Development of two Multiplex Immunoassays

By: Jiaxin Dong, Chris Melm, Holly Chapman, Farrah Fan, Dian Er Chen, Henry Duewel, Biowire Volume 10 Article 1

Preliminary Application in Determining Protein Depletion for Seppro® IgY14/SuperMix Depletion Columns


Depletion of Highly Abundant Protein (HAP) and Moderately Abundant Protein (MAP) from plasma or serum samples can be achieved through our Seppro® Tandem IgY14/ SuperMix columns.

A range of 77–129 MAPs has been identified from the bound fraction of Seppro SuperMix resin by LC/MS-MS method. However, the efficiency and lot-to-lot consistency of MAP depletion using SuperMix resin has not been determined and requires a robust method to monitor. By taking advantage of the Luminex® technology, we have developed two multiplex immunoassays to monitor protein targets of depletion or retention. The MAP-10PLEX assay can simultaneously quantify 10 MAPs (Ceruloplasmin, C4, Plasminogen, IgD, C1q, Antithrombin III, Hemopexin, α-Antichymotrypsin, CRP and Prealbumin) from plasma or serum samples depleted utilizing IgY14/ SuperMix columns. The LAP4PLEX assay, which is used to track loss of low abundant proteins (LAP) during depletion, is able to simultaneously quantify Adiponectin, soluble L-selectin, soluble ICAM-1 and TIMP-1. The preliminary assay data from the R & D, Operations and QC departments on Seppro IgY-SuperMix depleted samples has suggested the two assays to be accurate, fast and cost-saving methods to monitor depletion consistency from lot-tolot of the SuperMix resin.

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An avian IgY based immunodepletion technology is used for our Seppro® depletion products. The Human IgY14 Columns can remove 14 highly abundant proteins (HAP) and the unique Human SuperMix System is used to remove moderately abundant proteins (MAP). The combination of IgY14/SuperMix columns can deplete approximately 96–99% of total protein mass from human serum or plasma (Figure 1).

Figure 1 Seppro® SuperMix Depletion System

While the depletion of 14 HAPs has been well established, the further removal of MAPs is less well characterized. Previously, a range of 77-129 MAPs has been identified from the bound fraction of Seppro SuperMix resin by LC/MS-MS. However, the degree of specific depletion of MAPs as well as the non-specific impact on low abundant proteins (LAP) is still unclear. This also makes tracking the lot-to-lot depletion consistency of SuperMix products a challenge.

Regular ELISA methods are time-consuming and commercial ELISA kits are costly. Therefore, finding a fast and economical alternative analysis has appeared to be important. Luminex xMAP technology is a widely used multiplex platform to quantify multiple analytes from biological fluid samples. It combines the accuracy of ELISA and multiplicity of antibody arrays through protein-antibody interaction on the surface of microspheres and is characterized by speed and minimal consumption of samples (Figure 2). In order to evaluate Seppro SuperMix resins in the R & D department and to monitor the lot-to-lot consistency of materials produced by Operations in the QC department, we developed two Luminex immunoassays to simultaneously quantify 10 representative MAPs (Ceruloplasmin, C4, Plasminogen, IgD, C1q, Antithrombin III, Hemopexin, α-Antichymotrypsin, CRP and Prealbumin) and 4 representative LAPs (Adiponectin, soluble L-selectin, soluble ICAM-1 and TIMP-1) from depleted plasma via Seppro SuperMix columns.

Figure 2 Principle of Luminex Multiplexing Assay

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Materials and Methods

  1. Instrument: Luminex® 100 (Figure 2), Luminex Corp., Austin, Texas, USA
  2. Vacuum pump, 96-well microtiter filter plates and filtration manifold (Millipore Corp.)
  3. Capture antibodies were immobilized on Luminex® beads, detection antibodies were biotinylated before use, streptavidin-phycoerythrin conjugate (Cat. No. E4011)
  4. Data were calculated using STATLIA® software with a 5-parameter logistic curvefitting method (Brendan Scientific, CA).
  5. Depletion procedure: 50 or 100 μL human plasma (Cat. No. P9523) was loaded to Seppro IgY14-LC5/SuperMix- LC2 columns with tandem connection (Figure 3). Depletion was run in Waters 2695 HPLC System (Alliance). After collection of flowthrough from combined columns (SuperMix depleted sample), bound proteins were eluted from IgY14- LC5 and SuperMix LC2 column separately.
  6. Multiplex assay protocol: 50 μL standards or samples are mixed with 25 μL antibody conjugated bead mixture in 96-well filter plate. Shake 2 hr at RT or overnight at 4ºC and wash plate twice. Add 50 μL/well detection mixture, shake 1hr at RT and wash plate once. Add 50 μL/well SAPE, shake 30 min at RT and wash plate twice. Add 100 μL/well sheath fluid and shake 2–5 min at RT. Read plate on Luminex Instrumentation.


Click for Larger Image

Figure 3. Flowchart of Seppro® SuperMix Depletion Process and the Targets to Test.

The top 14 HAPs (plasma level ranges in 0.5-50 mg/ml) compose 95% of total plasma proteins. The 10 targets to test for SuperMix depleted plasma cover a wide range concentration (2.3 μg/ml for CRP, 0.5 mg/ml for Ceruloplasmin) of MAPs. The 4 LAP targets in normal human plasma ranges in 0.2-10 μg/ml.

Dilution linearity is commonly used to evaluate assay accuracy that is one of the most important requirements for immunoassays. A serial dilution of a pooled plasma was tested with MAP10PLEX and LAP4PLEX assay. The numbers in the table represent percentage of concentrations at each dilution point against start point. Both assays perform well. For normal human plasma samples, dilutions in the range from 5 x 104 to 5 x 105 for MAP10PLEX assay or 250 to 2000 for LAP4PLEX assay are suggested.

Figure 4 MAP 10PLEX and LAP 4PLEX assays

Performed with protocol described above and standard curves are plotted for each assay.

Figure 5 Depletion Efficiency of Seppro SuperMix Columns Tested by Luminex MAP 10PLEX Assay

Depletion efficiency of 10 representative MAPs is related to loading volume of plasma samples. >90% of all 10 MAPs can be depleted from 50 μL human plasma.

Figure 6 Depletion Efficiency of Seppro SuperMix Columns Tested by Indirect ELISA (for 6 targets only)

The same set of SuperMix depleted samples were tested by indirect ELISA and displayed a comparable result as Luminex MAP10PLEX assay.

Figure 7 Run-to-Run Depletion Consistency of SuperMix Columns

Three depletion runs were performed with 100 μL plasma utilizing Seppro IgY14 LC5/SuperMix LC2 columns and the depletion efficiency was determined by Luminex MAP10PLEX assay. Depletion of all 10 targets is consistent (CV%<15%)

Figure 8 Impact of SuperMix Depletion Process on LAP s

Four representative LAPs were tested by Luminex LAP4PLEX assay to monitor the loss of LAPs during SuperMix depletion process. Depending on different biological properties of the individual target, approximately 1-40% loss has been observed from SuperMix depleted plasma (Note: some proteins in bound fraction are unstable).

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  1. The MAP10PLEX and LAP4PLEX assays are able to simultaneously measure 10 MAPs or 4 LAPs from human plasma or serum samples within 4 hours.
  2. These two multiplex assays can be used to replace regular ELISA methods to determine MAP depletion and LAP retention from Seppro® IgY-14/SuperMix depleted samples.
  3. The preliminary test indicated that depletion of 10 MAPs from human plasma through the tandem IgY14/ SuperMix columns were reproducible and is consistent with Dr. Smith’s result based on spectral count data (Ref. 3).
  4. In order to remove > 90% of MAPs from plasma, columns with equal volume of IgY14 and SuperMix resins (IgY14/SuperMix ratio = 1:1) should be combined.

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The newly developed MAP10PLEX and LAP- 4PLEX assays using Luminex technology have been proven to be both time-and cost-saving solutions to monitor the robustness of protein depletions from lot-to-lot and sample-to-sample.

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  1. Seppro® Depletion Product Information
  2. Technical Bulletin, Luminex Corp., (2001).
  3. Qian W.J., et al., Molecular & Cellular Proteomics, 10, 1963-75 (2008).

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