian cells e.g. glycosylation of thyroid peroxidase expressed in insect Sf9 cells is different from that of the native human protein. In general it is believed that only mammalian cells can post-translationally modify an over-expressed mammalian protein in the appropriate manner.

Contamination by host cell proteins
Although many recombinant autoantigens exhibit a high degree of purity, the type (as opposed to the amount) of any impurity present is of great importance. Many healthy individuals possess antibodies to proteins of host cells (e.g. bacteria, yeast) in which recombinant proteins are often expressed. Testing the sera of such individuals for the presence of antibodies against a recombinant autoantigen will often give a false-positive result due to antibody reactivity with contaminating host cell proteins. As a general rule of thumb, it can be said that the further the host cell is phylogenically from the individual being tested (i.e. human), the greater the likelihood of a false positive result. Thus the incidence of false positive results will be as follows for autoantigens purified from the following sources:

Incidence of false positives: bacteria ≈ yeast >> insect cell > mammalian ≈ human

Fusion partners may cause interference
Most recombinant proteins are expressed as fusion partners with peptides or proteins which simplify the purification process. In many cases (e.g. ß-galactosidase, glutathione-S-transferase) healthy individuals have been found to have antibodies against the bacterial version of the fusion protein itself, thereby leading to false positive results. Other fusion partners (e.g. peptide epitopes, hexahistidine) are more innocuous, although there have been some reports in the literature stating that they interfere with protein structure.

Shouldn't recombinant autoantigens be more affordable?
Not necessarily, some reasons are:
Expression of autoantigens in economical expression systems (e.g. E. coli, yeast) has been found to yield antigens of deficient quality (false positives, incorrect conformation etc.) therefore more expensive eukaryotic expression systems (baculovirus, mammalian cells) need to be used.
The market for purified autoantigens is not so great to allow significant economies of scale. Therefore the high developmental costs for recombinant autoantigens need to be recuperated on a relatively small market volume, making the development premium charged per unit sold very high.