The overall goal of the following experiment is to convert a pre optimized Eliza assay for TNF Alpha cytokine to the Xap platform while evaluating alternative antibody pairs. This is achieved by coupling the capture antibody from the ELIZA kit, along with three other candidate capture antibodies to four different microsphere or bead sets when mixed together. These four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair.
Next two Xap assays are constructed with the two most optimal antibody pairs. The performance of the assays is then compared to that of the original EISA assay in regards to signal strength, dynamic range and sensitivity. The results show that similar antibodies can perform very differently under the same conditions and that several antibodies can be screened simultaneously using the Luminex Xap assay revealing that the assay has increased sensitivity and dynamic range when compared to EISA.
The main advantage of this technique over existing methods like EIS A is that xap based immunoassays allow the researcher to test multiple targets, or as in this case, test multiple antibodies simultaneously. To identify the best antibody demonstrated. The procedure will be Erica Lopez, an associate scientist at Luminex Corporation.
Begin this protocol with selection and preparation of four capture antibodies and four detection antibodies specific for human TNF alpha as described in the written protocol. Also, prepare one confirmation antibody specific for the host species of the capture antibodies. Bring the reagents in the antibody coupling kit to room temperature.
Label four reaction tubes with the bead region numbers selected for the coupling reaction. Transfer the contents of the four vials of mag plex microspheres into the four labeled reaction tubes. Wash each of the bead sets twice in 500 microliters of activation buffer and activate each bead set with the sulf ONHS and EDC solutions as described in the antibody coupling kit.
User manual following a 20 minute incubation on a rotator, repeat the previous wash step with the now activated beads a total of three times with 500 microliters of activation buffer. For each wash, prepare four separate 500 microliter solutions. Each containing 7.5 micrograms of capture antibody inactivation buffer.
Add these four capture antibody solutions to their respective reaction tubes. Vortex each tube immediately and incubate for two hours on a rotator. Repeat the wash step with the now couple beads a total of three times with 500 microliters of the wash buffer included with the antibody coupling kit.
After the final wash step, add 500 microliters of wash buffer to each reaction tube to provide a final stock concentration of 5 million antibody coupled beads per milliliter vortex, and then sonicate the reaction tubes to disperse the beads. Count the number of beads recovered after the coupling reaction using a cell counter or hemo cytometer. The recovery from the coupling reaction is typically over 90%Confirm the coupling reaction was successful by preparing test solutions of the coupled bead stocks for each set with a final concentration of 100 beads per microliter in assay buffer, prepare dilution of the FICO erything labeled antis species.
IgG confirmation antibody at four micrograms per milliliter in assay buffer aliquot 50 microliters of each test solution into four wells of a round bottom 96 well plate for a total of 16 wells. Then add 50 microliters of assay buffer into eight of the wells to measure background and 50 microliters of diluted confirmation antibody into the eight remaining wells. Mix the reactions gently by pipetting up and down several times with a multi-channel pipetter.
Cover the plate and incubate for 30 minutes At room temperature on a plate shaker. Place the plate on a magnetic plate separator for one to two minutes to draw the beads out of solution. Then remove the liquid by forcefully inverting the plate while on the separator over a waste receptacle.
Wash each well twice by adding 100 microliters of assay buffer and removing the SANE from the plate in a similar fashion. Using the magnetic plate separator resuspend the beads in 100 microliters of assay buffer by gently pipetting up and down five times with a multi-channel pipetter. Analyze on luminex xap instrument such as the magix instrument.
The intensity of the fluorescent signal of this reaction is directly proportional to the amount of protein on the surface of the beads, providing a rapid assessment of the relative amount of protein coupled to the beads. To determine the most effective antibody pair in the xap assay, prepare an initial mixture of all four bead sets by adding 10 microliters of each to 0.96 milliliters of assay buffer. Prepare the detection antibody solutions by diluting each antibody to one microgram per milliliter in assay buffer.
Also an assay buffer. Prepare the r and d systems TNF alpha protein standard at 2000 picograms per milliliter and dilute the Streptavidin R FICO eryn to eight micrograms per milliliter. Add 50 microliters of the antibody coupled bead mixture to each of 16 wells of a CoStar round bottom 96 well plate for the screening assay.
Then add 50 microliters of assay buffer to eight of the 16 wells to measure background. Next pipette 50 microliters of the TNF alpha standard to the other eight wells to measure the response. Incubate for one hour at room temperature protected from light while shaking on an assay plate shaker.
Then add 50 microliters of each of the four detection antibodies to four wells, two of which are background wells, and two of which are response wells. Incubate for 30 minutes under the same conditions following the addition of 50 microliters of the SAPE reagent to all wells. Incubate again for 15 minutes under the same conditions.
Place the plate on a magnetic plate separator for one minute. Then remove the liquid by forcefully inverting the plate while on the separator over a waste receptacle. Pipette 100 microliters of assay buffer to each of the 16 wells.
Then remove the liquid from the beads using magnetic separation. Next, add 100 microliters of assay buffer to each of the 16 wells. Read the plate with the Luminex mag picks instrument referring to the user's manual for proper operation.
Select an antibody pair that meets the desired signal strength. After selecting the best to capture antibody, dilute 100 microliters of that antibody coupled beat stock to 10 milliliters with assay buffer. Add 50 microliters of the diluted beads to 78 wells of two CoStar round bottom 96 well plates.
Each plate will be used to assess the performance of a different detection antibody. Next, prepare a 12 point standard curve beginning at 8, 000 picograms per milliliter and ending at four picograms per milliliter. With the r and d systems.
TNF Alpha standard add six 50 microliter replicates of each dilution to each of the plates, plus six wells with 50 microliters of assay buffer each as background for a total of 78 wells per plate. Incubate the plates for one hour at room temperature protected from light while shaking on an assay plate shaker. Then add 50 microliters of the first detection antibody to all 78 wells of the first plate.
Repeat for the second detection antibody on the second plate. Incubate the plates for 30 minutes under the same conditions. Then add the 50 microliters of the SAPE reagent to all wells of each plate.
After shaking the two plates for 15 minutes protected from light, place the plates on magnetic plate separators for one minute. Remove the liquid by forcefully inverting the plate while on the separator over a waste receptacle. Next, pipette 100 microliters of assay buffer to each of the 78 wells on the plates before removing the liquid from the magnetic beads is before.
After adding hundred microliters of assay buffer to the beads in each of the 78 wells on the plates, analyze the plates on the mag picks instrument referring to the user's manual for proper operation. Following the instructions included with the r and d systems, human TNF Alpha T NF SF one a DUO set ELI A kit. Measure the response generated by the standard provided with the r and d kit.
Repeat the ELI a assay three more times substituting the r and d systems capture and detection antibodies with the antibodies of the other vendors. For simplicity, pair the antibodies by vendor, evaluate each pair independently as required by the EISA format with each of the three TNF Alpha protein standards. Luminex Xap assays were performed as a multiplex to evaluate all four capture antibodies as a mixture by combining four sets of TNF alpha antibody coupled to Mag PLX microspheres, the capture antibodies were evaluated with each of the four biotinylated detection antibodies individually, such that the interaction of one detection antibody with each of the four capture antibodies could be determined simultaneously.
The results indicated that the antibody pair from the r and d systems DUO set performed best with a resulting response at 6, 183 median fluorescence intensity or MFI units. It was also observed that the detection antibodies from Millipore and ABAM provided a reasonable response in the XAP assay when combined with the RD systems capture antibody, the capture antibodies from Millipore, ACAM and Novus produced a less desirable response in the XAP assay. The r and d systems DUO set protocol was used to compare the four antibody pairs in an EISA format.
The r and d systems protocol was used with all antibody pairs because it is reflective of typical EIA protocols widely used today, and it is analogous to protocols used with xap technology. The EISA tests showed that the antibody pair from r and d systems again gave the best results. The antibody pair from Acam produced no response, and the antibody pairs from Millipore and Novas produced modest responses in order to assess any variation in antibody reactivity.
With the standard, all four antibody pairs were tested with three different recombinant TNF alpha protein standards from three different vendors. The data show that the recombinant TNF alpha protein standards from the three vendors gave equivalent results. The TNF alpha protein from r and d systems was used to generate standard curves with the ELSA and the XAP assays.
While the ELI A assay was done with the r and d system antibody pair, the xap assays utilized the r and d system's capture antibody and either the detection antibody from r and d systems or Millipore. The TNF alpha protein from r and d systems was diluted to produce a range of concentrations from 8, 000 to four picograms per milliliter only. The antibody pair from r and d systems produced the expected outcome in the Xap assay with a response greater than 20, 000 MFI as shown.
When the detection antibody from Millipore was used with the Xap assay in place of the r and d systems detection antibody, the response was about 30%of the response obtained with the detection antibody from RD systems. The green line on this chart represents the standard curve of the ELA, which had a manufacturer's recommended TNF alpha range of 16 to 1000 picograms per milliliter. Due to the limit of the spectrophotometer, it was not possible to increase the range of the Eliza assay.
Further, the dynamic range and sensitivity of both methods is better illustrated when plotted in a log scale. A clear distinction between the slope of the EISA response and the responses from the xap assays can be seen and further indicates a more limiting capability for detection of TNF alpha with the EL iza at both higher and lower concentrations. The limits of detection or LOD for the two functional TNF alpha xap assays were approximated by identifying the lowest TNF alpha concentration with an observed response level greater than background, plus three times its standard deviation or SD from six replicates.
Here it can be observed that when using the r and d systems pair, the lowest TNF alpha concentration at 3.91 picograms per milliliter produced a response of 66 MFI, which is greater than the response for the background plus three SD meeting. This criteria. When the millipore detection antibody was used with the r and d systems capture antibody, the limit of detection was less than 7.81 picograms per milliliter.
In this case, the second lowest TNF alpha concentration produced in acceptable response of 17 MFI greater than the response of the lowest TNF alpha concentration plus three times its standard deviation. Similarly, the limit of detection for the r and d systems duo set Eliza was estimated to be between 63 and 31 picograms per milliliter. Following this procedure, other steps like testing, various sample matrices or concentrations of antibody in reporter can be performed in order to further optimize the xap assay.
