We have developed unique techniques for coupling oligosaccharides to lipids and proteins with the aim of creating powerful tools for use in a range of applications.
These include:

Production of Monoclonal Antibodies

Using as little as 0.1 mg of neoglycoprotein per animal, is sufficient for the generation of antibodies to the required antigen. For this application, the short spacer arm version has been recommended by some of our customers.

While we offer both Bovine Serum Albumin and Human Serum Albumin conjugates as standard catalogue items, a relatively inexpensive custom service is available for conjugate preparation to others such as Tetanus Toxoid or Keyhole Limpet Haemocyanin (KLH).

Special prices are available for bulk amounts or pyrogenfree material, and you are invited to contact us to discuss your requirements.

Enzyme Acceptors

Experience has shown that the use of our unique 14-atom spacer arm when using neoglycoproteins as glycosyl transferase acceptors has proved to be advantageous. As shown below, the ability of 14 atom spaced 3'SLN-BSA to act as an acceptor for a fucosyl transferase was comparable to the Bovine Fetuin control. Using the shorter spaced version, was essentially ineffective.

We would therefore suggest that customers consider the steric influence of the protein prior to product selection.

ELISA

Many of our customers report that neoglycoproteins are ideal for ELISA. Please contact us for advice, or copies of technical bulIetins when ordering.

Quality Assurance

All of our neoglycoproteins are analysed by Matrix Assisted Laser Desorption Time Of Flight mass spectrometry (MALDITOF). This permits the determination of the number of covalently added molecules of antigen per molecule of carrier protein, together with the molecular weight distribution (maximum and minimum numbers of residues per molecule).

The traces obtained for three conjugates (NGPs 0334, 0501, and 0502) are illustrated below. The molecular weight for BSA is 66330 daltons. Comparing the degree of peak broadening of the neoglycoprotein relative to the BSA, gives an indication of the heterodispersity of the conjugate. Generally, neoglycoproteins have an average of 10 to 12 carbohydrates per molecule of protein, with a range of between 7 or 8 to 18 or 20.

Custom Neoglycoprotein Service 

We are able to link a wide range of carbohydrates to proteins and offer a custom service that can be tailored to meet clients’ specific requirements. For example, in the past we have made a considerable variety of neoglycoproteins using complex plantderived oligosaccharides and carbohydrate tumour antigens through to simple monosaccharides. Our neoglycoproteins are frequently used as the solid phase in ELISA assays and we can provide experimental details in this application if required. We can also manufacture our existing range of neoglycoproteins on a larger scale, for example 50 to 100mg or more for significantly lower unit prices.

Our present methods of choice link carbohydrates via the anomeric position to the ε-amino groups of lysine residues or to sulphydryl groups and we offer a choice of two linker arms. One is 3-atoms and sometimes is the choice for antigen presentation while the other is 14-atoms, frequently preferred as an enzyme acceptor. We aim to link at least eight carbohydrate residues per protein molecule if the target amino acid is lysine, but we cannot guarantee the number above this. As a general rule, the coupling ratio decreases with increasing molecular weight of carbohydrate due to a combination of poorer reactivity and increasing steric hinderance. If the target group is sulphydryl, then all free SH groups on the protein are usually reacted. We can discuss other coupling options, depending on specific applications.

The analytical method of choice to determine the coupling ratio is MALDI mass spectrometry. If this technique cannot be used because the protein is unstable, dissociates or forms aggregates, then we have an alternative colorimetric assay, but the accuracy is lower. There are other methods such as immunoassays that may also be considered.

In terms of protein the vast majority of requests are for BSA, HSA or other species’ serum albumins. We can use other proteins depending on their amenability to analysis and the availability of reactive groups.

There are some restrictions that apply to the present techniques. The link between carbohydrate and protein is always β. A number of carbohydrates, such as oligosaccharides with a fucose proximal to the anomeric residue will only react poorly. Our present minimum scale will yield 5 to 10mg of neoglycoprotein.

The cost of this service is influenced by a number of factors such as the efficiency of coupling, purification and product analysis. In general costs tend to rise with the size of the oligosaccharide being reacted because of the initial cost of the oligosaccharide and the reduction in coupling efficiency that often occurs with increased molecular weight. As elsewhere, scale is a major factor in pricing and larger quantities can be made at significantly lower unit costs. As a guideline the charge for attaching a carbohydrate, purification, analysis by MALDI mass spectrometry and supplying the minimum quantity of 5 to 10 mg of neoglycoprotein will be in the range of US$1800 to $3000 for the 3-atom linker arm and $3000 to $4000 for the 14-atom linker. These costs are exclusive of the cost of the carbohydrate, which may either be supplied by the client or will be charged at their catalogue price. We are happy to discuss your requirements in this area, so please don’t hesitate to contact us.

Blood Group and Lewis Antigen Neoglycoconjugates