A fully synthetic self-adjuvanting globo H-Based vaccine elicited strong T cell-mediated antitumor immunity

Fully synthetic, self-adjuvanting monophosphoryl lipid A–globo H conjugate elicited strong T cell-mediated immunity that could target and kill breast cancer.


S3
The sugar calibration curve was prepared using a standard solution of fucose, galactose, Nacetylgalactosamine, and glucose (1.2 mg/mL in 1/3/1/1 molar ratio) in distilled water. Aliquots were transferred to 10 dry 10 ml tubes in 5 μL increments ranging from 5 to 50 μL. In another 10 mL test tube, accurately weighed samples of the glycoconjugates to be analyzed were placed. At this point, all the tubes should contain between 5 to 50 μg of sugar, and one should contain an unknown amount of sugar to be determined. To all of the tubes were sequentially added 500 μL of 4% phenol and 2.5 mL of 96% sulfuric acid. The glycosyl linkages were cleaved and a colored complex was formed in this step. Solutions were transferred from the test tubes to cuvettes and measured at the wavelength of 490 nm. The calibration curve was obtained by plotting A490 against the weight (μg) of sugar in the standard samples. The amount of sugar present in each unknown sample was calculated based on the A490 of the unknown sample against the calibration curve, while the free proteins KLH and HSA were used as blank controls for conjugates 2 and 3, respectively. The carbohydrate loading of each glycoconjugate was calculated according to the following equation, and the results for KLH conjugate 2 and HSA conjugate 3 were 8.0% and 14.0%, respectively.
Carbohydrate loading % = sugar weight in a tested sample/total weight of the sample × 100%

V. Assays of MPLA-and KLH Specific Antibodies
MPLA-specific antibody titer was determined by ELISA, similar to that used to measure other antibodies but using MPLA as the capture reagent to coat the NUNC PolySorp TM 96-well plates.
The MPLA derivative S2 used for coating plates was dissolved in 0.2% triethylamine to get a final concentration of 0.03 mg/ml. After the solvent was evaporated, the plates were treated with blocking buffer following the normal protocol of ELISA. The MPLA-specific total antibody titer of the day 38 serum pooled from mice immunized with Globo H-MPLA conjugate 1 was 59,666, as compared to the Globo H-specific total antibody titer of 63,038 ( Figure S6).

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KLH-specific antibody titer was determined by ELISA, similar to that used to measure other antibodies but using KLH as the capture reagent to coat the plates. KLH powder was dissolved in the coating buffer (0.1 M bicarbonate, pH 9.6) to get a final concentration of 2 μg/mL. Each well of the plates was treated with 100 μL of KLH coating solution at 37 °C for 1 h and then with a blocking buffer following the normal protocol of ELISA. The KLH-specific antibody titer of the day 38 antiserum pooled from mice immunized with Globo H-KLH conjugate 2 was 293,919, and its Globo H-specific antibody titer was 23,177 ( Figure S7). Evidently, the KLH conjugate provoked much stronger anti-KLH antibody response than the anti-globo H antibody response.