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Gelatin veronal buffer

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Gelatin veronal buffer is a laboratory reagent used to prepare a buffered solution for various applications in biochemistry and molecular biology. It is a mixture of sodium barbital, sodium chloride, and gelatin that helps maintain a stable pH environment for experiments involving proteins, enzymes, and other biomolecules.

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Lab products found in correlation

Lyophilized guinea pig complement, by Cedarlane (4 mentions) Neutravidin 1 μm beads, by Thermo Fisher Scientific (1 mentions) 1300exi cytometer, by Stratedigm (1 mentions) Guinea pig complement, by Cedarlane (1 mentions) Anti c3 fitc, by MP Biomedicals (1 mentions) Ez link sulfo nhs lc lc biotin, by Thermo Fisher Scientific (1 mentions) Fluosphere neutravidin conjugated beads, by Thermo Fisher Scientific (1 mentions) Neutravidin beads, by Thermo Fisher Scientific (1 mentions) Fluorescein conjugated goat anti guinea pig complement c3, by MP Biomedicals (1 mentions) Lsr 2, by BD (1 mentions) Carbodiimide nhs ester coupling, by Thermo Fisher Scientific (1 mentions) Fluorescein conjugated goat igg fraction to guinea pig complement c3, by MP Biomedicals (1 mentions) Luminex beads, by DiaSorin (1 mentions) Calcium and magnesium, by Boston BioProducts (1 mentions) Ez link sulfo nhs lc lc, by Thermo Fisher Scientific (1 mentions) Red neutravidin conjugated microspheres, by Thermo Fisher Scientific (1 mentions) Reconstituted guinea pig complement, by Cedarlane (1 mentions) Ique flow cytometer, by Sartorius (1 mentions) Anti guinea pig c3 antibody conjugated to fitc, by MP Biomedicals (1 mentions)

9 protocols using gelatin veronal buffer

1

ADCD Assay for Complement Deposition

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The ADCD assay was adapted from Fischinger et al.84 (link). Antigen was coupled to non-fluorescent Neutravidin 1 μm beads (Invitrogen, #F8777) as described for ADCP. Immune complexes were formed by incubating 10 μL of coupled beads with 10 μL of diluted sample for 2 h at 37 ˚C. Plates were spun down, and immune complexes were washed with PBS. Lyophilized guinea pig complement (Cedarlane, #CL4051) was resuspended in cold water, diluted in Gelatin Veronal Buffer, Boston BioProducts, IBB-290X) and added to the immune complexes. The plates were incubated for 50 min at 37 ˚C and the reaction was stopped by washing the plates twice with 15 mM EDTA in PBS. To detect complement deposition, plates were incubated with Fluorescein-conjugated goat anti-guinea pig complement C3 (1:50 diluion) (MP Biomedicals, #0855385) for 15 min in the dark. Fluorescence was acquired with a Stratedigm 1300EXi cytometer.
Roy V., Jung W., Linde C., Coates E., Ledgerwood J., Costner P., Yamshchikov G., Streeck H., Juelg B., Lauffenburger D.A, & Alter G. (2023). Differences in HPV-specific antibody Fc-effector functions following Gardasil® and Cervarix® vaccination. NPJ Vaccines, 8, 39.
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2

ADCD Assay for Complement Activation

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The ADCD assay was performed as previously described (Fischinger et al., 2019 (link)). Spike biotinylation, bead coupling, and immune complex formation was performed as described for ADCP, using red Neutravidin beads (Invitrogen) and 1:10 dilution of serum and 1:1 dilution of breastmilk. Following immune complex formation, plates were washed and guinea pig complement (Cedarlane) diluted in gelatin veronal buffer supplemented with calcium and magnesium (Boston BioProducts) was added. Plates were incubated for 20 minutes at 37°C. Plates were washed twice with 15mM EDTA in PBS and C3-deposition was detected by staining with anti-C3 FITC (MPbio). Fluorescence was acquired using an iQue (Intellicyt) and C3-deposition is reported as the median fluorescence intensity of FITC.
Pullen K.M., Atyeo C., Collier A.R., Gray K.J., Belfort M.B., Lauffenburger D.A., Edlow A.G, & Alter G. (2021). Selective functional antibody transfer into the breastmilk after SARS-CoV-2 infection. Cell Reports, 37(6), 109959.
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3

Antibody-Dependent Complement Deposition Assay

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ADCD was conducted as previously described.82 (link) Briefly, NVX-CoV2373 Spike protein was biotinylated using EZ-link™Sulfo-NHS-LC-LC-Biotin (Thermo Fisher), and then coupled to red FluoSphere™ NeutrAvidin™-conjugated beads (Thermo Fisher). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum (ADCD 1:10 dilution) for 2 h at 37°C, and then washed to remove unbound antibody. The immune complexes were then incubated with lyophilized guinea pig complement (Cedarlane) and diluted in gelatin veronal buffer with calcium and magnesium (Boston Bioproducts) for 30 min. C3 bound to immune complexes was detected by fluorescein-conjugated goat IgG fraction to guinea pig Complement C3 (MP Biomedicals). Flow cytometry was performed to identify the percentage of beads with bound C3. Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1) (Figure S1).
Gorman M.J., Patel N., Guebre-Xabier M., Zhu A.L., Atyeo C., Pullen K.M., Loos C., Goez-Gazi Y., Carrion R J.r., Tian J.H., Yuan D., Bowman K.A., Zhou B., Maciejewski S., McGrath M.E., Logue J., Frieman M.B., Montefiori D., Mann C., Schendel S., Amanat F., Krammer F., Saphire E.O., Lauffenburger D.A., Greene A.M., Portnoff A.D., Massare M.J., Ellingsworth L., Glenn G., Smith G, & Alter G. (2021). Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination. Cell Reports Medicine, 2(9), 100405.
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4

Opsonic Killing Assay for Staphylococcal Enterotoxin E

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To determine opsonic killing of SEE, bacterial cultures were routinely grown overnight at 37°C on chocolate-agar plates, and then killing assessed using modifications of previously described protocols [14 ]. Modifications included use of EasySep™ Human Neutrophil Isolation Kits (Stem Cell Technologies Inc., Cambridge, Massachusetts, USA) to purify PMN from blood, and use of gelatin-veronal buffer supplemented with Mg++ and Ca++ (Boston Bioproducts, Ashland, Massachusetts, USA) as the diluent for all assay components. Final assay tubes contained, in a 400-μl volume, 2 X 105 human PMN, 10% (final concentration) SEE-absorbed horse serum as a complement source, 2 X 105 SEE cells and the serum dilutions. Tubes were incubated with end-over-end rotation for 90 minutes then diluted in BHI with 0.05% Tween and plated for bacterial enumeration.
Cohen N.D., Cywes-Bentley C., Kahn S.M., Bordin A.I., Bray J.M., Wehmeyer S.G, & Pier G.B. (2020). Vaccination of yearling horses against poly-N-acetyl glucosamine fails to protect against infection with Streptococcus equi subspecies equi. PLoS ONE, 15(10), e0240479.
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5

Complement Deposition Assay for Antigen-Antibody Complexes

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The ADCD assay was adapted from a previously described method (46 (link)). Briefly, biotinylated antigen was bound to 1 μm fluorescent Neutravidin beads (Invitrogen). Immune complexes were formed by incubating 10 μL of antigen-bound beads with 10 μL of diluted hyperimmune IgG for 2 hours at 37˚C. The immune complexes were spun down and washed with PBS. Lyophilized complement from guinea pig (Cedarlane) was resuspended in cold water, diluted in Gelatin Veronal Buffer (Boston BioProducts) and added to the immune complexes. The samples were incubated for 50 minutes at 37˚C and the reaction was quenched by washing the plates twice with 15mM EDTA in PBS. For complement deposition detection, samples were incubated for 15 min with Fluorescein-conjugated goat anti-guinea pig complement C3 (MP Biomedicals) in the dark and the samples were analyzed by flow cytometry (BD LSRII). The level of complement deposition was calculated as the ratio relative to naïve levels, which were set to 1.
Fox J.M., Roy V., Gunn B.M., Bolton G.R., Fremont D.H., Alter G., Diamond M.S, & Boesch A.W. (2023). Enhancing the therapeutic activity of hyperimmune IgG against chikungunya virus using FcγRIIIa affinity chromatography. Frontiers in Immunology, 14, 1153108.
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6

Multiplexed Complement Activation Assay

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ADCD was conducted using a 384-well based customized multiplexed assay. Protein antigens were coupled to magnetic Luminex beads (Luminex Corp) by carbodiimide-NHS ester-coupling (Thermo Fisher). OPS and LPS antigens were modified by 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium and conjugated to Luminex Magplex carboxylated beads Schlottmann et al., 2006 (link)). To form immune complexes, a mix of four antigen-coupled beads was incubated for 2 h at 37°C with diluted samples (1:10) and then washed to remove unbound immunoglobulins. Lyophilized guinea pig complement (Cedarlane) was resuspended according to manufacturer’s instructions and diluted in gelatin veronal buffer with calcium and magnesium (Boston BioProducts). Resuspend guinea pig complement was added to immune complexes and incubated for 20 min at 37°C. Post incubation, C3 was detected with Fluorescein-Conjugated Goat IgG Fraction to Guinea Pig Complement C3 (Mpbio).
Bernshtein B., Ndungo E., Cizmeci D., Xu P., Kováč P., Kelly M., Islam D., Ryan E.T., Kotloff K.L., Pasetti M.F, & Alter G. (2022). Systems approach to define humoral correlates of immunity to Shigella. Cell Reports, 40(7), 111216.
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7

Complement Deposition Assay for Antibodies

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ADCD was conducted as previously described [82 (link)]. Briefly, spike or RBD protein was biotinylated using EDC (Thermo Fisher Scientific) and EZ-link Sulfo-NHS-LC-LC (Thermo Fisher Scientific) and then coupled to red Neutravidin-conjugated microspheres (Thermo Fisher Scientific). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum (ADCD 1:10 dilution) for 2 hours at 37°C and then washed to remove unbound antibody. The immune complexes were then incubated with lyophilized guinea pig complement (Cedarlane - Burlington, Ontario, Canada) and diluted in gelatin veronal buffer with calcium and magnesium (Boston BioProducts - Milford, Massachusetts, USA) for 30 minutes. C3 bound to immune complexes was detected by fluorescein-conjugated goat IgG fraction to guinea pig Complement C3 (MP Biomedicals - Irvine, California, USA). Flow cytometry was performed to identify the percentage of beads with bound C3 and a complement deposition score was calculated (% beads positive × median fluorescent intensity of positive beads). Flow cytometry was performed with an LSRII (BD) and analysis was performed using FlowJo V10.7.1.
Zhu D.Y., Gorman M.J., Yuan D., Yu J., Mercado N.B., McMahan K., Borducchi E.N., Lifton M., Liu J., Nampanya F., Patel S., Peter L., Tostanoski L.H., Pessaint L., Van Ry A., Finneyfrock B., Velasco J., Teow E., Brown R., Cook A., Andersen H., Lewis M.G., Lauffenburger D.A., Barouch D.H, & Alter G. (2022). Defining the determinants of protection against SARS-CoV-2 infection and viral control in a dose-down Ad26.CoV2.S vaccine study in nonhuman primates. PLoS Biology, 20(5), e3001609.
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8

Antibody-Mediated Complement Activation Assay

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Recombinant SUDV GP was biotinylated and coupled to 1 µm red fluorescent Neutravidin beads. Serum from vaccinated guinea pigs were diluted 1:10, 1:100, and 1:200 in culture medium and incubated with GP-coated beads for 2 h at 37 °C followed by the addition of reconstituted guinea pig complement (Cedarlane Labs) diluted in gelatin veronal buffer containing magnesium and calcium (Boston Bioproducts) and incubation for 20 min at 37 °C. Beads were washed twice with phosphate buffered saline containing 15 mM EDTA, and stained with an anti-guinea pig C3 antibody conjugated to FITC (1:100, MP Biomedicals cat #0855385). C3 deposition onto beads was analyzed on a Sartorius iQue flow cytometer, and a minimum of 10,000 events were recorded and analyzed. C3 deposition was calculated according to the following formula: (geometric mean fluorescent intensity of FITC of all beads) x (% of FITC + beads)/10,000.
Malherbe D.C., Domi A., Hauser M.J., Atyeo C., Fischinger S., Hyde M.A., Williams J.M., Alter G., Guirakhoo F, & Bukreyev A. (2022). A single immunization with a modified vaccinia Ankara vectored vaccine producing Sudan virus-like particles protects from lethal infection. NPJ Vaccines, 7, 83.
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9

Sheep RBC Hemolysis Assay

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This assay measures the release of hemoglobin from sensitized sheep RBCs lysed upon exposure to CP-activated serum. Double distilled water and assay buffer were used as positive and negative controls, respectively. TT32 activity was accessed as the extent to which TT32 inhibits CP-mediated hemolysis of sheep RBCs (SRBCs). Sensitization of sheep RBC (Bioreclamation, Westbury, NY) was achieved by incubation with rabbit anti-sheep hemolysin (Cedarlane Labs) in the presence of gelatin-veronal buffer (Boston Bioproducts) for 30 min at 37°C. Next, the optimal serum concentration to lyse erythrocytes via CP activation was determined by incubating sensitized SRBCs with serial dilutions of complement-preserved human serum and measuring hemoglobin release at 541 nm. Lastly, TT32 and other inhibitors were spiked into serum at the dilution determined as described above, and incubated with sensitized SRBC, followed by washing step and reading the absorbance at 541 nm. Percent lysis was calculated as (A541sample-A541negcontrol)/(A541H20-A541negcontrol)*100. All data calculations and analyses were performed using Microsoft Office Excel 2007 (Microsoft Corp.) and GraphPad Prism version 5. Serum AP and CP activities were reported as % activity (Sample – Neg. Control/(Pos. Control – Neg. Control) x 100 and normalized to the activity in serum.
Fridkis-Hareli M., Storek M., Or E., Altman R., Katti S., Sun F., Peng T., Hunter J., Johnson K., Wang Y., Lundberg A.S., Mehta G., Banda N.K, & Michael Holers V. (2018). The Human Complement Receptor Type 2 (CR2)/CR1 Fusion Protein TT32, a Novel Targeted Inhibitor of the Classical and Alternative Pathway C3 Convertases, Prevents Arthritis in Active Immunization and Passive Transfer Mouse Models. Molecular immunology, 105, 150-164.
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