Effect of lyophilization on HRP–antibody conjugation: an enhanced antibody labeling technology

Monoclonal antibodies (mAb) and recombinant antigens

Purified mAb’s and antigens of dengue were procured from Biogate laboratories, Canada (Cat No. DN1-9, AG 02-01-4, AG 02-01-5, AG 02-01-3, AG 02-01-6). These purified antibodies and antigens were used for the labeling of enzymes for immunoassays.

Enzymes, chemicals and consumables

Horseradish peroxidase (HRP) was purchased from Sigma Aldrich (Cat. No. P6782) and was stored at 2–8 °C until use. The substrate used for HRP was 3,3′,5,5′-Tetramethylbenzidine (TMB) purchased from Bangalore Genei (Cat. No.62161018010A). Sodium metaperiodate (Cat. No. S1878) and Sodium cyanoborohydride (Cat. No.156159) were purchased from Sigma Aldrich (United States). Dialysis membrane (4 Spectra/Por; Reorder No. 132700) used for desalting purposes was purchased from Spectrum Laboratories (United States) and dialysis tubing closures (LA 404), coomassie brilliant blue R-250 (MB 153) from Hi-media Laboratories (India). ELISA high binding plates (EP2-20X10NO) were purchased from Hi-media (India). All other general chemicals and consumables used for preparation of the buffers and reagents were procured from Sigma Aldrich and Hi-media.

Enhanced labeling procedure

Labeling of HRPO was done using the modified version of classical periodate method by incorporating the process of lyophilization in-order to obtain better sensitivity [1]. The process of conjugation involved two steps; adoption of classical method from the periodate method. The step one involves the activation of HRPO was done using 0.15 M Sodium metaperiodate. The activated HRPO was then desalted by using dialysis method with 1× phosphate buffered saline (PBS) for 3 h at Room temperature. Post dialysis, the HRPO was frozen at − 80 °C (New Brunswick) for 5–6 h. The step two involves the overnight lyophilization of frozen HRPO. After this process, 1:4 molar ratios of antibody to HRP taken for the conjugation process. Where stock concentration of antibody were 7.78 mg/ml diluted to 1 mg/ml concentration for conjugation experiment and mixed with previously lyophilized HRPO and incubated at 37 °C for 1 h in a thermo mixer comfort (Eppendorf). Finally, to the conjugate Schiff’s base reaction carried out by adding 1/10th volume of sodium cyanoborohydride and then incubated at 4 °C for another 2 h (Additional file 1: Figure S1). The conjugate thus formed was kept for overnight dialysis against 1× PBS in room temperature. Any commercially available stabilizers can be added to the conjugate for long-term stability. This conjugate can be stored at 4 °C for 6 months and at − 20 °C for long-term storage.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE experiment performed based on previously reported method by Green and Sambrook [12]; 10% resolving gel was prepared using recommended volumes of SDS-PAGE components and 1 ml butanol was overlaid to form a uniformly layered gel. After polymerization, the gel was washed several times with distilled water to remove traces of un-polymerized acrylamide and butanol. The stacking gel was prepared and poured it over the polymerized resolving gel. The samples were then mixed with sample buffer containing β-mercaptoethanol and heated at 95 °C for 10 min for reducing and for non-reducing samples are mixed with sample buffer alone preceding to load on to the gel. A total volume of 10 µl of samples were mixed 2 µl sample buffer to load on to the gel along with prestained protein Ladder (MBT092 from Hi-media). An initial voltage of 85 V applied to the gel and after the dye front moved into the resolving gel, the voltage gradually increased to 120 V. The gel was run until the dye front reaches the bottom of the resolving gel. The gel was washed with distilled water and stained using coomassie brilliant blue (CBB) staining solution for 1 h. The stained gel was washed using distilled water and soaked in de-staining solution for 2–4 h. The gel image was photographed using Bio-Rad gel documentation system (Gel Doc™ EZ System #1708270).

Ultraviolet and visible (UV–Vis) spectrophotometer analysis

A Shimadzu UV-Pharmaspec 1700 recording double beam UV–visible spectrophotometer with data processing system was used. UV spectra of reference and sample solutions were recorded in 10-mm quartz cells (Hellma Analytics, Art. No. 115-10-40) at a wavelength scanning speed of approx. 3000–10 nm/min. The concentration of conjugated and control in its solutions were determined in between in wavelength ranging from 280 to 800 nm.

Enzyme linked immunosorbent assay (ELISA)

Direct ELISA was performed using the conjugates prepared by the enhanced technique. 100 µl of antigen of 100 ng per well concentration was coated on to the high binding uncoated ELISA plate using carbonate buffer (pH 9.2) followed by overnight incubation at 4 °C. The plates were then washed using PBST buffer and blocked with 2% skim milk powder for 1 h at 37 °C. The conjugate was serially diluted from 1:100 onwards in 1× PBS containing 2% bovine serum albumin (BSA) and added to the antigen coated plate and incubated at 37 °C for 1 h in dark. Plates were washed using PBST followed by taping on dry tissue paper to remove completely any trance of solution and TMB substrate was added and further incubated for 20 min in dark. Reaction was stopped by adding stop solution and the absorbance was read at 450 nm using Tecan Infinte 200 Pro ELISA reader. Similar concentration of antigen and ELISA protocol was used for comparison purpose conjugate prepared by the existing classical method.

To study sensitivity of the dengue antibody–HRP conjugates we have performed direct ELISA by coating different concentrations of dengue antigen starting from 100 to 1.5 ng. Followed by blocking as suggested above protocol and 100 µl of conjugate preparation with dilution factor 1:1000 was added to each well. Followed by rest of the protocol for ELISA same as above-mentioned and absorbance read to 450 nm.

Statistical analysis

The statistical analysis of the assays were done by plotting dilution response curve with Graph Pad software (Graph pad instat 3.1 version) using the secondary antibody conjugates prepared by both the methods. Turkey Kramer multiple comparison tests expressed as mean of three independent experiments. Statistical analysis was done using one-way annova followed by graph pad software to find out the significance of the results obtained. p value < 0.001 was considered to be statistically significant.