Immuno-PCR Imperacer
Imperacer® combines the ELISA-based method with the qPCR technique to amplify the artificial DNA, conjugated to the detecting molecule75,76,77,78. Below the Imperacer method developed for MACIVIVA, some additional information and instructions can be obtained at Chimera, following a service request. DNA-labeled P1 and DNA-labeled rgp41 were used in bridging assays for quantification of specific IgG and IgA antibodies and DNA-labeled IgG anti-IgG or anti-IgA for quantification of total IgG and IgA antibodies in sandwich Imperacer assay. This method is very sensitive, specific, and species-independent, as it can detect a broad range of antibody concentrations of any isotype and from any animal origin.
Assay volume for both specific and total antibody detection was 30?µL/well done in duplicate. The following amount of a serum sample for a given time point that was required for preparing the dilutions for the assay: 11?µL for detecting specific anti-P1, 3?µL for detecting specific anti-gp41, <1?µL for detecting total IgG and IgA, respectively. Unlabeled P1 (1?µg/mL) or rgp41 (0.5?µg/mL) provided by Mymetics SA were diluted in coating buffer (Chimera Biotec, catalogue no. C-010) and coated on Imperacer® microplate modules (Chimera Biotec, catalogue no. C-001) for at least 16?h at 4?°C. The coated microplates were washed automatically (HydroFlex, Tecan) three times with buffer A (no detergent) at pH 7.35 (Chimera Biotec, catalogue no. C-011), blocked for preventing unspecific interaction (Chimera direct block, Chimera Biotec, catalogue no. C-013), followed by three washing steps with buffer B (with detergent) at pH 7.35 (Chimera Biotec, catalogue no. C-012), and subsequent incubation with pre-immune or immune samples. For P1-specific antibody detection, one volume of serum sample (11?µL) was mixed with 5 volumes (55?µL) of DNA-labeled P1 (conjugate “CHI P1”, Chimera Biotec, catalogue no. 11-313, diluted 1:300 in SDB5MAC, the sample dilution buffer, Chimera Biotec, catalogue no. C-093) for obtaining about 66?µL volume, which is sufficient for 2?×?30?µL per well for duplicate analysis. For rgp41-specific antibody detection, samples were first prediluted 1:12 (3?µL serum?+?33?µL with PBS-Tween 20 0.05%, pH 7.33) and subsequently mixed 1:2 (33?µL?+?33?µL) with a DNA-labeled gp41 (conjugate “CHI GP41”, Chimera Biotec, catalogue no. 11–292) diluted 1:300 in SDB6000 buffer (“Sample Dilution Buffer” Chimera Biotec, catalogue no. C-017), respectively. Following an incubation period of at least 16?h at 4?°C (for anti-P1) or 45?min at room temperature (for anti-gp41), plates were washed three times with buffer B, followed by two final washing steps with buffer A. PCR-Mastermix (Chimera Biotec, catalogue no. C-022: including DNA-label specific primers and real-time PCR probe; DNA label and primer sequence property of Chimera Biotec) were finally added to each well and the sealed plate was placed in a real-time PCR instrument (Chimera Biotec, catalogue no. 25-002) for signal generation. A bound antibody in the bridging assay connects its first single Fab part to the coated unlabeled antigen (“capture”), while another Fab part binds DNA-labeled P1 or rgp41 antigen acting as a “detector”. The DNA which is thereby immobilized due to the presence of specific antibodies was amplified during real-time PCR (50 cycles; each cycle?=?12?s, 95?°C; 30?s, 50?°C; 30?s, 72?°C). As reference material, human anti-P1 2F5 mAb (Polyimmun Scientific, catalogue no. AB001) from 218.7 to 0.1?ng/mL, and human anti-gp41 mAb 5F3 (Polyimmun Scientific, catalogue no. AB010) from 1028 to 0.01?ng/mL were utilized for the preparation of a standard curve.
For IgG and IgA total antibody quantification, capture antibodies at 2?µg/mL in coating buffer (Chimera Biotec, catalogue no. C-010) were immobilized on the plate (goat anti-monkey IgG, Alpha Diagnostics, catalogue no. 70023; Goat anti-monkey IgA, KPL, catalogue no. 071-11-011). After at least 16?h coating at 4?°C, the microplate was washed (buffer A), blocked, and washed with buffer B as described above. The coated wells were subsequently incubated for 45?min at room temperature with diluted pre-immune or immune samples. Samples were diluted with PBS-Tween 20 0.05% at pH 7.33. Dilution was 1:30,000 for IgG detection and 1:300,000 for IgA detection, respectively. Following another three times washing step with buffer, antibody DNA detection conjugates were added to each well. For IgG detection, samples were incubated with DNA-labeled anti-IgG (CHI monkey IgG, Chimera Biotec, catalogue no. 11-324) diluted 1:300 in “Conjugate Dilution Buffer” (CDB, Chimera Biotec, catalogue no. C-020). For IgA detection, DNA-labeled anti-IgA was applied (CHI monkey IgA, Chimera Biotec, catalogue no. 11-323), also diluted 1:300 in CDB. Incubation was carried out for 45?min at room temperature. After a final washing step (three times with buffer B, followed by two times with buffer A, as above), PCR-Mastermix was added and PCR was carried out as described above. Real-time PCR signals were converted to approximate antibody concentrations (ng/mL) by analysis against a reference antibody curve. These antibody concentrations were provided only as indicative values.
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