A Highly Sensitive, Reproducible Assay for Determining 4-hydroxynonenal Protein Adducts in Biological Material

Materials and Reagents

1. Paper towel
2. 96-Well Plate (Clear Flat-Bottom, MaxiSorp, Thermo Fischer Scientific, catalog number: 442404)
3. 4-hydroxynonenal (Caymen Chemical, catalog number: 32100)
4. Bovine Serum Albumin solution, sterile (Sigma-Aldrich, catalog number: A9576)
5. Immuno Shot-Platinum (Cosmo Bio, catalog number: CSR-IS-P-500)
6. Anti-HNE antibody (Percipio Biosciences, catalog number: 24327)
7. Goat Anti-Mouse HRP conjugated antibody (Santa Cruz Bio Technology, catalog number: 170-5047)
8. Amplex UtraRed Reagent (Thermo Fischer Scientific, catalog number: A36006)
9. Sodium Phosphate Buffer (50 mM, pH 7.4)
10. NaCl (Sigma-Aldrich, catalog number: S9888)
11. KCl (Sigma-Aldrich, catalog number: P3911)
12. Na₂HPO₄·2H₂O (Sigma-Aldrich, catalog number: 71643)
13. KH₂PO₄ (Sigma-Aldrich, catalog number: 795488)
14. Tween 20 (Sigma-Aldrich, catalog number: P1379)
15. RIPA Buffer (Thermo Fischer, catalog number: 89901)
16. Tris Base (Roche, catalog number: TRIS-RO)
17. EDTA (Sigma-Aldrich, catalog number: EDS)
18. EGTA (Sigma-Aldrich, catalog number: E3889)
19. Triton X-100 (Sigma-Aldrich, catalog number: X100)
20. H₂O₂
21. 1x Phosphate Buffered Saline (PBS, see Recipes)
22. 1x ELISA Wash Buffer (see Recipes)
23. 1x Amplex UltraRed Solution (see Recipes)
24. 1x TEE-T Buffer (see Recipes)
    

Equipment

1. Incu-Mixer (Benchtop Scientific, model: MP4)
2. Standard Lab Rocker (any brand)
3. Fluorescent multi-well plate reader
4. Duall Tissue Grinder (Kimble, model: 3432N07)
   

Procedure

1. Sample Preparation
   
   1. Serum
      Dilute serum 1:10 in PBS (see Recipes) before plating.
      
   2. Tissue Homogenate
      Using a Duall Tissue Grinder (Kimble) and a 1 g: 10 ml weight to volume ratio, homogenize heart tissue in TEE-T buffer (see Recipes).
      
   3. Cell Lysate
      Lyse cells using RIPA buffer at a ratio of 1 ml buffer: 10⁶ cells.
      
   4. Positive Controls
      For cell culture: Add 100 μM H₂O₂ to cells for 2 h, and prepare cell lysate in RIPA buffer. Include those samples on ELISA plate.
      For serum or tissue homogenate: After homogenization and prior to antigen adsorption step (Step 2), add 1.25 μM HNE-BSA adduct (from Step 2) to at least 2 unknown samples, and include those samples on ELISA plate.
2. Preparations of HNE-adduct solutions for standard curve
   
   1. A standard curve is prepared fresh, one day prior to each ELISA, with serial dilutions of HNE in 1% BSA (diluting BSA solution in 50 mM PBS, pH 7.4). While keeping BSA constant, HNE is varied serially (0-0.019-0.0375-0.075-0.15-0.31-0.625-1.25-2.5-5 μM) in a 200 μl solution of 1% BSA.
   2. Incubate at 37 °C for 24 h.
3. Antigen adsorption
   
   1. Add 100 μl of standard curve solutions and 100 μl of unknown samples to wells, in triplicate, and cover with adhesive seal.
   2. Incubate overnight at 4 °C while rocking.
4. Plate wash
   
   1. Empty plate and remove excess fluid by blotting on paper towel.
   2. Add 200 μl of ELISA wash buffer to each well.
   3. Repeat Steps 4a and 4b until the plate has been washed a total of 6 times.
5. Blocking
   
   1. Add 200 μl of Immuno Shot-Platinum blocking agent to each well and cover with adhesive seal.
   2. Incubate for 4 h at room temperature while rocking.
6. Plate wash
   Wash plate as described in Step 4.
7. Primary antibody incubation
   
   1. Add 100 μl of a 1:100 dilution of primary antibody (Percipio Biosciences) in PBS to each well and cover with adhesive seal.
   2. Incubate for 2 h at 37 °C with agitation in the Incu-Mixer.
8. Plate wash
   Wash plate as described in Step 4.
9. Secondary antibody incubation
   
   1. Add 100 μl of a 1:7,500 dilution of secondary antibody (Santa Cruz BioTechnology,) in PBS to each well and cover with adhesive seal.
   2. Incubate for 2 h at 37 °C with agitation in the Incu-Mixer.
10. Plate wash
    Wash plate as described in Step 4.
11. Developing and reading fluorescence (Amplex UltraRed Solution)
    
    1. Add 100 μl of Amplex UltraRed Solution (prepared fresh) to each well and cover with adhesive seal.
    2. Incubate at room temperature, protecting from light.
    3. Using a multi-well plate reader, measure fluorescence in each well at ~560 nm/590 nm (ex/em spectrum for Resorufina, the oxidized product formed in the reaction of H₂O₂ with Amplex Red, catalyzed by HRP). Do this 15, 30, and 60 min after addition of Amplex UltraRed Solution.

Data analysis

1. Enter fluorescent values for each sample into analytical software (e.g., Excel, Graphpad Prism, SigmaPlot).
2. Fluorescent values for each time-point (15, 30, 60 min) must be compared with the values for the HNE-BSA standards at the same time-point. In some cases, 15 min may not be long enough, or 60 min may be too long. This needs to be individually optimized for each laboratory, and may vary based on sensitivity and availability of fluorescent plate-reader, and/or other factors.
3. Standard curve is fit to proper equation (typically 3^rd order polynomial).
4. Values in the unknown sample wells are interpolated using equation for standard curve.
5. Concentration of HNE-adducts in the unknown sample wells should be normalized to the protein concentration in each well.
   
   

Results

A representative HNE-adduct ELISA from our laboratory using tissue lysate prepared from human atrial biopsy is shown in Figure 1, and has previously been published (Katunga et al., 2015b). Lysate is prepared using TEE-T buffer as described in the Recipes below. The interpolated data shown in Figure 1C is then normalized to the protein concentration for each sample. As is clear from these data, sensitivity of the assay scales to the low nanomolar range for tissue lysate, but these values must be normalized to protein concentration within the well for final presentation. Final units are typically expressed as “nmol HNE-adduct/mg protein (or μmol/g).”


Figure 1. Representative data set of HNE-adduct ELISA used to measure HNE-adducts in human cardiac tissue homogenate/lysate prepared from right atrial appendage biopsy obtained at time of surgery (as described in Anderson et al., 2014; Katunga et al., 2015a). Samples were prepared as described in the protocol, and diluted 1:10 into PBS. In panel A is the standard curve of known concentrations of HNE-BSA adducts, in panel B is raw fluorescent data captured from samples on the same plate, and in panel C is the interpolated data from same samples. The standard curve was fitted using a third order polynomial equation (cubic), with 95% confidence intervals shown in dashed lines.

Recipes

1. 1x Phosphate Buffered Saline (1 L, pH 7.4)
   8 g NaCl
   0.2 g KCl
   1.44 g Na₂HPO₄·2H₂O
   0.24 g KH₂PO₄
2. 1x ELISA Wash Buffer (1 L)
   0.5 ml of Tween 20
   999.5 ml of PBS
3. 1x Amplex UltraRed Solution (10 ml, for full 96-well plate)
   50 μl of Amplex UltraRed (10 mM in DMSO)
   23 μl of 3% H₂O₂
   9.925 ml of PBS
   Note: Prepare immediately prior to Step 11.
4. 1x TEE-T Buffer (1 L, pH 7.5)
   1.21 g Tris base
   292 mg EDTA
   380 mg EGTA
   5 ml Triton-X
   995 ml distilled water
   

Acknowledgments

This work was supported by funding from National Institutes of Health (HL122863, AG057006) and Department of Defense (PR181276) to E.J.A.

Competing interests

Authors have no conflicts of interest to disclose.

References

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