Frequently Asked Questions

Kode™ Technology

What is Kode™ Surface Modification Technology?
Kode™ Surface Modification Technology consists of the preparation and use of Kode™ Constructs to add functionalities to surfaces, both biological (e.g. cell membranes) and synthetic (e.g. filter membranes, microspheres).

What are Kode™ Constructs?
Kode™ Constructs are Function-Spacer-Lipid (FSL) constructs that are readily dispersible in water, i.e. biocompatible, yet spontaneously and stably incorporate into cell membranes.

How are Kode™ Constructs different from other bioconjugates?
In addition to being dispersible in water and spontaneously incorporating into membranes, antibodies to the spacers (S) of Kode™ Constructs appear to be absent from the antibody profiles of mammals, including humans. The likelihood of non-specific cross-reactivity is therefore reduced when using Kode™ Constructs as opposed to other bioconjugates utilizing conventional bioconjugation chemistries, e.g. polyethylene glycol (PEG).

Are Kode™ Constructs toxic?
Kode™ Constructs have been used to modify cells, microbes, zebrafish and embryos without any observed adverse effect on vitality or development. Kode™ Constructs have also been administered intravenously to small mammals at rates of 200 mg/kg bodyweight with no observed adverse effects.

How scalable is the use of Kode™ Technology?
Kode™ Technology has been and continues to be used in the development and commercialization of products for use in laboratories throughout the world. All methodologies used for the synthesis of Kode™ Constructs can be scaled and in several instances have already been scaled for commercial product demands.

R&D Tools

How can I find out more about how to use the technology and Kode™ Constructs that are available?
The easiest way to get generic information about the use of Kode™ biosurface engineering technology is to download the following document or view the video. For information concerning Kode™ Constructs currently available for R&D use visit the Technology and Sales sections at Kode Biotech Store

Where can I order Kode™ Constructs for R&D use?
The Kode™ Constructs currently available for R&D-use may be ordered directly from Sigma-Aldrich or from Kode Biotech Materials Limited.

How can I obtain FSL Constructs of my own design?
There are two ways to obtain FSL constructs comprising a functional moiety (F) selected by the user. For FSL constructs where F is a peptide use the Kode™ FSL-RFG (Maleimide) FSL Construction Kit (#960819-1-R&D, KBML). For the custom synthesis of an FSL construct of your design contact us directly.

Are Kode™ Constructs toxic?
Kode™ Constructs have been used to modify cells, microbes, zebrafish and embryos without any observed adverse effect on vitality or development. Kode™ Constructs have also been administered intravenously to small mammals at rates of 200 mg/kg body-weight with no observed adverse effects.

How scalable is the use of Kode™ Technology?
Kode™ Technology has been and continues to be used in the development and commercialization of products for use in laboratories throughout the world. All methodologies used for the synthesis of Kode™ Constructs can be scaled and in several instances have already been scaled for commercial product demands.

Methodology

How easy is it to use Kode™ Technology?
Mix a solution of Kode™ Constructs with a suspension of cells or virus particles and incubate. Wash and re-suspend in buffer to provide your “kodecytes” or “kodevirions”.

Do I need a clean-up step following "koding" of cells or viruses?
Provided an excess of Kode™ Constructs has not been used and the mixture has been incubated for at least 1 h no further purification is required. Flow cytometry gating is adequate to separate free Kode™ Constructs from “kodevirions”.

Can I print Kode™ Constructs?
Yes. A simple desktop inkjet printer with refillable ink cartridges may be used to print solutions of Kode™ Constructs. To help visualize the “koded” surface 0.025% (w/v) of bromophenol blue may be included in the print solution. The dye is lost during incubation while the Kode™ Constructs are retained in subsequent washing steps. A wide range of standard printer papers have been used, but coated or higher quality papers tend to provide better resolution.

Are the Kode™ Constructs used for “koding” cells and inkjet printing the same?
Yes. Kode™ Constructs may be used for “koding” of both biological surfaces, e.g. the membranes of cells, and non-biological surfaces, e.g. paper and filter membranes.

Can I use Kode™ Constructs in media containing protein?
Yes. The “koding” process is unaffected by most proteins, including albumin.

Can I use Kode™ Constructs in media containing lipids?
Yes, but in general a higher concentration of Kode™ Construct will be required. Insertion in plasma requires about 40 times more Kode™ Construct to achieve the same level of insertion as will occur in phosphate buffered saline (PBS). Washing cells free of lipids prior to “koding” is recommended.

Can I store “kodecytes” in lipid containing media?
No. Kode™ Constructs tend to partition into media containing significant concentrations of lipids.

How stable are Kode™ Constructs?
Stability of a Kode™ Construct (FSL) is primarily dependent on the stability of the functional moiety (F), but in general the Kode™ Construct are stable and can be transported and managed as dry powders at room temperature. Solutions of Kode™ Constructs in water are less stable than solutions of Kode™ Constructs in saline.

What concentration of Kode™ Constructs should be used when “koding” cells?
The concentration will depend on the Kode™ Construct. The concentration is usually in the range 1 to 100 μg/mL for Kode™ Constructs (FSL) where F is a peptide and up to 1000 μg/mL for Kode™ Constructs (FSL) where F is a carbohydrate. Higher concentrations may start to affect the integrity of the membrane.

Can I target circulatory cells in vivo?
No. Kode™ Constructs will insert into cells non-specifically, but may show a preference for some cell types.

Can I make two different cells adhere to each other using Kode™ Surface Modification Technology?
Yes. A number of methods of promoting cell-cell adherence are currently available. In one method a Kode™ Construct (FSL) where the functional moiety F is biotin (#187786-1-R&D or 416662-1-R&D, KBML) is used in “koding” two populations of cells. One of the populations is then avidinylated by the addition of an excess of avidin before washing and mixing with the other population of biotinylated “kodecytes”.

What is the CMG spacer?
The CMG spacer (S) is semi-rigid spacer designed to optimize presentation of functional moieties (F), e.g. antigens, at the surface of a cell or virus as well as imparting good solubility to the Kode™ Construct (FSL) as a whole.

Why is DOPE used as the lipid tail?
The diacyl-lipid 1,2-O-dioleoylphospatidylethanolamine (DOPE) has proven to be most suitable for insertion in to red cell membranes. The cis-double bond of the acyl chains is believed to prevent Kode™ Constructs packing too tightly together. Nevertheless, other Kode™ Constructs employing steryl as the lipid moiety have also been developed.

Is the diacyl nature of DOPE important?
Yes. Monoacyl FSL constructs do not appear to spontaneously and stably incorporate in to cell membranes.

Can I use Kode™ Surface Modification Technology with fixed cells?
Yes. Kode™ Constructs will incorporate into fixed cells. Alternatively, kodecytes can be fixed after “koding” subject to the functional moiety (F) of the Kode™ Construct being compatible with the fixative. The fixing process must be alcohol, solvent and detergent free if fixing “kodecytes”.

Can I observe “kodecytes” using standard histological techniques?
Yes. In freeze cut or formalin fixed freeze cut tissues the Kode™ Constructs should be retained. Kode™ Constructs (and other glycolipids) will be leached from the “kodecytes” in paraffin imbedded samples during the deparaffination steps.

Can I use FSL constructs to separate live cells from dead cells?
Yes. The concentration of Kode™ Constructs in the membranes of living “kodecytes” reduces more rapidly than the concentration of Kode™ Constructs in the membranes of dead “kodecytes” due to the difference in membrane turnover and cell division.

How many cells or virus particles can be “koded” using one vial of Kode™ Constructs?
Typically a 1 mg vial of a Kode™ Construct is used to prepare between 1 and 10 mL of working solution, each 1 mL of working solution being able to modify 1 mL of packed cells/viruses. Once prepared solutions of most Kode™ Constructs can be frozen and stored for up to 1 year.

In-vivo Studies

Are Kode™ Constructs biocompatible?
It is a basic requirement for an FSL construct to be dispersible in water for it to be a Kode™ Construct. Solutions of Kode™ Constructs are generally prepared in saline.

Do Kode™ Constructs exhibit cross reactivity with serum antibodies?
The spacer (S) of a Kode™ Construct (FSL) has been selected so as to have negligible cross-reactivity with serum antibodies and substantially reduce the incidence of false positives in diagnostic applications.

Is the modification of cells and viruses using Kode™ Constructs stable?
Yes. The “koding” of cells and viruses is stable (subject to the rate of turnover of the membrane components).

Can Kode™ Constructs be used to attach cells and viruses to surfaces?
Yes. The “koding” of cells and virus particles using a Kode™ Construct comprising the appropriate functional moiety (F), e.g. biotin, (#187786-1-R&D or 416662-1-R&D, KBML) permits the “koded” cells (“kodecytes”) or virus particles (“kodevirions”) to be retained on an appropriately selected surface, e.g. avidinylated beads.

Can Kode™ Constructs be used to label bacteria?
Yes. Bacillus and coccus have been labeled with FSL constructs.

Are "kodecytes" incorporating Kode™ Constructs indistinguishable from natural cells?
No. For example, Kode™ Constructs where F is a carbohydrate function as synthetic glycolipids and, in common with natural glycolipids, are laterally mobile in the membrane. Such “kodecytes” therefore present a higher proportion of their glycan structures in the form of mobile glycolipids (cf. glycoproteins).

Can the biological half-life of a functional moeity (F) be altered?
Yes. Some otherwise rapidly cleared functional moieties (bioactives) appear to be retained in the circulation much longer when administered as a Kode™ Construct.

Can Kode™ Constructs be administered via the pulmonary route?
Yes. Nebulized solutions of Kode™ Constructs in the size range 3 to 12 microns have been prepared.

Can you use Kode™ Constructs to monitor the biodistribution of cells and viruses in real time?
Yes. The distribution of “kodecytes” prepared using Kode™ Constructs where F is a fluorophore (721472-1-R&D, KBML) has been used to monitor the biodistribution of cells in zebra fish. The distribution of “kodevirions” prepared using Kode™ Constructs where F is tyrosine has been used to monitor the biodistribution of oncolytic viruses in mice.

What is the shelf-life of a suspension of "kodecytes"?
Kode™ Constructs will remain in the membrane of inactive cells (e.g. red blood cells) for the life of the cell provided it is stored in lipid free media.

What is the half-life of "kodecytes" in the circulation?
Kode™ Constructs are observed to be lost a rate of about 1% per hour in the peripheral circulation. The initial “koding” dose and the minimum level required for detection determine how long the presence of “kodecytes” in the circulation can be monitored. For red blood “kodecytes” reliable monitoring of the presence of the “kodecytes” for up to 3 days post intravenous administration has been demonstrated in small mammals.

What is the fate of Kode™ Constructs in the circulation?
Solutions of Kode™ Constructs (as opposed to suspensions of “kodecytes”) infused into the circulation are cleared within 1 to 2 days and appear to be broken down in the liver.

Can you see a difference between "kodecytes" and unmodified cells by scanning electron microscopy (SEM)?
None observed for red blood “kodecytes” compared with red blood cells.

How do Kode™ Constructs migrate within a membrane?
The migration of Kode™ Constructs within the membrane appears to be influenced by the composition of the lipid moiety (L) of the FSL construct. Some Kode™ Constructs may cluster due to the characteristics of the functional moiety (F).

Do Kode™ Constructs pass through the plasma membrane?
Kode™ Constructs may enter the interna of cells via membrane invagination and endocytosis.

Do Kode™ Constructs participate in signal transduction across the plasma membrane?
As the Kode™ Construct (FSL) is anchored in the membrane via a lipid tail (L) it is believed they do not participate in signal transduction, but may act as agonists or antagonists of the initial binding event.

Have any therapeutic applications of Kode™ Technology been pursued?
Selected Kode™ Constructs have been demonstrated to be capable of reducing virus infection of cells and neutralizing toxins and circulating antibodies.

In-vitro Studies

Can Kode™ Surface Modification Technology be used to increase sensitivity of assays?
Yes. The presentation of the antigen (F) in the form of a Kode™ Construct appears to increase the sensitivity of ELISA based assays.

Can Kode™ Surface Modification Technology be used to increase specificity of assays?
Yes. If the functional moiety (F) of the Kode™ Construct has been chosen correctly, specificity will increase. Sometimes more than one functional moiety (F) will be required to cover the range of specificities required of the assay.

What is the correlation between assays using Kode™ Surface Modification Technology and conventional immunoassays?
Usually very good. However, because Kode™ Constructs typically present a more discrete part of the antigen (peptide versus protein) in common with other peptide based immunological assays a more restricted antibody profile may be observed.

Do "kodecytes" react with both IgG and IgM antibodies?
There does not appear to be a difference between IgM and IgG antibodies directed against Kode™ Constructs (FSL) where the functional moiety (F) is a carbohydrate antigen. It appears that some Kode™ Constructs where the functional moiety (F) is a monomeric peptide antigen react poorly with IgM antibodies.

Do Kode™ Constructs where the functional moeity (F) is a di-, tri-, or tetra- saccharide of the ABO blood groups cross-react differently?
Yes. Polyconal serum contains both antibodies that will react with the di- and tri- saccharides, but not the tetra- saccharides, and true ABO antibodies that will react with the tri- and tetra- saccharides, but not the di- saccharides. Therefore, “kodecytes” prepared using Kode™ Constructs (FSL) where F is a trisaccharide can be used to detect both ABO-like and true ABO antibodies while “kodecytes” prepared using Kode™ Constructs (FSL) where F is a tetrasaccharide can be used to detect only true ABO antibodies.

Are there any sensitivity differences when using Kode™ Constructs (FSL) with spacers (S) of different lengths?
Yes. Using Kode™ Constructs (FSL) with a longer spacer (S) to prepare red blood “kodecytes” has been shown to improve sensitivity by 2-fold in agglutination based assays using such “kodecytes”.

Will monoclonal antibodies react with Kode™ Constructs?
Sometimes. FSL constructs may have a restricted antigen/epitope. If the monoclonal antibody and the Kode™ Construct are not complementary then they will not react.

Other

Is Kode™ Surface Modification Technology proprietary?
Yes. The preparation and use of Kode™ Constructs in a range of applications is the subject of patent applications and granted patents filed and maintained in many of the major markets of the world.

How do I become a licensee?
Kode™ Constructs are available for use in R&D under the terms of a standard end user license. Partners wishing to obtain an option to commercialize Kode™ Surface Modification Technology in a particular field should contact Kode Biotech Limited.

Does Kode Biotech Limited have an R&D pipeline?
Yes. Kode Biotech Limited is continuously developing Kode™ Constructs for both general R&D use and field specific use in collaboration with its existing Research Partners.

Are Kode™ Constructs manufactured to quality assured standards?
Yes. All Kode™ Constructs manufactured for use in the preparation of commercial products are manufactured under license by Authorized Kode™ Construct Manufacturers according specified quality standards.

What is the difference between Kode Biotech Limited (KBL) and Kode Biotech Materials Limited (KBML)?
KBL is the licensor of Kode™ Surface Modification Technology. KBML is an Authorized Kode™ Construct Supplier.

Is Kode Biotech Limited owned by AUT University?
No. KBL is an independent company. AUT University is a shareholder and a collaborative partner. KBL is physically based on the AUT University city campus.