One half of a milliliter of freshly separated plasma was mixed CEMX174 cells, about 1-2x106 and incubated on ice for 2 hours for virus isolation from plasma (cell free virus). After incubation, 10 ml of complete media (RPMI 1640) was added and centrifuged at 1000 rpm for 10 minutes to pellet the cells. The supernatants were discarded and cells resuspended at a concentration of 1 x 106 cells/ml and cultured in complete media (RPMI 1640). Development of CPE was monitored for upto 28 days before, cultures were terminated and supernatants taken for p27 antigen ELISA.
3.14 Antigen Capture ELISA Technique
To test for viral antigen in tissue culture fluid, a SIV core (p27) antigen assay commercial kit (Advanced Bioscience Laboratories, Inc, 5510 Nicholson Lane, Kensington, MD 20896-1078) was used. This kit utilizes an anti-SIVmac core antigen p27 monoclonal antibody. The kit was removed from the cold room at 4oC and allowed to warm at room temperature before use. Twenty five microlitres of disruption buffer (provided with kit) was added into each antibody- coated well except the blank well. Then, 100 µl of each diluted SIV p27 standard was added in duplicate into microelisa wells containing disruption buffer. To serve as negative control, a 100 µl of complete tissue culture media (+10% FBS) was added to 4 wells containing disruption buffer. Then, 100 µl of the prepared test samples were added into microelisa wells containing disruption buffer. The microelisa plate was covered with sealer and then incubated at 37oC ± 0.5
o
After the incubation, the microelisa was washed by filling the wells with 300 µl of the diluted wash buffer and soaked for 15 seconds, and then aspirated. The wash procedure was repeated for a total of four washes. After the last aspiration, the microelisa plate was tapped firmly on absorbent paper (paper towel). Then, 100 µl of conjugate solution was added to each well. The microelisa plate was covered with plate sealer and incubated at 37oC ± 0.5 oC for 60 ± 2 minutes. Then the microelisa plate was washed as previously described. 100 µl of prepared peroxidase substrate was added to each well and then incubated uncovered for 30 ± 1 minutes at room temperature (19-23 oC). In the same order that the peroxidase substrate was added, 100 µl of stop solution was added and absorbance read at 450 nm in a microelisa plate reader within 20 minutes. A reaction was considered positive if the OD value was above a cut-off value of 0.6.
3.15 Analysis of antibody responses in ELISA
Genscreen HIV-1/2 version 2 commercial kit (Bio-Rad, 3, bd Raymond Poincaré, 92430 marnes-la-coquette-France) was utilized for this assay. Genscreen HIV-1/2 version 2 is an enzyme immunoassay based on the principle of the two-step sandwich technique for the detection of the various antibodies associated with HIV-1 and/or HIV-2 virus in human serum or plasma. The kit is based on the use of a solid phase method whereby the immunoplates are coated with purified recombinant proteins derived from HIV-1 (envelope glycoproteins, gp160, and nucleocapsid, p25) and HIV-2 (a peptide mimicking the immunodominant epitope of the envelope glycoprotein). Thus, the antibodies to envelope and nucleocapsid of HIV-1 and those to envelope of HIV-2, if any, would bind to the antigens immobilized on the wells of the immunoplate.
Briefly, the serum samples and control sera to be assayed were diluted 1:4 with diluent buffer provided with the kit, and 100 µL pipetted into microplate wells. The wells were washed, and then incubated with peroxidase labelled goat anti-human IgG and IgM. A detailed procedure was provided together with the kit. The kit was removed from the cold room at 4oC and allowed to warm at room temperature 15 minutes before use. The plasma samples to be assayed and control sera (provide with the kit) were pipetted into the microplate well (100µl/well) in a dilution of 3:4.
Samples and controls were diluted in sample diluent provided with the kit. The plate was sealed, incubated at 37oC for 30 minutes and thereafter washed three times. The plates were incubated at 37oC for 30 minutes with conjugate after seal and washed after the incubation as described above.
The plates were incubated in the dark for 30 minutes (without sealing) at room temperature with peroxidase substrate and tetramethylbenzidine (TMB) as chromogen. The reaction was stopped by adding 100µl/well of 1N sulphuric acid solution. In addition, absorbance was read at 450nm with a reference wavelength at 620-700nm using plate reader. Samples with absorbance values equal or greater than the cut-off value were considered positive. The cut-off value was defined as one tenth of the mean absorbance of the cut-off control serum provided.
3.16 Analysis of antibody responses in Western blot
To detect and confirm anti-SHIV antibodies in SHIV- infected animal, a New Lav –Blot I (Bio- Rad 3, bd Raymond Poincaré, 92430 marnes-la-coquette-France) HIV-1 western Blot assay was performed according to the manufacturer’s instructions. The test is based on the principle of indirect ELISA technique on a nitrocellulose strips containing all the HIV-1 constituent proteins and an internal anti-IgG control. The nitrocellulose strips, alkaline phosphatase-labelled anti- human IgG antibody, colour development reagents and an internal anti –IgG control were provided in the kit. These strips were rehydrated and incubated with sample to be confirmed or control serum.
The kit was removed from the cold room at 4oC and allowed to warm at room temperature 30 minutes before use to allow regents to stabilizes at room temperature (18 -30 oC). Two milliliters of the reconstituted buffer solution /diluent was added into each cell with the strips and incubated at room temperature for 5 ± 1 minutes (18 - 30 oC) under shaking. 20 µl of each sample or control serum were added into corresponding cell. This was followed by incubation for 2 hours ± 5 minutes at room temperature (18 - 30 oC) under shaking. The strips were dried and rinsed under tap aspiration carefully to avoid sample cross-contamination.
The strips were washed with 2 ml of the reconstituted buffer solution/diluent. Each strip was washed twice, allowing the contact for 5 minutes, under shaking, 2 ml of the reconstituted buffer solution/diluent (i.e. a total 3 wash steps). Two (2 ml) of the conjugate was dispensed into each
cell; the conjugate solution was previously stabilized at room temperature. The strips were incubated for 1 hour ± 5 minutes at room temperature (18 -30 oC) under shaking. Washing was repeated and 2 microlitres of colour development solution was dispensed into each cell.
This was followed by incubation at room temperature for 5 minutes under shaking and monitoring the appearance of the coloration. The reaction was stopped by removing the colour development solution and strips rinsed 3 times in distilled water. The strips were dried between 2 sheet of absorbent paper at room temperature (18 -30 oC). The appearance of specific coloured bands indicated the presence of anti-HIV-1 or cross-reactive antibodies in the serum to be detected. The bands that appeared in the positive control serum were classified as env (gp 160, gp 110/120, gp 41) gag (p 55, p 40, p 24/25, p 18/17) pol (p 68/66, p 52/51, p 34/31).
NB Accounting to the kit instruction, a test sample is define as positive if it developed at least 2 env ± gag ± pol in all the three regions (env, gag& pol), this interpretation by WHO criteria and if there was 1env+ (gag or pol) according to Consortium for Retrovirus Serology Standardization (CRSS) and indeterminate where there was 1env ± gag ± pol or gag+ pol, gag and pol (WHO) and when gag+ pol, gag, pol and env by CRSS. According to the manufacturer’s instructions, a test sample is defined positive if it develops at least one band in the three regions (i.e. env, gag & pol). A sample was defined as negative if no band develops or if the bands appearance could not be classified.
3.17 Cell homogenization for RNA preparation
Cell sample was isolated by gentle centrifugation and removal of supernatant. The pellet was resupended in 400ul of RNA lysis buffer. The sample mixture was centrifuged at ≥12,000xg for 1 minute. The lysate was transferred to a Zymo-spin™ lllc column in a collection tube and centrifuged at 8,00xg for 30seconds Save the flow through.0.8 volume of ethanol (95-100%) was added to the flow – through in the collection tube and mixed well (e.g.,320 µl ethanol added to 400 µl flow- through).
The mixture was transferred to a Zymo-spin™ llc in a collection tube and centrifuged at ≥12,000xg for 1 minute without saving the flow through. 400 µl of RNA prep buffer was added to the column and centrifuged at ≥12,000xg for 1 minute. The flow–through was discarded and the Zymo-spin™ llc column was replaced back into the collection tube. 800 µl of RNA wash buffer was added to the column and centrifuged at ≥12,000xg for 30 seconds. The flow-through was discarded and Zymo-spin™ llc column placed back into the collection tube.
The wash step was repeated with 400µl RNA wash buffer. Zymo-spin llc column was centrifuged at ≥12,000xg for 2 minute in the empted collection tube to ensure complete removal of wash buffer. Zymo-spin llc column was placed into an RNase-free tube and added ≥25µl DNase/RNase- free water directly to the column matrix and allowed to stand at room temperature for 1minute. To elute the RNA from the column Zymo-spin llc column was centrifuged at 10,000xg for 30 second.RNA was stored at ≤-70oc.