The overall goal of the following experiment is to interrogate RIC HIV one envelope glycoproteins or ENV confirmation with monoclonal antibodies. This is achieved by first transecting adherence cells to express chimeric HIV one E NV proteins at the cell surface. As a second step, the cells are incubated with anti ENV monoclonal antibodies, which will bind ENV depending on epitope exposure.
Next, an HRP conjugated secondary antibody is added in order to quantify antibody interaction by a chemiluminescence assay. Results are obtained that show relative levels of VNV bound antibodies based on the relative light units acquired for each. Well, The main advantage of this technique over existing methods, such as facts-based tying, is that this technique can be done with low amount of antibodies and at high throughput, a postdoc in the lab will be performing this technique.
Human osteosarcoma cells are used in this experiment one day prior to transfection plate, two times 10 to the fourth cells per well. In an opaque 96 well cell culture plate suitable for luminescence reading. Used delcos modified eagle medium supplemented with fetal bovine serum and penicillin streptomycin incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide on the following day.
Prepare the transfection mixture. First, set out two tubes, two bay and two B to two bay. Add five microliters of DMEM supplemented with 25 millimolar hippies, followed by 10 nanograms of tat and coating plasmid, and 150 nanograms of chimeric HIV one envelope glycoprotein encoding plasmid.
Note that the TAT encoding plasmid is only required when using tat dependent HIV one envelope glycoprotein encoding plasmid to two B at five microliters of DMM and 450 nanograms of polyethaline or PEI. The quantities of the reagents and DNA should be adjusted according to the number of wells that are to be transfected with the same HIV one envelope glycoprotein. Next, add the contents of two B to two bay and mix thoroughly by vortexing for 10 seconds.
Incubate the transfection mix for 10 minutes at room temperature. After 10 minutes, add 10 microliters of the transfection mix per well of the 96 well plate incubate for 48 hours at 37 degrees Celsius and 5%carbon oxide. The Ali SA to study the recognition of chimeric HIV one envelope glycoprotein by monoclonal antibodies is performed two days after the transfection of the human osteosarcoma cells.
Prepare 250 milliliters of washing buffer per plate being used at the same time. Prepare 125 milliliters of blocking buffer per plate by adding 1%non-fat dry milk and five millimolar of tris pH 8.0 to the washing buffer. Remove the cell culture media and transfection mix from the 96 well plate.
Add 100 microliters of blocking buffer per well and incubate for 20 minutes. At room temperature, remove the supernatant and add 50 microliters of antibody per well diluted to the appropriate concentration in blocking buffer. Incubate for one hour at room temperature.
Wash three times with 100 microliters of blocking buffer and then repeat the washing process three times. Wash three times with 100 microliters of blocking buffer and then wash three times with 100 microliters of washing buffer after the last wash. Remove the sate.
Add 100 microliters of blocking buffer and incubate for five minutes. At room temperature, remove the supernatant and add 50 microliters of secondary antibody diluted one to 3000 in blocking buffer. Incubate for 40 minutes at room temperature after 40 minutes.
Wash three times with 100 microliters of blocking buffer, followed by three washes with 100 microliters of washing buffer. To prepare the samples for data acquisition, remove the supernatant from the 96 well plate and add 30 microliters of one x enhanced chemiluminescence substrate per well. Acquire chemiluminescence signal for one second per well on a suitable plate reader.
According to the manufacturer's instructions, the impact of soluble CD four or S CD four on the exposure of CD four I epitopes on envelope glycoproteins or ENV was assay interaction of CD four with ENV induces ENV confirmational changes that expose 17 B and 48 D epitopes that overlap the co-receptor binding site, but does not affect the outer domain recognizing antibody two G 12. To assess the impact of point mutations in the NV confirmation, two ENV mutants were used H 66 A, which has a decreased propensity to spontaneously sample the CD four bound confirmation and S3 75 W, which predisposes the NV to the CD four bound state. Normalizing the raw data according to expression levels, reveals that H 66 A diminishes CD four I 17 B recognition, whereas S3 75 W enhances the 17 B signal and is sufficient to restore the phenotype of the H 66.
A mutant assaying cells cot transfected with increasing amounts of a CD four expressor. Together with the NV blue bars revealed increasing signals for CD four I monoclonal antibodies, A three, two, and C 11, which recognized discontinuous epitopes in the inner domain of the ENV glycoprotein. GP one 20 ENV recognition by the two G 12 antibody was not affected.
Lastly, the raw data was normalized to two G 12, the absence of a 32 and C 11 modulation when ENV was cot transfected with a CD four mutant with decreased ability to interact with ENV indicates that the increased signals depended on E NV CD four interaction Once mastered, this technique can be done in four hours.
