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Immunocapture 6

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ImmunoCapture 6.4 is a software application designed for the analysis and processing of data generated from immunoassay experiments. The software provides tools for data acquisition, analysis, and reporting.

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Lab products found in correlation

Streptavidin alkaline phosphatase, by Southern Biotech (2 mentions) Lectin pokeweed mitogen, by Merck Group (2 mentions) Anti iga biotin, by Mabtech (2 mentions) Cowan strain, by Merck Group (2 mentions) Nbt bcip, by Thermo Fisher Scientific (2 mentions) Immunospot s6 micro analyzer, by Cellular Technology (1 mentions) Complete freund s adjuvant cfa, by Merck Group (1 mentions) Mouse cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Immunospot s6 analyzer, by Cellular Technology (1 mentions) Freund s adjuvant, by Thermo Fisher Scientific (1 mentions) Aec substrate, by BD (1 mentions) Immunospot s6 micro analyzer elispot reader, by Cellular Technology (1 mentions) Multiscreen hts plate, by Merck Group (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Streptavidin hrp, by BD (1 mentions) Phytohemagglutinin (pha), by Merck Group (1 mentions) Elispot plate, by Merck Group (1 mentions) Elx50 microplate strip washer, by Agilent Technologies (1 mentions) Ctl immunospot s6 micro analyzer, by Cellular Technology (1 mentions) Anti mouse ifn γ ab, by BD (1 mentions)

8 protocols using immunocapture 6

1

Quantifying Antigen-Specific CD4+ T Cell Responses

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C57BL/6 mice were immunized with e1a2 peptide (GEGAFHGDAEALQRPVASDF, 2 mg/ml) or 2W1S peptide (EAWGALANWAVDSA, 0.2 mg/ml) emulsified in Complete Freund’s Adjuvant (CFA) (Sigma-Aldrich, St. Louis, MO) and harvested two weeks later. CD4+ T cells were enriched using a mouse CD4 T cell Isolation Kit (Miltenyi, Bergisch Gladbach Germany). Naïve mouse splenocytes were harvested, irradiated and mixed at a 1:1 ratio with the immunized CD4+ T cells in wells coated with IFN-gamma capture antibody (AN-18) at a concentration of 15μg/mL. Synthetic overlapping peptides that spanned the BCR-ABL p190 fusion (Genscript, Piscataway, NJ) were added to individual wells. 2W1S peptide and Concanavalin A were used as positive controls. Samples were developed using a biotinylated IFN-gamma detection antibody (R4-6A2) and streptavidin-Alkaline Phosphatase with 5-Bromo-4-chloro-3′-indolyphosphate (BCIP) and Nitro-blue tetrazolium (NBT) developing agents and photographed and analyzed with a ImmunoSpot S6 Microanalyzer using the ImmunoCapture 6.3 and ImmunoSpot 5.0 Pro DC software (Cellular Technology Ltd. (Shaker Heights, OH).
Manlove L.S., Berquam-Vrieze K.E., Pauken K.E., Williams R.T., Jenkins M.K, & Farrar M.A. (2015). Adaptive immunity to leukemia is inhibited by cross-reactive induced regulatory T cells. Journal of immunology (Baltimore, Md. : 1950), 195(8), 4028-4037.
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2

Pn3P-OVA T Cell Activation

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Groups of BALB/c mice were immunized with 10ug of Pn3P-OVA emulsified in Freund’s adjuvant (Thermo Scientific) by subcutaneous injection. CD4+ T cells were isolated from lymph nodes of Pn3P-OVA immunized mice and stimulated in vitro in the presence of irradiated APCs pulsed with indicated antigens. After 24hr stimulation, IL-2 production was detected by ELISPOT assay using mouse ELISPOT reagent set (Cellular Technology Ltd) according to the manufacturer’s protocol. The ELISPOT samples were analyzed by ImmunoSpot S6 analyzer using the ImmunoCapture 6.3 and ImmunoSpot 5.0 Pro DC software (Cellular Technology Ltd).
Middleton D.R., Sun L., Paschall A.V, & Avci F.Y. (2017). T Cell Mediated Humoral Immune Responses to Type 3 Capsular Polysaccharide of Streptococcus pneumoniae. Journal of immunology (Baltimore, Md. : 1950), 199(2), 598-603.
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3

ELISPOT Assay for Virus-Specific T Cell Responses

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Frozen PBMC were thawed in a water bath at 37°C, washed twice with thawing media (RPMI 1640 and 20% FBS), resuspended in culture media [RPMI 1640, 10% FBS, 1% antibiotic-antimycotic, and 55 μM 2-mercaptoethanol (Gibco)], and rested for 2 hours. Single cells isolated after lung digestion were not cryopreserved but used immediately. PBMC or lung cells were plated at 0.5 or 1 million live cells per well in 96 well MultiScreen HTS plates (Millipore, Billerica, MA) pre-coated with anti-IFN-γ (P2G10, BD Biosciences). The cells were then incubated at 37°C for 48 hours with 5 x 105 TCID50 of UV-inactivated TX98 or CA04 virus particles or virus-free UV-treated MDCK supernatant. Afterwards, the plates were developed using a biotin-conjugated anti-IFN-γ mAb (P2C11, BD Biosciences), streptavidin-HRP (BD Biosciences), and AEC substrate (BD Biosciences), according to manufacturer instructions. The number of spots in each well was read using an ImmunoSpot S6 Micro Analyzer ELISPOT reader with ImmunoCapture 6.4 software (Cellular Technology Ltd., Shaker Heights, OH). The data are presented as the number of spots per 106 PBMC or lung cells after subtracting the average number of spots in wells cultured with virus free MDCK supernatant.
Artiaga B.L., Morozov I., Ransburgh R., Kwon T., Balaraman V., Indran S.V., De Carvalho Madrid D.M., Gu W., Henningson J., Ma W., Richt J.A, & Driver J.P. (2022). Evaluating α-galactosylceramide as an adjuvant for live attenuated influenza vaccines in pigs. Animal Diseases, 2(1), 19.
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4

SARS-CoV-2 Spike Protein ELISpot Assay

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MBC stimulations were performed on peripheral blood mononuclear cells (PBMCs) collected from subjects in the convalescent cohort. To induce MBC differentiation into antibody secreting cells, 1×106 PBMCs were stimulated with 10 ng/ml Lectin Pokeweed Mitogen (Sigma-Aldrich), 1/100,000 Protein A from Staphylococcus aureus, Cowan Strain (Sigma-Aldrich), and 6 μg/ml CpG (Invitrogen) in complete RPMI in an incubator at 37°C/5% CO2 for 5 days. After stimulation, cells were counted and added to ELISpot white polysterene plates (Thermo Fisher) coated with 4 μg/ml of SARS-CoV-2 spike that were blocked with 200 μl of complete RPMI. ELISpot plates were incubated with cells for 16 hours overnight in an incubator at 37μC/5% CO2. After the overnight incubation, plates were washed and incubated with anti-IgG-biotin and/or anti-IgA-biotin (Mabtech) for 2 hours at room temperature. After secondary antibody incubation, plates were washed and incubated with streptavidin-alkaline phosphatase (Southern Biotech) for 2 hours at room temperature. Plates were washed and developed with NBT/BCIP (Thermo Fisher Scientific) for 2–10 minutes, and reactions were stopped by washing plates with distilled water and allowed to dry overnight before counting. Images were captured with Immunocapture 6.4 software (Cellular Technology Ltd.), and spots were manually counted.
Guthmiller J.J., Stovicek O., Wang J., Changrob S., Li L., Halfmann P., Zheng N.Y., Utset H., Stamper C.T., Dugan H.L., Miller W.D., Huang M., Dai Y.N., Nelson C.A., Hall P.D., Jansen M., Shanmugarajah K., Donington J.S., Krammer F., Fremont D.H., Joachimiak A., Kawaoka Y., Tesic V., Madariaga M.L, & Wilson P.C. (2020). SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike. bioRxiv.
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5

Quantifying SARS-CoV-2 Spike-Specific Antibodies

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MBC stimulations were performed on PBMCs collected from subjects in the convalescent cohort. To induce MBC differentiation into antibody secreting cells, 1x106 PBMCs were stimulated with 10 ng/ml Lectin Pokeweed Mitogen (Sigma-Aldrich), 1/100,000 Protein A from Staphylococcus aureus, Cowan Strain (Sigma-Aldrich), and 6 µg/ml CpG (Invitrogen) in complete RPMI in an incubator at 37µC/5% CO2 for 5 days. After stimulation, cells were counted and added to ELISpot white polystyrene plates (Thermo Fisher) coated with 4 µg/ml of SARS-CoV-2 spike that were blocked with 200 µl of complete RPMI. ELISpot plates were incubated with cells for 16 hours overnight in an incubator at 37°C/5% CO2. After the overnight incubation, plates were washed and incubated with anti-IgG-biotin and/or anti-IgA-biotin (Mabtech) for 2 hours at room temperature. After secondary antibody incubation, plates were washed and incubated with streptavidin-alkaline phosphatase (Southern Biotech) for 2 hours at room temperature. Plates were washed and developed with NBT/BCIP (Thermo Fisher Scientific) for 2–10 minutes, and reactions were stopped by washing plates with distilled water and allowed to dry overnight before counting. Images were captured with Immunocapture 6.4 software (Cellular Technology Ltd.), and spots were manually counted.
Stamper C.T., Dugan H.L., Li L., Asby N.W., Halfmann P.J., Guthmiller J.J., Zheng N.Y., Huang M., Stovicek O., Wang J., Madariaga M.L., Shanmugarajah K., Jansen M.O., Amanat F., Stewart I., Changrob S., Utset H.A., Huang J., Nelson C.A., Dai Y.N., Hall P.D., Jedrzejczak R.P., Joachimiak A., Krammer F., Fremont D.H., Kawaoka Y, & Wilson P.C. (2020). Distinct B cell subsets give rise to antigen-specific antibody responses against SARS-CoV-2. Research Square.
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6

Quantifying Antigen-Specific T Cell Responses

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Patient PBMC were thawed and resuspend at 3×106/ml in AIM-V® AlbuMAX® serum-free media supplemented with 1mM L-glutamine. The KTR responders were run in triplicate in media, test wells and Phytohemagglutinin (Sigma-Aldrich, St Louis, MO, USA) as a positive control. The MLR consisted of 3×105 thawed KTR PBMCs plated on an anti IFN-γ antibody (2G1, Thermo-Scientific, Woburn, MA, USA) coated ELISPOT plate (Millipore, Bellerica, MA, USA) co-incubated with 4 or 5 B cells lines at 5×104/per B cell line, 2 B cell lines per well (total of 1×105 B cells/well), or with the 5th B cell line at 1×105 B cells/well, for 24h. These B cell lines had either 17 or 21 disparate antigens, with the most similar cell lines paired. After which, the plates were handled as per previously published protocol23 (link), with half the plates washed utilising an ELx50 Microplate strip washer (BioTek, Winooski, VT, USA).
The images captured with Cellular Technology, Ltd (CTL)-ImmunoSpot® S6 Micro Analyzer (Cellular Technology, Ltd, Shaker Heights, OH, USA) with ImmunoCapture 6.4 software (Cellular Technology, Ltd). The spots counted using CTL ImmunoSpot 5.0 (Cellular Technology, Ltd) and quality controlled by inspecting every well and adjusting sensitivity. The IFN-γ ELISPOT plates were analysed at the Clinical Trials of Organ Transplantation (CTOT) center in Mount Sinai School of Medicine, New York.
Hope C.M., Fuss A., Hanf W., Jesudason S., Coates P.T., Heeger P.S, & Carroll R.P. (2015). Peripheral natural killer cell and allo-stimulated T-cell function in kidney transplant recipients associate with cancer risk and immunosuppression related complications. Kidney international, 88(6), 1374-1382.
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7

IFNγ ELISpot Assay for Mouse Splenocytes

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For ELISpot assay, flat-bottom 96-well plates were pre-coated with 10 g/ml anti-mouse IFNγ Ab (BD Biosciences, USA) overnight at 4°C and blocked for 2 h at RT. Fresh mouse splenocytes were added into the pre-coated 96-well plates (4 × 105 cells/well) and stimulated with each peptide pool (2 μg/ml for each peptide) for 20 h. Negative control wells were not stimulated with peptide pool. Phytohemagglutinin (PMA) was added to positive control wells. After stimulation, cells were removed from plates and the plates were probed with biotinylated IFNγ antibody, streptavidin-HRP conjugated antibody and 3-amino-9-ethylcarbazole (AEC) substrate. The development was stopped by thoroughly rinsing samples with deionized water when spots became visually observable. Finally, the numbers of spots were determined using CTL ImmunoSpot S6 Analyzer (CTL, USA) and image analysis software Immuno Capture 6.5.0 (Cellular Technology, USA).
Du P., Huang L., Fang Y., Zhao F., Li Q., Ma X., Li R., Chen Q., Shen H., Wang Q., Li H, & Gao G.F. (2024). Broad-spectrum Delta-BA.2 tandem-fused heterodimer mRNA vaccine delivered by lipopolyplex. PLOS Pathogens, 20(4), e1012116.
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8

SARS-CoV-2 BA.1 Neutralization Assay

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Serum sample from each rhesus macaque was heat inactivated (56°C, 30 min) and subjected to a three-fold serial dilution started at 1:50 using DMEM. Then, diluted sera were mixed with equal volume of SARS-CoV-2 BA.1 (1000 PFU/mL) and incubate for 1 h at 37°C. Then, the serum-virus mixtures were added on pre-plated Vero cells (2×105 cells/well) and incubated for 1 h at 37°C. After incubation, the inoculum was removed. The cells were washed once by PBS, added 1ml DMEM mixed with 0.8% Carboxymethylcellulose and cultured for 96 h. After washing by PBS, plates were fixed with 8% Paraformaldehyde (Solarbio Life science) for 1 h and stained with 0.05% crystal violet overnight. Plaques were captured and calculated by CTL ImmunoSpot S6 Analyzer (CTL, USA) and image analysis software Immuno Capture 6.5.0 (Cellular Technology, USA). The half maximal inhibitory concentration (IC50) was calculated based on plaque numbers by GraphPad Prism 9.0 (GraphPad Software Inc., USA).
Du P., Huang L., Fang Y., Zhao F., Li Q., Ma X., Li R., Chen Q., Shen H., Wang Q., Li H, & Gao G.F. (2024). Broad-spectrum Delta-BA.2 tandem-fused heterodimer mRNA vaccine delivered by lipopolyplex. PLOS Pathogens, 20(4), e1012116.
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