The phage-displayed Ubv library used in this study was re-amplified from Library 2 as previously described6 (link). Protein immobilization and subsequent phage selections were performed according to established protocols35 (link). Briefly, purified 53BP1 protein fragments were coated on 96-well MaxiSorp plates (Thermo Scientific 12565135) by adding 100 µL of 1 µM proteins and incubating overnight at 4 °C. Afterwards, five rounds of selection using the phage-displayed Ubv library were performed against immobilized proteins. A total of 96 phage clones obtained from the fourth and the fifth round of binding selections (48 from each round) were subjected to clonal ELISA to identify individual phages that bound to 53BP1. The sequences of phage-displayed Ubvs were derived by sequencing of the phagemid DNA35 (link). For phage ELISA, proteins in study (53BP1 and/or control proteins) were immobilized on 384-well MaxiSorp plates (Thermo Scientific 12665347) by adding 30 µL of 1 µM proteins for overnight incubation at 4 °C before adding amplified phages (1:3 dilution in PBS + 1% BSA + 0.05% Tween) and incubated overnight. Binding of phage was detected using anti-M13-HRP antibody (GE Healthcare 27942101).
Maxisorp plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States, Germany, United Kingdom, Canada
Maxisorp plates are a type of microwell plate designed for immunoassays. They feature a high-binding surface that allows for efficient capture of proteins and other biomolecules. The plates are made of polystyrene and are available in a variety of well configurations to suit different experimental needs.
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Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (28 mentions)
Bovine serum albumin (bsa), by Merck Group (23 mentions)
Kpl stop solution, by LGC (15 mentions)
Kpl tmb 2 component peroxidase substrate kit, by LGC (14 mentions)
Casein, by Thermo Fisher Scientific (12 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (11 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (11 mentions)
Tmb substrate reagent set, by BD (10 mentions)
Tmb substrate, by Merck Group (10 mentions)
Blocker casein, by Thermo Fisher Scientific (9 mentions)
Peroxidase labeled goat anti monkey igg, by LGC (9 mentions)
Tmb 2 component peroxidase substrate, by LGC (9 mentions)
Tmb microwell peroxidase, by LGC (7 mentions)
Tween 20, by Merck Group (7 mentions)
Alkaline phosphatase conjugated goat anti mouse igg, by Merck Group (7 mentions)
Erms da470, by Merck Group (7 mentions)
Calcein am, by Thermo Fisher Scientific (7 mentions)
Stop solution, by LGC (6 mentions)
Microplate reader, by Tecan (5 mentions)
P nitrophenyl phosphate, by Merck Group (5 mentions)
569 protocols using maxisorp plate
1
Phage Display Library Selection for 53BP1
2
Phage Display Library Selection for 53BP1
3
SARS-CoV-2 Spike Protein ELISA
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For ELISA experiments with NTD-targeted mAbs, 384-well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 2 μg/mL of S glycoprotein in 20 mM HEPES pH 8 and 150 mM NaCl. For ELISA experiments with RBD-targeted mAbs, 384-well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 4 µg/mL of hACE2-His in 20 mM Sodium Phosphate pH 8 and 100 mM NaCl. The antibodies that were used were purified previously13 (link),36 (link). Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher Scientific 37532) for one hour at 37 °C. Plates were slapped dry and mAbs were serially diluted in TBST with an initial concentration of 50 µg/ml. Plates were left for one hour at 37 °C and washed 4X with TBST, then 1:5000 Goat anti-Human (Thermo Fisher Scientific A18817) was added. Plates were left for 1 h at 37 °C and washed 4x with TBST, and then TMB Microwell Peroxidase (Seracare 5120-0083) was added. The reaction was quenched after 4 min with 1 N HCl and the A450 of each well was read using a BioTek plate reader.
4
Dengue Virus Particle and Envelope ELISA
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For whole virus particle ELISA Maxisorp plates (Nunc) were coated with 4G2 antibody and virions were captured from infected C6/36 cell supernatants. The following virus strains were used: DENV-1-05K2916, DENV-2-TSV01, DENV-3-VN/32 and DENV-4-2641Y08. For rE ELISA, Maxisorp plates (Nunc) were coated with 150 ng of purified rE protein in 100 ul coating buffer at 4 °C overnight. rE protein was produced in S2 cells as described previously.44 The sequences were derived from DENV-1-05K2916, DENV-2-TSV01, DENV-3-08K4141 and DENV-4-2641Y08. Abs were added at 1 μg/ml and binding was detected by adding anti-human IgG-HRP. TMB substrate was used for the color reaction.
5
ELISA Analysis of RBM-CH3(scaffold) Binding to ACE2
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The ELISA was performed to characterize the binding of RBM-CH3(scaffold) to ACE2. Maxisorp plates (Nunc) were coated with 100 μl of ACE2-Fc protein (2 μg/ml) or BSA as control in 1× carbonate/bicarbonate buffer, pH 9.6 for overnight at 4 °C. Next day, the plate was blocked by 250 μl of PBS containing 5% skimmed milk. Different concentration of RBM-CH3(scaffold) protein ranging from 16 μg/ml to 0.25 μg/ml in ½ fold serial dilution was added to the wells (100 μl/well) and incubated for 2 h at RT. The wells were then washed and HRP-conjugated anti-His antibody (Abcam) in 1:2000 dilution were used for developing ELISA.
For characterizing the binding of immunized sera with the RBM-CH3(scaffold), the RBD and the prefusion spike protein, Maxisorp plates (Nunc) were coated with 100 μl of the proteins at concentration of 2 μg/ml in 1× coating buffer for overnight at 4 °C. Next day, the plates were blocked using 250 μl of PBS containing 5% skimmed milk. The immunized sera were diluted serially 1:3 times in the dilution buffer with a starting concentration of 1:300 dilution and incubated for 2 h followed by washing and blocking. HRP-conjugated anti mouse secondary antibody (Jackson Immuno Research, USA) in 1:2000 dilution was used for developing ELISA.
For characterizing the binding of immunized sera with the RBM-CH3(scaffold), the RBD and the prefusion spike protein, Maxisorp plates (Nunc) were coated with 100 μl of the proteins at concentration of 2 μg/ml in 1× coating buffer for overnight at 4 °C. Next day, the plates were blocked using 250 μl of PBS containing 5% skimmed milk. The immunized sera were diluted serially 1:3 times in the dilution buffer with a starting concentration of 1:300 dilution and incubated for 2 h followed by washing and blocking. HRP-conjugated anti mouse secondary antibody (Jackson Immuno Research, USA) in 1:2000 dilution was used for developing ELISA.
6
Adhesion Assay for Cell Stimulation
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An adhesion assay was performed as previously described (19) . In short, 5 Â 10 6 cells/mL were incubated with calcein-AM (1 mmol/L; Molecular Probes) for 30 minutes at 37 C. Adhesion was measured in an uncoated 96-well MaxiSorp plate (Nunc). Note that 2 Â 10 5 calceinlabeled cells were stimulated with PMA (100 ng/mL; Sigma Aldrich), DTT (10 mmol/L; Sigma Aldrich), Pam3Cys (20 mg/mL; EMC Microcollections), C5a (10 nmol/L; Sigma Aldrich), and TNFa (10 ng/mL; Peprotech) and incubated for 30 minutes at 37 C with 5% CO 2 in an uncoated MaxiSorp plate (Nunc). Adhesiveness of the cells was determined in a Genios plate reader after lysis in 0.5% (w/v) Triton X-100 (Sigma Aldrich) for 5 minutes at room temperature at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
7
ELISA for Cryptosporidium-specific IgG Titration
8
ELISA assay for ESAT-6 nanobody binding
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ELISA experiments were performed as described previously (Weinstein et al., 2022 (link)). MaxiSorp plates (Invitrogen 442404) were coated with ESAT-6 at 5 µg/mL in PBS overnight at 4 °C. Plates were blocked in 2% BSA, 2% Polyvinylpyrrolidone (PVP), 0.1% Tween-20 in PBS (blocking buffer) for 30 min at RT. Dilutions of E11rv or isotype control VHH 52 were made in blocking buffer and added to plates for 1 hr at RT with shaking. Plates were washed with PBST three times for 5 min each and biotinylated anti-VHH antibody (Jackson Immuno 128-065-232) at 1:10,000 in blocking buffer was added for 1 hr at RT with shaking. Plates were washed again and incubated with 1:10,000 streptavidin-HRP (Jackson Immuno 016-030-084) in blocking buffer for 1 hr at RT. Plates were washed again and incubated with 50 µL of ODP (Thermo Fisher 34006), prepared according to the manufacturer’s instructions, for 15 min before stopping with 50 µL of 2 N H2SO4. A492 was measured using a CLARIOstar PLUS (BMG) plate reader. Raw data was background subtracted with wells lacking nanobody and normalized by dividing by the highest binding well, then fit to a 4PL sigmoid curve in Prism 10.0.0 (Graphpad) to determine the EC50.
9
Quantitative ESAT-6 Binding ELISA
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ELISA experiments were performed as described previously (Weinstein et al., 2022 (link)). MaxiSorp plates (Invitrogen 442404) were coated with ESAT-6 at 5 μg/mL in PBS overnight at 4°C. Plates were blocked in 2% BSA, 2% Polyvinylpyrrolidone (PVP), 0.1% Tween-20 in PBS (blocking buffer) for 30 minutes at RT. Dilutions of E11rv or isotype control VHH 52 were made in blocking buffer and added to plates for 1 hour at RT with shaking. Plates were washed with PBST 3 times for 5 minutes each and biotinylated anti-VHH antibody (Jackson Immuno 128-065-232) at 1:10,000 in blocking buffer was added for 1 hour at RT with shaking. Plates were washed again and incubated with 1:10,000 streptavidin-HRP (Jackson Immuno 016-030-084) in blocking buffer for 1 hour at RT. Plates were washed again and incubated with 50 μL of ODP (ThermoFisher 34006), prepared according to the manufacturer’s instructions, for 15 minutes before stopping with 50 μL of 2N H2SO4. A492 was measured using a CLARIOstar PLUS (BMG) plate reader. Raw data was background subtracted with wells lacking nanobody and normalized by dividing by the highest binding well, then fit to a 4PL sigmoid curve in Prism 10.0.0 (Graphpad) to determine the EC50.
10
Quantifying Tau Antibodies in Mouse CSF and Plasma
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AD-tau was coated onto 384-well MaxiSorp plates (Fisher Scientific) at 30 ng/well in 0.1 M sodium carbonate buffered to pH 9.6 at 4 °C overnight. Plates were blocked with BlockAce solution (Abd Serotec) at 4 °C overnight. CSF was collected from the cisterna magna of anesthetized mice following intraperitoneal injection of ketamine/xylazine. CSF samples were flash frozen on dry ice and kept at − 80 °C until analysis. Plasma was obtained from cardiac puncture blood treated with EDTA and centrifuged 2000 g 15 min 4 °C. CSF and plasma were incubated in AD-tau coated plates 2 h at RT and detected with HRP-conjugated anti-mouse secondary antibodies (Jackson Immunoresearch). Tau mAbs levels in CSF and plasma were determined by comparison to standard curves of purified DMR7 and SKT82.
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