Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

Proteins and amine-containing ligands can be covalently linked to polysaccharides activated by the cyanylation reagent, 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), to form covalent protein (ligand)-polysaccharide conjugates. This article describes an improved protocol for carrying out controlled CDAP activation at 0 °C and varying pH and performing subsequent conjugation of the activated polysaccharides.

Polysaccharides are poorly immunogenic unless linked to protein and need to be activated for derivatization or conjugation to proteins. In this method, CDAP is used as sand lading reagent. The CDAP activates polysaccharide hydroxyls for derivatization or to be linked to proteins for use in vaccines or diagnostic reagents.

CDAP has largely replaced cyanogen bromide as a polysaccharide sand lading reagent. CDAP is simpler to use, more efficient, and can be used at a lower pH than cyanogen bromide. It's a much better activating reagent.

CDAP can be used with most polysaccharides, unlike say reductive amination, which requires vicinal hydroxyls. The chemistry can be used to both directly and indirectly conjugate to proteins, and it can be used to make diagnostic reagents. The CDAP activation reaction, as originally described, was very rapid and difficult to control.

The protocol provided here makes the chemistry far easier to perform and much more reproducible. Before starting the experiment, adjust the pH of the polysaccharide solution to 9 by adding 200 microliters of dimethylaminopyridine stock solution in a dropwise manner while stirring. Keep the reaction chilled in an ice water bath for the entire process of the activation.

Proceed with cyano-dimethylaminopyridine-tetrafluoroborate or CDAP activation by transferring 100 microliters of CDAP to the polysaccharide solution while stirring. Start the timer and monitor the pH change during the entire activation for 15 minutes. Maintain the reaction at the target pH by promptly adding 10 microliter increments of 0.1 molar sodium hydroxide.

When the optimal activation time is reached, add two milliliters of 0.5 molar adipic dihydrazide all at once to the activated polysaccharide. Check that the pH is in the required range for hydroxide functionalization. After continuous stirring of the reaction mixture for at least one hour, transfer the reaction mixture to four degrees Celsius.

To reduce ADH concentration from the product, dialyze the crude derivatized polysaccharide solution using the dialysis device. Recover the derivatized polysaccharide from dialysis and determine the concentration of the polysaccharides and hydrazide to calculate the hydrazide to polysaccharide ratio. Prepare one milligram per milliliter unmodified polysaccharide solution to be used as the standard.

Preheat the heating block to 140 degrees Celsius for a minimum of one hour to achieve a stable uniform temperature. Use a protective pad underneath and surrounding the heating block in case of acid spills. Add varying volumes of one milligram per milliliter carbohydrate standard to the labeled 13 by 100 borosilicate test tubes in triplicates and make up the volume to 100 microliters with water to achieve the desired standard concentrations.

Similarly, set up sample assays by adding a volume containing five micrograms of the derivatized polysaccharide to three sample tubes. Add 100 microliters of freshly prepared six milligrams per milliliter resorcinol to each tube. Using a repeat pipetter, add 300 microliters of the prepared 75%sulfuric acid to each tube, vortex the tubes vigorously, pointing the tube away, then place all the tubes in a heater block at a steady pace in sequential order and immediately set the timer for three minutes.

At three minutes, remove the tubes in the same order and place them directly in a rack in an ice water bath. Remove the ice cold tubes to allow them to equilibrate to room temperature for five minutes. Read the absorbance of all the tubes on a UV-Vis spectrophotometer at 430 nanometers using a 10 millimeter path length cuvette.

To set up assay reactions, sort and arrange the labeled standard tubes in the tube rack in order of increasing concentration. Using a calibrated micropipette, add 100 microliters of the standards to each corresponding tube. For the zero standard, use 100 microliters of the sample buffer.

Similarly, prepare and arrange the sample tubes. Then using a calibrated 1, 000 microliter micropipette, add 875 microliters of assay buffer to all assay tubes. To start the assay reaction, add 25 microliters of 1%trinitrobenzene sulfonic acid to each assay tube in the predetermined order within five minutes.

Vortex the assay tubes for two seconds at high speed, making sure that the liquid swirls to a height of half an inch from tube opening. Record the assay start time and set the timer to two hours. Then place the assay tube rack in the dark at room temperature for two hours.

When the time is over, vortex the tubes and proceed to data collection. The ADH dextran was assayed for dextran using the resorcinol sulfuric acid assay. A typical standard tube using glucose as the sugar standard was plotted.

The hydrazide content in derivatized polysaccharide or dextran was determined using the TNBS assay. The level of derivatization was calculated as hydrides per 100 kilodaltons of dextran to facilitate the comparison between polymers of different average molecular weights. The protocol described here may need to be adapted for a specific polysaccharide.

CDAP chemistry can be used to functionalize polysaccharides for use with a wide variety of conjugation reagents. It can be used, for example, to biotinylate polysaccharides for use in ELISAs. Proteins can be directly linked to CDAP-activated polysaccharides, but this usually requires some optimization for a specific polysaccharide.

The conjugation yield can be over 75%The use of CDAP chemistry has enabled low-cost conjugate vaccines, like the ones made, for example, by the Serum Institute of India.