The aim of the following experiment is to determine the phagocytic activity of antigen specific antibodies in clinical samples at high throughput. This is achieved by first coating fluorescent beads with an antigen of choice to allow capture of the antigen-specific fraction of antibody present in complex clinical antibody samples. As a second step phagocytic, TP one cells are incubated with the ionized fluorescent beads, which then phagocytose the beads as driven by the number and FC domain characteristics of the antibodies bound.
Next, the fluorescence intensity of the THP one cells is determined by high throughput plate based flow cytometry in order to quantify the phagocytic activity of the antibodies present in the sample. Ultimately, the ability of antigen specific antibodies in the sample to induce phagocytosis based on differential uptake of the opsonize fluorescent beads due to either variable antibody titer or variable recognition by the antibody receptors expressed on the phagocytic cells can be evaluated. The main advantages of this technique over existing methods such as those using primary phagocytic cells is that throughput is increased and standardization is improved.
This method can help answer key questions about the basic immunology of the adaptive immune response, such as whether different vaccine regimens, induced antibodies with different activities. The implications of this technique extend toward therapy of infectious disease because antibodies make a major contribution to mounting an effective protective immune response. Though we have used this method to provide insight into the activity of antibodies in progressive HIV infection and vaccination, it can also be applied to other disease states such as influenza, autoimmunity, and dengue, where frago antibodies can exacerbate D disease rather than provide protection.
Begin by diluting the antigen in a buffer that does not contain primary amines. Then dissolve the sulfa NHS lc, biotin reagent in water. Next, immediately add the hydrated reagent to the antigen and allow the reaction to proceed for one hour at room temperature while mixing.
Occasionally when the reaction is complete, add the sample to the top chamber of a centrifugal concentration unit to remove any excess unconjugated biotin by buffer exchange. Now add PBS to bring the total volume up to the 15 milliliter fill line. Finally, centrifuge the sample at 4, 000 times G for five minutes at four degrees Celsius until the volume is brought down to approximately 1.5 milliliters.
Repeating this step three times to achieve near quantitative removal of free biotin. After spinning down 100 microliters of one micron fluorescent eut tradin beads in a micro centrifuge at 13, 000 times G for five minutes, resuspend and wash the beads in one milliliter of 0.1%P-B-S-B-S-A following the same centrifugation procedure. Then after Resus suspending the washed beads in 100 microliters of P-B-S-B-S-A, again eloqua the beads into 10 micro fued tubes.
Next, combine 10 microliters of the washed bead suspension with varying concentrations of biotinylated antigen into fold steps. Then incubate the bead and antigen suspensions overnight at four degrees Celsius in a micro centrifuge tube on a rotator. The next day.
Remove any unbound antigen by washing the suspensions twice with one milliliter of P-B-S-B-S-A and centrifuge the samples at high speed as before until the beads are pelleted. Finally, Reese suspend the antigen coated beads in a final volume of one milliliter of P-B-S-B-S-A after purifying the clinical antibody samples from plasma using a melon gel IgG purification kit. According to the manufacturer's instructions, determine the concentration of the purified antibodies by absorbance at a two 80.
Then dilute the antibodies to one milligram per milliliter in PBS, setting aside samples for the positive and negative monoclonal control antibodies as well. Next, resus, suspend the antigen saturated bead solution by vortexing and then transfer 10 microliters of the solution into each well of a round bottom 96 well plate take care to continually agitate the bead suspension to ensure that equal numbers of the fluorescent beads are added to each of the wells. Create a dose response curve for each control antibody by adding varying concentrations of the antibodies of interest in volumes no larger than 20 microliters to each.
Well now incubate the bead and antibody samples for two hours at 37 degrees Celsius to allow the antibodies to bind to the beads right before the beads are finished. Incubating dilute a sample of THP one cells at 2.5 times 10 to the five cells per milliliter in media. Then add 200 microliters of the cells to each.
Well finally incubate the cells with the antibody coated beads overnight at 37 degrees Celsius and 5%carbon dioxide in a stationary incubator, allowing the cells and beads to settle via gravity. The next day, remove 100 microliters of supinate from each well being careful not to disturb the cell pellet. Fix the cells in 100 of 4%para formaldehyde, and then pipette to resuspend and mix the cells.
Now load the plate onto the HTS plate reader of a high throughput flow cytometer, allowing automated data collection. The fluorescence intensity of HTHP one cell is captured and recorded. This fluorescence intensity is then used to determine the number of beads phagocytose by each cell, which defines the phagocytic activity of the antibody sample being tested.
There should be a clear differentiation of antibody samples from affected and unaffected subjects. In this figure. The facts histogram of an antibody sample from an HIV negative subject represented by the Black Trace and an HIV positive subject represented by the gray trace, demonstrate the increased phagocytosis driven by the presence of antigen-specific antibodies.
The optimal sensitivity of this assay is dependent on saturation of the beads with biotinylated antigen as seen here. Two micrograms antigen per microliter of beads was determined to be the saturating concentration for this antigen. This dose response curve demonstrates the differential capacity of subject antibody samples to induce phagocytosis over an antibody concentration range of 0.05 to five micrograms per milliliter.
This differential phagocytosis may be driven by either differences in titer or in FC domain properties such as the IgG subclass or the glycosylation state. When TP one cells are imaged by fluorescent microscopy, there is clear evidence of bead phagocytosis. This still image of TP one cells after incubation with antibody opsonize beads in green and non-op inized beads in red demonstrates the lack of phagocytic uptake in the absence of antibody as only the green beads were cyto by the TP one cells.
When time-lapse microscopy is performed, the antibody specific phagocytic uptake of fluorescent beads is even more striking. The green fluorescent beads seen here are antibody ionized. The red fluorescent beads provide the negative control Following this procedure, other tests of antibody function can be performed in order to answer additional questions about the characteristics of the antibody response to infection or vaccination.
After watching this video, you should have a good understanding of how to determine the phagocytic activity of antibody samples using eye throughput flow cytometry. Don't forget that working with clinical blood and plasma samples can be extremely hazardous and that precautions such as wearing proper personal protection and following safe handling procedures should always be taken while performing this procedure.
