Removal of Inhibitory Effects in a Serum Cardiac Troponin I Immunoassay (original) (raw)

Journal Article

,

Address correspondence to this author at:, National Institute of Standards and Technology, 100 Bureau Dr., Stop 8312, Gaithersburg, MD 20899-8312, Fax 301-330-3447, E-mail lili.wang@nist.gov

Search for other works by this author on:

,

Chemical Science and Technology Laboratory

, National Institute of Standards and Technology (NIST), Gaithersburg, MD

Search for other works by this author on:

,

Chemical Science and Technology Laboratory

, National Institute of Standards and Technology (NIST), Gaithersburg, MD

Search for other works by this author on:

Chemical Science and Technology Laboratory

, National Institute of Standards and Technology (NIST), Gaithersburg, MD

Search for other works by this author on:

Published:

01 November 2009

• PDF
• Split View
• • Article contents
  • Figures & tables
  • Video
  • Audio
  • Supplementary Data
• Cite

Cite

Lili Wang, David M Bunk, Hua-Jun He, Kenneth D Cole, Removal of Inhibitory Effects in a Serum Cardiac Troponin I Immunoassay, Clinical Chemistry, Volume 55, Issue 11, 1 November 2009, Pages 2055–2056, https://doi.org/10.1373/clinchem.2009.126011
Close

Navbar Search Filter Mobile Enter search term Search

To the Editor:

The measurement of cardiac troponin I (cTnI)1 has become the gold standard for the clinical diagnosis of myocardial infarction (1). Varieties of commercial sandwich-type immunoassays are used for the measurement of cTnI concentration in human serum or plasma. Several of these assays use pairs of monoclonal antihuman cTnI antibodies with high specificities for the invariant part of the cTnI molecule (amino acid residues 30–110), in accordance with the recommended guidelines (2). These assays are often based on magnetic beads or latex particles to take advantage of the ease of bead/particle washing, which can minimize assay interference by matrix proteins. Signal amplification by means of chemiluminescence or electrochemiluminescence allows the detection of serum cTnI at concentrations <0.05 μg/L.

As a part of the recent effort of the IFCC Working Group for Standardization of Troponin I to develop a cTnI reference measurement system (1), we have used various measurement techniques to investigate binding affinities between 6 monoclonal antibodies (mAbs) obtained from HyTest (each serving as either the capture or the detection antibody) and cTnI, in the form of either a reference material (NIST SRM 2921) or a cTnI-positive serum pool (PS). One of the techniques is the multiplexed bead array (3), which resembles the commercial bead- or particle-based immunoassays. The capture mAb molecules are covalently immobilized on different bead populations to capture cTnI. The bound cTnI is recognized by the biotinylated detection antibody, which is then bound to a fluorescent reporter, streptavidin–phycoerythrin (SA-PE). Although commercial cTnI assays show higher detection sensitivities, the ease of the assay multiplexing and the low cost of the instrumentation make the bead-array platform advantageous for optimization of antibody pairs for cTnI detection.

It is well known that a human serum matrix can introduce inhibitory effects that cause immunoassays to report lower concentrations of cTnI. These inhibitory effects could be due to the presence of heterophilic antibodies and/or autoantibodies in the serum (4). Finding the optimal conditions to minimize the inhibitory effects and enhance cTnI detection is crucial to obtain the most robust and accurate immunoassay results. Therefore, bead arrays were used in the testing of 18 different conditions, including depletion of serum samples before analysis with commercial antibody-based multiprotein depletion columns, magnetic beads coupled with protein G or protein A, and carboxylated beads covalently coupled to antimouse IgG. In addition, various antihuman antibodies to serum protein (IgG, IgA, IgM, complement component C3), mouse IgG, detergent (Triton X-100), buffer solutions with different pH values (4.0–7.4), EDTA, and a combination of both buffer solution and EDTA were added separately to serum samples before the cTnI-capture step. For both SRM 2921 spiked into normal (nonpathologic) human serum (NS) and a PS, the addition of both EDTA and a buffer solution at pH 5.2 to the sample substantially diminished the inhibitory effect of the matrix on bead assays performed with different antibody pairs. An optimized bead-array assay protocol is as follows: Initial addition of 12.5 μL of a solution of 15 mmol/L EDTA in sodium acetate buffer (0.1 mol/L), pH 5.2, is followed by addition of 10 μL NS, 7.5 μL SRM 2921 spiked into PBS blocking buffer [BB in the letter refers to 0.01 mol/L PBS (0.138 mol/L NaCl, 0.0027 mol/L KCl), pH 7.4; 1% BSA; 0.05% NaN3.], and 10 μL BB. After beads (14 μL) are added and incubated for 30 min, 10 μL biotinylated mAb (8.1 × 104 μg/L) is added. After 30 min, 10 μL SA-PE (1.0 × 105 μg/L) is added.

The left panel of Fig. 1 shows that for SRM 2921 spiked into NS, the addition of 12.5 μL of acetate-EDTA solution to a total sample volume of 40 μL before the addition of the capture mAb–coated beads produces a significant increase in assay response (▾), compared with the same dilution of the serum in BB (▴). This increase in signal was also observed for PS (right panel). The addition of this buffer component to the serum samples increases the sensitivity of cTnI detection and enables more reliable measurement of cTnI in serum. Considering that the strong interaction between cTnI and troponin C (TnC) is Ca2+ dependent (5) and that the high positive charge of cTnI (pI 9.87) increases interactions with negatively charged molecules such as TnC (pI 4.05), our result suggests that cTnI in both SRM 2921 and PS is largely associated with TnC, because both a low buffer pH and EDTA likely help to reduce the strong interaction between cTnI and TnC and thereby allow binding of the capture mAb to cTnI. Although this approach has not been evaluated on commercial cTnI immunoassay platforms, we speculate that it may also be beneficial to commercial platforms for enhancing the sensitivity and reliability of cTnI detection.

Figure 1.

Immunoassay results for spiked-in SRM 2921 (left panel) and a PS (right panel), with NS serving as the control and use of a bead array with a capture antibody against cTnI amino acid residues 83–93 and a detection antibody against residues 41–49 (both from HyTest).

Assay protocol: Addition of 12.5 μL acetate-EDTA solution is followed by addition of 10 μL NS, 7.5 μL SRM 2921 spiked into BB, and 10 μL BB. After beads (14 μL) are added and incubated for 30 min, 10 μL biotinylated mAb (8.1 × 104 μg/L) is added. After 30 min, 10 μL SA-PE (1.0 × 105 μg/L) is added. AU, arbitrary unit.

Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors’ Disclosures of Potential Conflicts of Interest:No authors declared any potential conflicts of interest.

Role of Sponsor: The funding organizations played a direct role in the design of the study, the review and interpretation of data, and the preparation and final approval of the manuscript.

Acknowledgments: We are indebted to Robert Christenson at the University of Maryland School of Medicine for supplying the cTnI-positive serum pool for the current investigation. The serum was provided with institutional review board approval and in accordance with CLSI document C37a.

Disclaimer: Certain commercial equipment, instruments, and materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.

1

Nonstandard abbreviations: cTnI, cardiac troponin I; mAb, monoclonal antibody; PS, cTnI-positive serum pool; SA-PE, streptavidin–phycoerythrin; NS, normal (nonpathologic) human serum; BB, PBS blocking buffer; TnC, troponin C.

References

1

Panteghini M, Bunk DM, Christenson RH, Katrukha A, Porter RA, Schimmel H, . for the IFCC Working Group on Standardization of Troponin Iet al. Standardization of troponin I measurements: an update.

Clin Chem Lab Med

2008

;

46

:

1501

-1506.

2

Panteghini M, Gerhardt W, Apple FS, Dati F, Ravkilde J, Wu AH. Quality specifications for cardiac troponin assays.

Clin Chem Lab Med

2001

;

39

:

175

-179.

3

Wang L, Cole KD, Peterson A, He HJ, Gaigalas AK, Zong Y. Epitope selection of monoclonal antibodies for interleukin-4 quantification using suspension arrays and forward-phase protein microarrays.

J Proteome Res

2007

;

6

:

4720

-4727.

4

Kim WJ, Laterza OF, Hock KG, Pierson-Perry JF, Kaminski DM, Mesguich M, et al. Performance of a revised cardiac troponin method that minimizes interferences from heterophilic antibodies.

Clin Chem

2002

;

48

:

1028

-1034.

5

Reiffert S, Jaquet K, Heilmeyer L, Herberg F. Stepwise subunit interaction changes by mono- and bisphosphorylation of cardiac troponin I.

Biochemistry

1998

;

37

:

13516

-13525.

© 2009 The American Association for Clinical Chemistry

Citations

Views

Altmetric

Metrics

Total Views 262

190 Pageviews

72 PDF Downloads

Since 1/1/2020

Citations

3 Web of Science

×

Email alerts

Citing articles via

• Latest

• Most Read

• Most Cited

More from Oxford Academic