Methods for IgA1 Fab profiling have previously been extensively detailed (3 (link), 4 (link)). Briefly, all IgA was captured using CaptureSelect IgA affinity matrix (Thermo Fisher Scientific). Human milk samples were assumed to contain 0.8 μg/μL SIgA and added to excess amount of bead slurry, PBS, and 200 ng of the monoclonals anti-CD20 mIgA1 (7D8-IgA1) and anti-cMET (5D5v2-IgA1). These monoclonals were used as internal standards for quantification, and were a gift from Genmab (Utrecht, NL). Samples were incubated followed by removal of the flow through, containing all non-IgA human milk components. The samples were then washed several times and IgA was digested overnight with the O-glycopeptidase from Akkermansia muciniphila, OgpA (OpeRATOR®, Genovis, Llund, Sweden). Digestion was performed using 40 U SialEXO (a sialidase cocktail to remove sialic acids from the O-glycans) and 40 U of OgpA enzyme, and incubated overnight at 37°C, in an Eppendorf thermal shaker (Eppendorf, The Netherlands). Following overnight digestion with OgpA, Ni-NTA agarose slurry was added to the samples to bind the enzyme and incubated for 30 min. Finally, the flowthrough, containing the IgA1 Fabs, was collected by centrifugation.
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6 protocols using thermal shaker
1
IgA1 Fab Profiling Protocol
2
Selective Cleavage of IgA1 Fab Fragments
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For the hinge region digestion of IgA1 we relied on the O-glycopeptidase from Akkermansia muciniphila, OgpA; OpeRATOR®, Genovis, Llund, Sweden). The enzyme docks at O-glycans, by preference non-sialylated core 1 (thus GalNAc-Gal), and then cleaves the protein N-terminally of the glycan. As these O-glycans are unique for IgA1 (and not present in IgA2) exclusively the Fab molecules of IgA1 are cleaved off.
Based on previous analysis of these human milk samples, by MS-based proteomics and ELISA, the expected IgA1 concentrations in the human milk samples were assumed to be 0.2 - 1.4 mg/mL (17 (link)), and therefore contain at least 10 µg in 50 µL. Based on these concentrations we added to each spin column 50 µL PBS containing 40 U SialEXO (a sialidase cocktail to remove sialic acids from the O-glycans) and incubated for 1 h at 37°C with continuous shaking at 750 rpm. Then 1 µL (40 U) of OgpA enzyme was added, and incubation was continued overnight, in and Eppendorf thermal shaker (Eppendorf, The Netherlands). Following overnight digestion with OgpA, 20 µL of pre-washed Ni-NTA agarose slurry (1:1 in PBS) was added to the spin columns in order to capture the His-tagged enzymes. The incubation was continued for 30 more minutes. Then the plug was removed from the column, and the flowthrough, containing the IgA1 Fabs, was collected by centrifugation for 1 min at 500 × g, RT.
Based on previous analysis of these human milk samples, by MS-based proteomics and ELISA, the expected IgA1 concentrations in the human milk samples were assumed to be 0.2 - 1.4 mg/mL (17 (link)), and therefore contain at least 10 µg in 50 µL. Based on these concentrations we added to each spin column 50 µL PBS containing 40 U SialEXO (a sialidase cocktail to remove sialic acids from the O-glycans) and incubated for 1 h at 37°C with continuous shaking at 750 rpm. Then 1 µL (40 U) of OgpA enzyme was added, and incubation was continued overnight, in and Eppendorf thermal shaker (Eppendorf, The Netherlands). Following overnight digestion with OgpA, 20 µL of pre-washed Ni-NTA agarose slurry (1:1 in PBS) was added to the spin columns in order to capture the His-tagged enzymes. The incubation was continued for 30 more minutes. Then the plug was removed from the column, and the flowthrough, containing the IgA1 Fabs, was collected by centrifugation for 1 min at 500 × g, RT.
3
Affinity Capture of IgA from Milk
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All IgA was captured using CaptureSelect IgA affinity matrix (ThermoFisher Scientific). 40 µL bead slurry was added directly to Pierce spin columns with screw cap (ThermoFisher Scientific). The beads were then repeatedly washed with 150 µL PBS by centrifugation at 500 × g, room temperature (RT). After the third wash, a plug was inserted to the bottom of the individual spin columns and 100 µL PBS was added to the beads. Subsequently 50 µL of human milk and 1 µL IgA solution containing 200 ng of 7D8-mIgA1 were added. Samples were then incubated for 1h while shaking at 750 rpm at RT in and Eppendorf thermal shaker (Eppendorf, The Netherlands). Following the incubation, the plugs were removed from the spin columns, the human milk/PBS dilution was collected by centrifugation for 1 min at 500 × g, RT. Then the beads were washed four times by addition of 200 µL PBS and subsequent centrifugation for 1 min at 500 × g, RT. After the fourth wash the plugs were reinserted into the bottom of the spin columns.
4
Rapid DNA Extraction from Solid Tissues
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Example 3
Incubation time required for sample lysis and nucleic acid purification is decreased from 60 min to 5 min) and yields are increased by incubating ChargeSwitch™ beads at the recommended temperature during vigorous shaking. Samples were incubated at 55C on an Eppendorf thermal shaker at max rpm. All samples were incubated in Invitrogen ChargeSwitch™ Lysis Buffer (L13) buffer and purified using ChargeSwitch™ magnetic beads according to the manufacturer's protocol. These experiments discovered a method to increase the lysis step for complex solid tissues prepared using the ChargeSwitch™ method. The standard protocol yielded 10.2 ng/ul of DNA from 20 mg of tissue after a 1.5 hour incubation at 55 C. In contrast, thermomixing yielded a mean of 10.0 ng of DNA/ul from 20 mg of tissue after only 10 minutes. Additional thermomixing (e.g. 20 min) also yielded 10.8 ng DNA/ul, indicating that the system (e.g. number of beads) reached the maximum binding capacity. These experiments indicate that the maximum yield had been reached by 10 min and that the time could be further reduced.
5
Efficient Nucleic Acid Purification from Solid Tissues
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Example 3
Incubation time required for sample lysis and nucleic acid purification is decreased from 60 min to 5 min) and yields are increased by incubating ChargeSwitch™ beads at the recommended temperature during vigorous shaking. Samples were incubated at 55 C on an Eppendorf thermal shaker at max rpm. All samples were incubated in Invitrogen ChargeSwitch™ Lysis Buffer (L13) buffer and purified using ChargeSwitch™ magnetic beads according to the manufacturer's protocol. These experiments discovered a method to increase the lysis step for complex solid tissues prepared using the ChargeSwitch™ method. The standard protocol yielded 10.2 ng/ul of DNA from 20 mg of tissue after a 1.5 hour incubation at 55° C. In contrast, thermomixing yielded a mean of 10.0 ng of DNA/ul from 20 mg of tissue after only 10 minutes. Additional thermomixing (e.g. 20 min) also yielded 10.8 ng DNA/ul, indicating that the system (e.g. number of beads) reached the maximum binding capacity. These experiments indicate that the maximum yield had been reached by 10 min and that the time could be further reduced.
6
Stability of Gallium-67 Chelate in Plasma
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The stability of 67Ga–DFO–HPG in mouse plasma was determined over 8 days using ITLC. For this purpose 50 μL of 67Ga–DFO–HPG tracer in PBS was incubated with 250 μL of mouse plasma at 37 °C (Eppendorf thermal shaker, 650 rpm). Aliquots were taken at designated time points (1 h, 1 d, 4 d and 8 d) and measured by ITLC using 0.1 M EDTA (pH 7.4) as eluent. Radioactive intensities on the ITLC were integrated and compared (Rf (67Ga–DFO–HPG) = 0, Rf (67Ga–EDTA) = 1).
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