Isolation of antigen-specific nanobodies

Separation of peripheral blood lymphocytes: 5 ml of anticoagulant fresh blood was taken and mixed with 0.9% Nacl solution (Nuclease free water) in a 15ml falcon tube. 2.5ml of Hisep 1077 (Sigma Aldrich) was added slowly in a 15 ml falcon tube. Blood mixture overlapped to the Hisep 1077. Care was taken to keep the blood mixture and Hisep in a separate layer. Нe mixture of blood was carefully centrifuged at 2800 rpm for 20 min at room temperature. We obtained three distinguished layer in which top layer was plasma, interphase appeared white ring which was peripheral blood lymphocyte and the bottom layer was Hisep. Нe interphase was collected to a new 1.5 ml eppendorf tube. Equal volume of freshly prepared PBS was added (1X with Nuclease free water) for wash. Centrifuged at 2200 rpm for 10 min at 4°C [9,10,22] and decanted the supernatant aіer centrifugation. Cell count was done with the help of Hemocytometer. Cell can be stored at -20°C in RNA later for long time without freezing and thawing [22-24].

RNA isolation from peripheral blood lymphocyte: Sample was taken from -20°C and kept at room temperature for 10 min. 250 µl of sample was taken and added to 750 µl of Trizol. Re-suspended the sample by pipetting several times. Incubated the sample for 5 min at room temperature. 200 µl Chloroform was added in 1 ml Trizol (Invitrogen) cap tube. Shaken vigorously or vortexed for 20-30 sec. Incubated at room temperature for 5 min. Centrifuged at 12,000 rpm for 15 min at 4°C. Only 300 µl sample (upper aqueous phase) was taken carefully and kept it in a new tube. Added 100% chilled alcohol and the volume made up to 1.5 ml. Sample kept at -20°C for further use. Next day, sample was centrifuged at 13,000 rpm for 10 min at 4°C. Supernatant was removed and the pellet was leі. 400 µl 75% chilled alcohol was added to the pellet. Нe sample was inverted for 4-5 times and centrifuged at 9000 rpm, at 4°C for 5 min. Нe supernatant was discarded very carefully by pipetting. All the tubes were kept inside the laminar air flow for air drying and 20 µl of nuclease free water was added [25]. Eluted sample was treated with DNase I enzyme (Invitrogen). RNA sample was taken 1 µg (10 ul), DNase reaction buوٴer was added (10X) 2 µl, DNase I enzyme was added 1 ul, Nuclease free water was added 7 µl. Total volume was 20 µl. Incubated sample kept at room temperature for 15 min and then 1µl of 25 mM EDTA solution was added. Нe enzyme activity was inactivated at 65°C for exactly 10 min [25]. First-strand c-DNA synthesis: Total RNA sample was taken 8 µl, added Random Hexamer (Bioline) and mixed properly, incubated at 70°C for 10 min, 25°C for 15 min, and stored at 4°C. In this mixture, 6 µl strand buوٴer (Invitrogen, 5x), DTT (Invitrogen, 0.1 M) 3 µl, dNTPs (Taqara, 2.5 mM) mixture 6 µl, nuclease free water (Bioline) 1 µl, and reverse transcriptase (Invitrogen, 200 U/ult) 1ul were added. Нe PCR was set at 25°C for 10 min, 37°C for 60 min, 42°C for 60 min, 80°C for 15 min and finall\ stored at 4°C. Sample was stored at -20°C for long time storage without freezing and thawing [16]. First PCR reaction: Buوٴer was taken (10X)-2 µl, dNTPs (2.5 mM) 2 µl, Forward Primer (Call 001)-1 µl , Reverse Primer (Call 002)-1 µl , Taq Polymerase (Invitrogen, 5 U/ult ) 0.5 µl , Nuclease free water added 11.5 µl. Total reaction volume was 20 µl. PCR temperature was set at 95°C for 5 min, 95°C for 45 Sec, 57°C for 45 sec, 72°C for 45 sec, 72°C for 10 min and 4°C for storage. PCR reaction was for 35 cycles. Each reaction product checked by 1% agarose gel. 5 µl sample was loaded in each well. PCR product was 700 bp [16].

Gel elute first PCR product: Prepared washing buوٴer-��� µl gel wash buوٴer was mixed properly with 1000 µl of ethanol. 700 bp product with the help of sharp knife was cut and kept it in an eppendorf tube. 100 µl gel binding buوٴer was added in the same tube and was kept it in the water bath. Нe temperature maintained at 55°C for 10 min. Нe gel solution was mixed continuously. Нe whole solution was poured in a fresh column and centrifuged at 12,000 rpm for 2 min 30 sec at 27°C. 300 µl gel binding buوٴer was added and centrifuged at 12,000 rpm, for 2 min 30 sec at 27°C. Mix buوٴer was discarded and 600 µl washing buوٴer was added. Centrifuged again at 12,000 rpm for 2 min 30 sec at 27°C. Centrifuged empty column at 12,000 rpm, for 2 min at 27°C. In a new column 20 µl elution buوٴer was added and centrifuged at 12,000 rpm, at 4°C, for 2 min 30 sec. Only liquid was taken into micro centrifuge tube and the sample was stored at -20°C. Second PCR: 1.5 µl of sample was taken and 1.5 µl of forward primer pstI, 1.5 µl of NotI, 3µl of 10X reaction buوٴer, 3 µl of dNTPs, 1µl of Taq Polymerase enzyme, 18.5 µl of nuclease free water were added. Temperature was set at 95°C for 5 min, 95°C for 45 sec, 61°C for 45 sec, 72°C for 45 sec,72°C for 10 min and 4°C for storing [16]. 1% agarose gel was checked and 400 bp product was found. Total sample was loaded and elute 400 bp product followed by Himedia 3urification kit. Ligation reaction: 400 bp product was purified and ligated with Pgem® -T Easy vector (Pgem(R)-T Easy vector System I, Promega). 5.5 µl eluted second PCR product (12 ngm/ µl), vector 1 µl (50 ngm/µl), ligation buوٴer 7.5 µl, T4 ligation enzyme 1 µl were added. Total reaction volume was 15 µl. Reaction mixture kept at 17°C, overnight in BOD incubator (Care was taken so that temperature distributed equally). Competent cell preparation: 200 µl of DH5α bacteria culture was inoculated in 5 ml LB Broth. No antibiotic was added. Kept the tube in BOD shaker overnight for 37°C at 150 rpm. Turbidity of the bacterial culture was observed next day to confirm the growth. 0.1% bacterial culture was inoculated in to 50 ml LB broth in a 250 ml conical flask and kept in BOD shaker incubator at 150 rpm for 50-60 min. O.D at 600 nm was checked every 30 min. When O.D was 0.3, the flask was kept immediately in ice to stop further growth. Measured 50 ml culture in a centrifuge tube. Centrifuged at 2700 rpm, for 10 min, at 4°C. Supernatant was discarded and precipitate was dried with the help of paper towel used to drain out media. Chilled 0.1 M Cacl2 was added kept it in ice for 20 min. Centrifuged at 2,700 rpm, for 10 min at 4°C and dissolved the precipitate as much as required in 1.5 ml Nuclease free water. One eppendorf tube rack was taken, filled with some water and all competent cells tube kept in a rack, so that temperature equally distributed. Kept it at 4°C for overnight for transformation [16,25,26]. Transformation: Нree competent cell tubes were taken. 3 tubes were labeled as ligated product, positive control and negative control. DNA was added all 3 competent cell tubes inside the hood. Ligated product was Pgem® -T Easy vector (Pgem(R)-T Easy vector System I, Promega) and desired gene product, Positive control was Pgem® -T Easy vector only, Negative control was nothing. Gently tapped for mixing. Kept in ice chilled bucket for 30 min. Heat shock maintained at 42°C. All eppendoوٴ tubes closed with parafilm. Kept all the tubes at 42°C for exactly 90 sec. Tubes were kept in ice for 15 min. In each tube 900 µl LB broth was added for recovery of the cell. Kept it at 37°C for 1 hr in a BOD shaker, with 150 rpm. 200 µl transformed cells were taken and put on LB agar plate containing Ampicillin and thoroughly spreaded with the help of sterilized spreader. Kept it inside the laminar air flow for drying. Kept all the tubes inverted for overnight growing at 37°C in BOD. Next day colonies were observed and compared with positive and negative control [25]. Colony screening: Positive colonies were picked up by sterilized wooden tooth-pick. Individual Colony with tooth-pick was inoculated in to 6 ml LB broth with ampicillin and kept it at 37°C for overnight growth rpm in BOD shaker [16,27]. Plasmid isolate from the positive clone: Maxi preparation-(i) Alkaline solution I: 50 mM Glucose, 25 mM Tris-Cl (pH 8.0), 10 mM EDTA (pH 8.0). Prepared solution-I from standard stocks in batches of 100 ml autoclave for 15 min, at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4°C. (ii) Alkaline solution II: 0.4 N NaOH (freshly diluted from 10 N stock), 2% (W/V) SDS. (iii) Alkaline Solution III: 5 M Potassium Acetate-60 ml, Glacial Acetic Acid-11.5 ml, Nuclease free water-28.5 ml. Нe resulting solution was 3M with respect to Potassium and 5 M with respect to Acetate. Stored the solution at 4°C and transferred it to an ice bucket just before use. (iv) STE Buوٴer� Sodium Chloride-Tris-EDTA Buوٴer, 10X-pH 8.0. 10X solution containing 100mM Tris-HCl, 10 mM EDTA and 1 M NaCl. Cell harvested-5 ml of primary culture was inoculated at 37°C in BOD shaker at 160 rpm. Care was taken for decontamination. On the same day evening, turbidity for bacterial growth was checked. 0.5% inoculum was inoculated to 50 ml LB ampicillin medium. Kept it in BOD shaker at 160 rpm for overnight culture. Next day, 50 ml of culture media took in a sterilized centrifuge tube inside the laminar air flow and centrifuged the culture media at 4000 rpm for 6 min at 4°C temperature. Supernatant was discarded and took the pellet. 10 ml STE buوٴer was added. Нe tube was vortexed for 1 min so that precipitate dissolved and centrifuged at 9000 rpm for 6 min at 4°C. Only pellet was taken, 10 ml alkaline Solution-I was added and freshly prepared 1ml of Lysozyme was added (stock 10 mg/ml, Amresco) and then vortexed for 1 min. Нen incubated for 10 min in an ice bucket. Aіer incubation, 20 ml of freshly prepared alkaline solution-II was added and inverted several times for mixing. Tubes were kept in ice exactly for 5 min. 15 ml chilled solution-III was added. Reagents were mixed properly. Incubated for 30 min (maximum) in ice and centrifuged at 13,000 rpm for 15 min, at 4°C. Supernatant was taken by filter paper. Around 40 ml supernatant was taken. Added 0.7-0.8 volume of supernatant, 32 ml Isopropanol. Mixed by inverted position by 2-3 times. Incubated at room temperature for 20 min and centrifuged at 13,000 rpm for 15 min at room temperature. Supernatant was discarded. Only pellet was taken and added 5 ml 70% alcohol (used Marck 100% alcohol, added Nuclease free water). Vortexed the sample so that the pellet was partially dissolved. Centrifuged at 13,000 rpm for 15 min at 4°C. Alcohol was decanted and pellet was completely dried. Pellet was stored at -20°C for overnight. Next day, 500 µl nuclease free water was added and thoroughly mixed. 30 µl Rnase A (Invitrogen, 20 mg/ml) was added and kept in BOD incubator for 3 hrs at 37°C [25]. Digestion by pst1 and Not1: Plasmid was confirmed by electrophoresis run in 1% agarose gel. Product size was further confirmed by 100-1200 bp marker. Desire product digested by Pst1, Not1 enzyme. Prepared master mix of pst1 and Not1 enzyme each 1 µl (NEB, 20U/µl), BSA 1 µl, DNA 20 µl, NEB Buوٴer 3.5 µl, total volume was 30 µl. Incubated exactly for 45 min at 37°C in BOD. Immediately digested product kept at 4°C. For long time storage it was kept at -20°C. Aіer incubation, digested product was run in 1% agarose gel. Digested 400 bp product was separated out from the pGEM-T vector. Desired fragment was further purified by Himedia purification kit. Plasmid isolation (PHEN4 vector): maxi preparation (i) Alkaline solution I: 50 mM Glucose, 25 mM Tris-Cl (pH 8.0), and 10 mM EDTA (pH 8.0). Prepared solution-I from standard stocks in batches of ~100 ml autoclave for 15 min, at 15 psi (1.05 kg/cm2 ) on liquid cycle, and stored at 4°C. (ii) Alkaline solution II: 0.4 N NaOH (freshly diluted from 10 N stock), 2% (W/V) SDS. (iii) Alkaline Solution III: 5 M Potassium Acetate-60 ml, Glacial Acetic Acid-11.5 ml, Nuclease free water-28.5 ml. Нe resulting solution was 3M with respect to Potassium and 5 M with respect to Acetate. Stored the solution at 4°C and transferred it to an ice bucket just before use. (iv) STE Buوٴer� Sodium Chloride-Tris-EDTA Buوٴer, 10X-pH 8.0. 10X solution containing 100 mM Tris-HCl, 10 mM EDTA and 1 M NaCl. Cell harvested-5 ml primary culture was inoculated at 37°C in BOD shaker at 160 rpm. On that same day evening we have checked the turbidity for bacterial growth. 0.5% inoculum was inoculated to 50 ml of LB ampicillin medium and kept it in BOD shaker at 160 rpm for overnight culture. Next day, 50 ml of culture media was taken in a sterilized centrifuge tube inside the laminar air flow. Centrifuged the culture media at 4000 rpm for 6 min at 4°C temperature. Supernatant was discarded and only pellet was collected. 10 ml STE buوٴer was added. Vortexed the tube for 1 min to dissolve the precipitate and centrifuged at 9000 rpm for 6 min at 4°C. Only pellet was taken, 10 ml alkaline Solution-I and freshly prepared Lysozyme 1ml (stock 10 mg/ml, Amresco) was added, then vortexed for 1 min and incubated for 10 min in an ice bucket. Aіer incubation, 20 ml freshly prepared Alkaline solution-II was added and mixed properly by inverting several times. Tubes were kept in ice exactly for 5 min and 15 ml chilled solution-III was added. Нe reagent was mixed for three times and incubated for 30 min (maximum) in ice. Centrifuged at 13,000 rpm for 15 min at 4°C. Around 40 ml of supernatant was collected by filter paper. Added 0.7-0.8 volume of supernatant, 32 ml Isopropanol. Mixed by inverted position by 2-3 times and incubated at room temperature for 20 min. Centrifuged at 13,000 rpm for 15 min at room temperature and discarded the supernatant. Only pellet was collected and 5 ml of 70% Alcohol was added. Vortexed the sample to dissolve the pellet partially and centrifuged at 13,000 rpm for 15 min at 4°C. Decanted alcohol and pellet was completely dried. Pellet was stored at -20°C for overnight. Next day, 500 µl of Nuclease free water was added and thoroughly mixed up. Added 30 µl Rnase A (Invitrogen, 20 mg/ml). Kept in BOD incubator for 3 hrs at 37°C [25]. Plasmid purification by phenol-chloroform-isoamyl alcohol: 500 µl of sample was taken in an eppendorf tube and equal amount of saturated Phenol:Isoamyl alcohol:Chloroform-25:24:1 ratio was added. Tubes were vortexed vigorously at least 3 min and centrifuged at 13,000 rpm for 15 min at 4°C. Нere were three distinguished layer. Aqueous phase was taken. Нis was repeated twice. 3 M 0.1 volume of Sodium Acetate, pH 5.2 was added and incubated sample for 10 min at room temperature. 0.8 volume of Isopropanol was added and incubated for 20 min. Sample was Kept at -20°C for overnight. Next day centrifuged the sample 13,000 rpm, for 15 min at 4°C. Supernatant was decanted and 500 µl of 70% alcohol was added in to the pellet. Нe sample was vortexed and centrifuged at 13,000 rpm for 15 min. Alcohol was decanted and kept in laminar air flow for drying. Pellet should not dry completely. 500 µl of nuclease free water was added and dissolved the pellet. Checked in 1% agarose gel [25]. PHEN4 vector digested by PstI and NotI: 2 µl (2 µgm/µl) of DNA sample was taken, added PstI NEB enzyme, NEB Cut smart buوٴer-�.� µl (10X), BSA-1 µl, and finall\ nuclease free water-18.5 µl was added. Total volume was 25 µl. Kept at 37°C inside the BOD incubator for exactly 15 min. Immediately aіer digestion, mixture was kept at 4°C. Prepared master mix for NotI enzymatic digestion. NotI enzyme 2 µl, BSA 1 µl, NEB buوٴer (10X)-3 µl were taken. 24 µl of nuclease free water was added. Total reaction volume was 30 µl and distributed in 6 eppendoوٴ tubes, each tube contained 5 µl of master mix. 5 µl of previously digested product (PstI) was added in each tube and kept at 37°C in the BOD incubator for 30 min. Tubes were immediately kept at 4°C. 1 µl XhoI (NEB, 10 U/µl) was added in each tubes and kept it in the BOD incubator for another 30 min. Sample was then stored at -20°C [16,28]. Gel elute digested phen4 product: Prepared washing buوٴer-��� µl of gel wash buوٴer was mixed with 1000 µl of ethanol. Both reagents were mixed properly. Electrophoresis of PHEN4 digested product was done in 1% freshly prepared agarose gel. Around 4500 bp product with the help of sharp knife was cut and kept it in an eppendorf tube. 100 µl gel binding buوٴer was added in the same tube and kept it in the water bath at 55°C for 10 min. Gel solution was mixed continuously to dissolve it. All solution was poured in a fresh column and centrifuged at 12,000 rpm for 2 min 30 sec. at 27°C. 300 µl of gel binding buوٴer was added and centrifuged at 12,000 rpm, for 2 min 30 sec at 27°C. Mix buوٴer was discarded and 600 µl of washing buوٴer was added. Centrifuged again at 12,000 rpm for 2 min 30 sec at 27°C. Centrifuged empty column at 12,000 rpm, for 2 min at 27°C. 20 µl of elution buوٴer was added in a new column and centrifuged at 12,000 rpm, at 4°C, for 2 min 30 sec. Only liquid was collected in to micro centrifuge tube and sample was stored at -20°C.

Ligation reaction: Added eluted digested Phen4 Vector 1 µl (50 ngm/µl), 2nd PCR double digested product (around 12 ngm/µl) 3 µl, Ligation Buوٴer 5 µl, T4 Ligation enzyme 1 µl. Total reaction volume was 10 µl. Reaction mixture kept at 17°C, overnight in BOD. Next day, reaction mixture was taken and kept at -20°C [16]. Competent cell preparation of TG1: 200 µl of TG1 bacterial culture was inoculated in 5 ml of LB Broth. No antibiotic was added. Kept the tube in BOD shaker overnight at 37°C at150 rpm. Next day, turbidity of the bacterial culture was observed to confirm the growth. 0.1% bacterial culture was inoculated in to 50 ml of LB broth in a 250 ml conical flask. Kept in BOD shaker incubator at 150 rpm for 50-60 min. O.D at 600 nm was checked every 30 min. When O.D was 0.3, the flask was kept immediately in the ice to stop the further growth. Measured 50 ml of culture in a centrifuge tube and centrifuged at 2,700 rpm for 10 min at 4°C. Supernatant was discarded and precipitate was dried. Chilled 0.1 M CaCl2 was added and kept it in ice for 20 min. Centrifuged at 2700 rpm for 10 min at 4°C. Precipitate was dissolved as much as required in 1.5 ml nuclease free water. All competent cell tubes were kept in a rack to maintain the equal temperature and kept it at 4°C for overnight for transformation experiment [16,25,26]. Transformation: Нree competent cell tubes were taken. Ligated product, positive control and negative control were labeled and DNA was added. Ligated product was Phen4 vector and desired gene product, positive control was Phen4 vector only, negative control was nothing. Gently tapped to mix and kept in ice chilled bucket for 30 min. Heat shock-maintained temperature exactly at 42°C. All eppendoوٴ tubes were closed with parafilm and kept all the tubes at 42°C for exact 90 sec. Tubes were kept in ice for 15 min exactly and 900 µl of LB broth was added in each tube for recovery of the cell. Tubes were kept at 37°C for 1 hr in a BOD shaker, with 150 rpm. 200 µl of transformed cells were taken and inoculated on LB agar plate containing Ampicillin and spreaded thoroughly with the help of sterilized spreader. Kept it for some time for drying inside the laminar air flow. Kept all the plates at 37°C in the BOD incubator. Next day, colonies were observed and compared with the positive and negative control [25]. Colony screening: Ten colonies were picked up from the ligated plate as it is mentioned before. Individual colony was inoculated in to 6 ml LB broth and ampicillin mixture. Kept it at 37°C for overnight growth at 150 rpm in BOD shaker [16,27].

Colony PCR using GIII primer: PCR was performed on 10 independent colonies in order to determine the vector with the right insert size. Combined dNTPs (2.5 mM), universal reverse primer and GIII primer (20 uM), Taq DNA polymerase (Invitrogen 2.5 u/µl), PCR buوٴer and volume made up to 20 µl. Single colony was taken using sterile tooth pick. Cells were taken in to the PCR reaction by stirring the tooth pick in the reaction mix. PCR reaction was set for 35 cycle and the reaction temperature was 95°C for 5 min, 95°C for 45 secs, 57°C for 45 secs, 72°C for 45 secs, 72°C for 10 min and 4°C for storing the samples. Used 5 µl of each reaction for analytical gel electrophoresis on a 1% agarose gel in 1 × TAE buوٴer. DNA smart ladder was used as DNA molecular weight marker. Нe PCR product from the vector with a full VHH sequence were 700 base pairs [16,29]. Preparation of phagemids: 100 µl library stock was inoculated in to 100 ml of TY containing ampicillin (100 ugm/ml) and glucose (2%). Kept for growth at 37°C for 2-3 hrs with shaking at 150 rpm. Added 1012 pfu M13KO7 helper phage (NEB). Incubated for 30 min without shaking at room temperature. Centrifuged at 4000 rpm for 10 min. Cells re-suspended in 300 ml 2 × TY containing ampicillin and kanamycin (70 µgm/ml). Incubated overnight at 37°C, with shaking at 150 rpm. Centrifuged at 8000 rpm, for 30 min at 4°C. Transferred supernatant containing phages to a fresh tube. Added 1/6th volume to 50 ml PEG/NaCl solution. Mixed well and placed in ice for 30 min. Centrifuged at 4000 rpm for 30 min at 4°C. Supernatant was removed and allowed the tubes to drain on paper tissue. Phage pellet was resuspended in PBS to make it final volume 1 ml. Centrifuged at 13000 rpm for 5 min to remove bacterial cells, debris. Transferred supernatant to a fresh micro centrifuge tube [8,11,13]. Screening by ELISA: For recombinant phage clone, 96 well microplate was coated using 100 µgm/ml antigen. Нe plate was incubated for 1-2 hours at room temperature in a humidified container. Each well of the microplate was filled with 1X blocking buوٴer. Нe wells of the plate were dried with paper towel. 200 µl of the recombinant phage antibody supernatant was added to each antigen coated well, and incubated for 1-2 hours at room temperature. Aіer washing and blotting, HRP/anti-M13 monoclonal conjugate (GE Healthcare) was added to each appropriate well and kept for incubation for 1 hour at room temperature. Aіer washing, ABTS solution was added and reading was taken at ELISA plate reader at 405-415 nm. Нe value was three times higher than the negative control. Expression of recombinant antibody: TG1 clone bacteria inoculated in to 10 ml of LB-Ampicillin media and incubated at 37°C till O.D reaches 0.6. IPTG 1 mM was added and kept overnight at 28°C. Periplasmic fraction of the bacteria was further confirmed by SDS PAGE and Western blot. Membrane fraction solubilized protein were transformed from 10% SDS-PAGE gel to Polyvinylidene difluoride (PVDF) membrane and blocked with 5% nonfat dried milk at 4°C. Нe membrane was treated with Rabbit anti-HA antibody (Invitrogen) in 1:1000 ratio for overnight. Next day, secondary antibody was added and kept in shaker at room temperature for 2 hours. Visualization of AP-VHH specific bands was done by ECL Chemiluminescence (BioVision).